[Parenteral nutrition and concentrated amino acid solution]. The authors report on their experience with a solution of amino acids concentrated at 25 gomegaN/liter in eighty patients on exclusive and total parenteral nutrition (TPN). The average duration of the TPN is 35.08 days. Azonutril 25 was given as nutritive melange, maintaining a calorie-nitrogen ratio of 150 to 200 calories for every added gram of nitrogen. The clinical and biological studies provide a series of arguments in favor of such an amino acid solution in total parental nutrition in patients with acute or chronic digestive insufficiency. No signs of renal or hepatic intolerance have appeared during this study. The improvement in nitrogen balance in fifteen patients is strongly significant (P less than 0.001) and can be correlated with weight gain and with the classic immunological tests.


[Four cases of tracheoesophageal fistula associated with prolonged ventilation]. On the basis of four cases of oesophago-tracheal fistulae developing in ventilated patients, the authors describe the pathogenesis of these lesions, related to circulatory disturbances in the tracheal mucosa in contact with the balloon of the balloon of the tracheal tube. The spontaneous course is rapidly fatal and the results of surgical treatment remain disappointing. Prevention of these lesions is essential but difficult. It is based above all on the use of "large volume and low pressure" balloons, with periodic exsufflation. The diagnosis of these lesions must be make early and is base upon the development of tracheal dilatation, even minimal and seen radiologically by the appearance of the tube balloon. Such a finding should lead to endoscopic exploration of the tranchea and oesophagus, in order that treatment may be strated rapidly.


[Visual identification of Doppler sounds in gas embolisms]. The method of choice for the early detection of air metabolism during neurosurgical operations in a sitting position is that of continuous cardiac auscultation using Doppler effect ultrasound apparatus. In the present study, involving 15 patients, an air embolism was produced by the injection of 2 ml of CO2 into the right heart and acoustic changes were recorded on paper using a photographic process. These optical changes, although variable, were invariably present and characteristic of aire emboli and represent an additional element in early diagnosis.


[Traumatic intoxication by an anticholinesterase agent]. An unusual observation is reported concerning intoxication by an anticholinesterasic. In this case a traumatic blow was caused by an industrial liquid projected under high pressure and resulted in a true parenteral injection. This product, presumed not to be toxic, lead to the delayed appearance of a massive and eventually fatal intoxication. The problemes of prevention, diagnosis and treatment of this type of intoxication are commented on.


[Anesthetic evidence of pheochromocytoma]. The authors report the clinical case of a patients who underwent four anaesthetic inductions during a period of 3 months. Two were associated with severe acute hypertension making the intended operation impossible at that time. Thorough investigations led to a diagnosis of phaeochromocytoma. Reviewing the protocol of the four anaesthetic inductions, the authors discuss the mechanism of provocation of paroxysmal hypertension which was seen on two occasions.


Calcium ion regulation in barnacle muscle fibers and its relation to force development. It is possible to use the photoproteins aequorin and obelin within the slowly responding striated muscle films to provide both qualitative and quantitive information as to the nature of the calcium-dependent reactions for force development. It seems that these reactions are not in equilibrium with the Ca2+ during a transient response. In addition, these experiments suggest that kinetically at least two calciums are required per functional unit for force development and that this active site is competed for by Mg2+, H+, and K+ ions.


Intracellular and extracellular calcium ions in transmitter release at the neuromuscular synapse. The theme of this presentation has been to show that the control of transmitter release at the neuromuscular synapse is achieved by extracellular and intracellular calcium. For the fast information transfer represented by the end-plate potential, the electrochemical gradient for calcium across the presynaptic membrane and the associated calcium conductance seem to play the primary role. For slower processes such as tetanic and posttetanic potentiation, the combined effect of both sources for calcium determine the amount of transmitter liberated.


[Cutaneous periarteritis nodosa. Critical study about 4 cases (author's transl)]. Cutaneous form of periarteritis nodosa. Four personal cases are added to 70 cases of the previous literature. The clinical and biological picture of the cutaneous form of P.A.N. are different from the systemic one by the lack of visceral involvement and a noteworthy good long-term prognosis. Venous (superficial thrombophlebitis) and distal arterial involvement in some cases, raise doubts about diagnosis and classification. The solution is in an accurate definition of the diagnostic clues and specially of the histopathological ones.


The graft-versus-host reaction in chickens: determination of some parameters. Some aspects of the ""Graft-Versus-Host'' (GVH) reaction were studied in White Leghorn chickens by assessing the splenic response of recipients to an injection of lymphocytes from donors. The responses varied according to the origin of recipient embryos as well as to the source of the donor lymphocytes. Blood lymphocytes induced a higher splenomegaly in recipients than spleen lymphocytes, at least in the first seven weeks of life. The influence of the thymus on GVH reactions was also investigated. Lymphocytes from thymectomized chickens induced stronger responses than lymphocytes from non-thymectomized ones. These results support the conclusion that in the chicken T-lymphocytes may not be the only cells involved in the development of GVH reactions and cast some doubts on the reliability of GVH reactions to check the T specificity of immunological cellular responses.


A comparative study of three beta 1-adrenoreceptor blocking drugs with different degree of intrinsic stimulating activity (metoprolol, practolol and H 87/07) in patients with angina pectoris. Three beta1-selective beta-blocker (metoprolol, practolol and H 87/07) were compared in 29 patients with stable angina pectoris. The main pharmacological difference between the three beta-blockers was their intrinsic stimulating activity (I.S.A.), metoprolol being devoid of I.S.A., practolol having moderate I.S.A. and H 87/07 having high I.S.A. Each drug was given in randomized order and the length of each cross-over period was 2 weeks. Daily activity was measured by an automatic step-counter, and subjective symptoms and nitroglycerin consumption were registered on a diary-card. Objective data, such as ECG changes and exercise capacity, were obtained by bicycle ergometer tests performed at the end of each period. At rest, the heart rate was significantly lower on metoprolol than on practolol or H 87/07. During exercise, the heart rate was significantly higher on H 87/07 than on practolol or metoprolol. No other haemodynamic differences were found between the three beta-blockers. No differences were found between the three test periods with regard to daily activity, expressed as the number of steps walked, while on the beta-blocker with high I.S.A., H 87/07, the attack rate and nitroglycerin consumption were significantly higher than when the patients were on metoprolol and practolol. No difference was found between the three beta-blockers with regard to total work or exercise time until 1 mm of S-T segment depression. Except for one patient who experienced a severe exanthema on practolol, the three beta-blockers were equally well tolerated.


[Nisin formation by immobilized cells of the lactic acid bacterium, Streptococcus lactis]. The problem of microbial cell immobilization at present attracts the ever increasing attention of the scientists, since such organisms may be the source of various enzymes. Production of nizin by the immobilized cells of Str. lactis was studied. It was found that the cells of Str. lactis incorporated into polyacrylamide gel produced nizit on definite media. Still, the amount of the antibiotic was 2-3 times lower than in case of using free cells. The effect of a number of factors on the process of immobilization was studied and the influence of some factors, such as temperature, pH, aeration on nizin synthesis by the immobilized cells of the streptococcus was elucidated. Optimal conditions for nizin biosynthesis by the immobilized cells of Str. lactis were developed.


Efficiency of beef extract for the recovery of poliovirus from wastewater effluents. The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.


Protection of rats from compound 48/80-induced lethality. A simple test for inhibitors of mast cell-mediated shock. The dose of 0.5 mg/kg i.v. of compound 48/80 was lethal in 97.2% of the injected rats. Observations before death and at autopsy were in accordance with the basic effect of compound 48/80 in rats i.e. the sustained release of mast cell mediators, whose action on the cardiovascular system leads to circulatory collapse. The administration of drugs with various pharmacological effects before the intravenous challenge with compound 48/80 allowed us to conclude that the following effects are not sufficient to prevent the lethal shock: inhibition of prostaglandin biosynthesis; H2-histamine antagonism; cholinergic, alpha- or beta-adrenergic blockade; beta-adrenergic stimulation; CNS-effects of antidepressants, hypnotics, sedatives, neuroleptics or narcotic analgesics; ganglion blockade; glucocorticoid or cromoglycate-like activity. Dose-dependent protection from the lethal reaction was obtained with compounds known to exert a single or several actions of the following types: oxatomide-like inhibition of mast cell mediator release; h1-histamine antagonism; serotonin antagonism. Quantitatively, however, when measured in in vitro systems these effects are poorly related to the protection from lethal compound 48/80 challenge. The new test offers the advantage of a simple, comprehensive measure of the potency of a compound to prevent mast cell-mediated shock.


Anticholinesterase activity of some butyrophenones. The anticholinesterase (antiChE) activity of haloperidol, droperidol and trifluperidol was studied by employing dog plasma as a cholinesterase source and changes in arterial blood pressure, elecited by a mixture of acetylcholine (Ach) and dog plasma with or without those drugs, as an indicator of their antiChE activity. When butyrophenones were present in concentrations of 0.5 up to 5 X 10(-4)M in plasma containing Ach the hypotensive effect of the latter was preserved. Such findings provide experimental evidence that butyrophenones may exert antiChE activity.


Effects of indenolol (YB-2), a new beta-adrenergic blocking agent, and its dextro isomer on the central nervous system of mice and rabbits. Central depressant actions of a new beta-adrenergic blocking agent, indenolol (YB-2), and its dextro isomer were studied in mice and rabbits. d-Indenolol was less active than dl-indenolol in lowering of methamphetamine group toxicity and calming of fighting behavior, though muscle relaxant, hypolocomotive and anticonvulsant effects of d-indenolol were virtually equal to those of the racemate. dl-Propranolol showed a similar profile of central depressant actions to dl-indenolol, whereas dl-practolol revealed no significant central effects in mice. The dose-response relationship between beta-blocking and central effects in the same animal species suggested that both taming and anti-methamphetamine group toxicity effects of dl-indenolol s.c. do not directly relate to either the peripheral beta-blocking or the membrane stabilizing activity. Intravenous as well as intracerebroventricular administration of dl-indenolol resulted in suppression of pressor responses to the electrical stimulation of the brain stem of unanesthetized rabbits. Neither d-indenolol nor dl-practolol was effective against the pressor responses, suggesting that these two compounds acted on the CNS in a manner different from dl-indenolol even when intracerebroventricularly administered. A possible central beta-adrenergic blocking effect of dl-indenolol was discussed in the light of mechanisms of an antihypertensive action of this compound.


[Experimental analysis of interference between alpha-adrenergic stimulants with anti-hypertensive activity and the narcotic activity of pentobarbital]. The interactions of 3 classical alpha-adrenergic antihypertensives of prevalently central type (St 155 or clonidine St 600; BR 750 or guanabenz) with the narcotic effects of pentobarbital have been investigated in the Mus musculus. Interferences between convulsant drugs and spontaneous and coordinatory motor activity were also analysed in comparative studies.


Influence of aminophylline or beta-adrenoceptor blockade on glucagon release by a vasoactive adenosine analogue. The glucagon-releasing activity of adenosine-5'-ethyl-carboxamide (744-96) was studied in conscious dogs pretreated either with aminophylline or with bupranolol, a beta-adrenoceptor blocking drug. The adenosine antagonist, aminophylline, inhibited both the glucagon-releasing and the blood pressure lowering effects of 744-96 (10 microgram/kg i.v.). The actions of the adenosine analogue on glucagon release and haemodynamics were not affected by bupranolol pretreatment. These results indicate that the adenosine analogue mediates glucagon release by interaction with so-called adenosine receptors. A beta-adrenoceptor mediated release secondary to 744-96-induced hypotension and baroreceptor-activation can be excluded.


[Importance of measuring blood volume by an isotope method in essential hypertension]. Measurement of the blood volume in cases of essential hypertension has shown a negative correlation with increase in blood pressure. We have been able to distinguish a group of moderate hypertensives who are hypervolemic, and whose systemic effect is more marked than in the moderate hypovolemic type. Such cases respond poorly to single drug therapy with beta blockers, but well to diuretics. The authors propose a general treatment scheme for essential hypertension: single therapy (beta blockers or diuretics) in mild cases of hypertension; single therapy by diuretics in the moderate types with the increased volume; single therapy by beta blockers in the moderate types with low volume, multiple treatment with the drugs together in cases of severe hypertension.


Neuroleptic-induced prolactin level elevation and breast cancer: an emerging clinical issue. This article reviews the evidence that neuroleptics may increase the risk of breast cancer via their effects on prolactin secretion. All available neuroleptics, including reserpine, raise serum prolactin levels. Elevated serum prolactin level increases the incidence of spontaneously occurring mammary tumors in mice, and increases the growth of established carcinogen-induced mammary tumors in rats. Caution is necessary in extrapolating this relationship to human mammary tumors because human and rodent tumors differ in some important characteristics, including hormone responsiveness. Serum prolactin levels in women with, or at risk for, breast cancer have generally been normal, and only a minority of human mammary tumors respond to changes in serum prolactin levels. Epidemiologic studies have failed to demonstrate an increased risk of breast cancer associated with the use of neuroleptics or reserpine. Thus, although some human mammary tumors are prolactin dependent, the available evidence does not demonstrate an increased risk of breast cancer in women receiving neuroleptics. We conclude that (1) additional epidemiologic studies of the incidence of mammary tumors in women treated with neuroleptics are desirable; (2) it is premature to mandate warning patients of an unknown and undemonstrated increase in the risk of developing breast cancer associated with neuroleptic treatment; (3) detection of existing mammary tumors by breast examination prior to administration of neuroleptics is desirable; and (4) development of antipsychotic drugs that do not increase serum prolactin level may be indicated.


Delusional unipolar depression: description and drug response. This retrospective study describes the response of 13 delusional unipolar depressives to combination antipsychotic-tricyclic antidepressant drug therapy. The Research Diagnostic Criteria were used by two independent raters to verify the presence of a delusional, primary, unipolar, major depressive disorder. Thirteen patients met the criteria and received combined antipsychotic-antidepressant pharmacotherapy. Twelve of the 13 responded. This contrasts sharply with the low response rate described for delusional depressives when treated with tricyclic antidepressants alone. Descriptive characteristics of the delusional depressives were compared with those of a group of nondelusional unipolar depressives. Delusional depressives had histories of a greater number of prior depressive episodes and more of the delusional depressives demonstrated a psychomotor disturbance.


Benzodiazepines in depressive disorders. Some investigators have found benzodiazepines effective in the treatment of anxious depression and thus have argued that benzodiazepines were "antidepressants." We reviewed the literature on benzodiazepines in depressive disorders. Comparative studies indicate they are less effective than standard antidepressants in the treatment of several types of depressive illnesses. Although they display definite anxiolytic properties and may elevate mood, they exert limited effect on the core symptoms of endogenous depression. An argument is made that benzodiazepines are primarily anxiolytic rather than antidepressant.


Use of antidepressant drugs in schizophrenia. This review surveys the therapeutic efficacy of tricyclic antidepressants and monoamine oxidase inhibitors in schizophrenic patients. In general, the use of these drugs alone was found not to be warranted in schizophrenia, except perhaps in the so-called pseudoneurotic subgroup. In most cases, combinations of antidepressants and phenothiazines were not more beneficial than phenothiazines alone. In particular, the conditions of agitated patients and patients with histories of social deviance dating back to childhood were often made worse by the addition of an antidepressant. However, when the patients who demonstrated symptoms of clinical depression other than anergia were isolated from several of these studies, it was found that they constituted a subgroup that was often benefited by use of these combinations. Favorable and unfavorable clinical response patterns are discussed, and recommendations for future research are outlined.


The effect of methylphenidate on serum growth hormone: influence of antipsychotic drugs and diagnosis. Intravenously administered methylphenidate, 0.5 mg/kg, causes a consistent rise in human serum growth hormone level, with peak values usually occurring 30 minutes after infusion. This rise is attenuated in patients receiving various antipsychotic medications administered on a long-term basis and is decreased in schizophrenic and drug-dependent patients. Methylphenidate causes increases in talkativeness, blood pressure, and pulse that generally parallel increases in serum growth hormone level. However, in contrast to the methylphenidate-induced rise in serum growth hormone level, methylphenidate-induced changes in cardiovascular variables and talkativeness are not altered by antipsychotic medications or diagnostic classification.


Clinical significance of an increased or decreased serum alkaline phosphatase level. An increase in the level of serum alkaline phosphatase (ALP) may be caused by a wide variety of pathologic lesions that involve multiple organs, since the enzyme is an ubiquitous one. Before one attempts to identify a significant pathologic abnormality, the clinician should consider the possibility of a physiologic or spurious cause for an increased level of ALP. A decreased ALP level also has diagnostic value.


Metal chelate affinity chromatography of hamster interferon. Syrian hamster interferon, produced by benzo(a)pyrene-transformed embryo cells does not display an affinity for Zn++ chelate-agarose. However, the interferon binds to Cu++ chelate-agarose and can be displaced from the column by a falling pH-gradient. The chromatography of crude hamster interferon preparation on a tandem of columsn: Zn++ chelate-agarose in equilibrium Cu++ chelate-agarose results in a 25-fold purification of interferon.


Orthodontic seminar: a realistic approach construction, adjustment and repair of appliances. Details of the design and construction of removable and simple fixed appliances are presented. Equipment, instruments, and supplies are listed. Welding, soldering, banding and bonding are illustrated. Finally, step-by-step descriptions are given of first aid measures for the repair of fixed appliances applicable to the general practice situation.


Myocardial infarction--the problems and prognosis: Paper 1. Deaths from coronary heart disease and myocardial infarct may occur in previously undiagnosed subjects (the so called pre-hospital phase), in hospital following admission to a coronary care unit, or following discharge. Mortality in the pre-hospital phase has been estimated at 35--40 per cent. Within Australia the mortality in the coronary care unit is 11--17 per cent. Following discharge, the mortality is about 20 per cent in the first 12 months and then occurs at about four to five per cent per year for the first five years.


Factors influencing the infarct: the place of beta-blockade in limiting infarct size. The mechanisms whereby myocardial infarction in man is precipitated are not understood--largely because the event is usually well established by the time medical help is called, but also because the techniques available have not yet permitted analysis of the earliest changes. On the other hand, the consequences of myocardial infarction for the heart and circulation are now well understood, and the need for therapy better appreciated.


The role of long term beta-blockade after myocardial infarction: Paper 1. The opinion is emerging that beta-blocking drugs have an important role in management of patients following acute myocardial infarction. Already beta-blocking drugs are accepted as the treatment of choice in hypertension and in angina pectoris--in the major risk factor and consequence respectively of coronary atherosclerosis, and both commonly recognized in patients who survive acute myocardial infarction. But beta-blocking drugs also may be of benefit in reducing the incidence and risk of subsequent infarction, and so may be of value for long term treatment of patients who have no symptoms whatever following acute infarction.


The role of long term beta-blockade after myocardial infarction: Paper 2. Acute myocardial infarction is associated with pain, tissue damage and circulatory impairment. As a result of these changes, there is increased sympathetic drive to the heart in the form of increased sympathetic nerve activity and increased circulating catecholamines.


The role of long term beta-blockade after myocardial infarction: Paper 3. For many years the search has gone on for suitable drug therapy to improve survival following myocardial infarction. Most recently, beta-adrenergic blockade has entered the lists. Naturally beta-blockade is used in the conventional way to combat angina pectoris following an infarct, and one can expect that 70 per cent of patients will obtain significant relief.


The role of long term beta-blockade after myocardial infarction: Paper 4. The place of beta-blockade following myocardial infarction should be considered in two phases; the acute infarction, and the long term management after infarction.


Susceptibility of orbiviruses to low pH and to organic solvents. Six orbiviruses, Corriparta, Wallal, Eubenangee, D'Aguilar, Warrego and Mitchell River, were inactivated within 30 min in solutions of pH less than 6. All but Wallal virus resisted treatment with ether at 1 degrees for 22 h but only Corriparta virus resisted treatment with chloroform at 1 degrees for 22 h. The genomes of the orbiviruses, including Corriparta virus, after electrophoresis in acrylamide gels separated into basically similar patterns which were distinct from the pattern of reovirus RNA.


Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae. Data from steady-state kinetic analysis of yeast K+-activated aldehyde dehydrogenase are consistent with a ternary complex mechanism. Evidence from alternative substrate analysis and product-inhibition studies supports an ordered sequence of substrate binding in which NAD+ is the leading substrate. A preincubation requirement for NAD+ for maximum activity is also consistent with the importance of a binary enzyme-NAD+ complex. Dissociation constant for enzyme-NAD+ complex determined kinetically is in reasonable agreement with that determined by direct binding. The order of substrate addition proposed here differs from that proposed for a yeast aldehyde dehydrogenase previously reported. Different methods of purification produced an enzyme that showed similar kinetic characteristics to those reported here.


Electron-paramagnetic-resonance studies on the redox properties of the molybdenum-iron protein of nitrogenase between +50 and -450 mV. The midpoint potentials, Em, for the oxidation of the characteristic e.p.r. signal with g values near 4.3, 3.7 and 2.01, of the nitrogenase Mo-Fe proteins from a number of bacteria were measured. They were 0mV for Clostridium pasteurianum, -42mV for Azotobacter chroococcum and Azotobacter vinelandii, -95mV for Bacillus polymyxa and -180mV for Klebsiella pneumoniae Mo-Fe proteins at pH 7.9. The oxidations were thermodynamically reversible for the proteins from A. chroococcum, A. vinelandii and K. pneumoniae and the Em was independent of protein activity for this last protein. The protein from C. pasteurianum required a lower potential for reduction than for oxidation, and the oxidation of the protein from B. polymyxa was only 70% reversible. The apparent Em of the latter protein was decreased by 40mV in the presence of 60mM-MgCl2. The pH-dependence of the Em of the protein from K. pneumoniae was interpreted in terms of a single ionization, not directly associated with the e.p.r.-active centre, with a pKa of 7.0 in the oxidized form of the protein and a pH-independent region at low pH (Em = 118 +/- 6.3 mV). Approx. 20% increase in activity after oxidation was observed for the proteins from B. polymyxa, A. chroococcum and K. pneumoniae. The significance of the above results and their relationship to other published data are discussed.


Elastin cross-linking in vitro. Studies on factors influencing the formation of desmosines by lysyl oxidase action on tropoelastin. The formation of isodesmosine and desmosine in vitro by the action of lysyl oxidase on tropoelastin was studied. The synthesis of desmosines occurred in the absence of additional substances. The formation of desmosines was not affected by removal of molecular O2 from the reaction medium nor was it affected by the lack of proline hydroxylation in tropoelastin. However, there was virtually no desmosine formation at 15 degrees C, a temperature not conducive to coacervation, indicating that coacervation is an important prerequisite for cross-linking.


Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified from ox liver, and obtained essentially free from 3-oxoacyl-CoA thiolases. The kinetic behaviour, like that of the synthases from chicken liver and yeast, is compatible with a reaction pathway involving condensation of an acetyl-enzyme with acetoacetyl-CoA. The Km for acetoacetyl-CoA, less than 1 micronM at pH 7.8, may possibly be low enough to permit rapid ketogenesis under physiological conditions without the need for a binary complex between the synthase and oxoacyl-CoA thiolase.


Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes. A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.


Use of the sulphite adduct of nicotinamide-adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase. 1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).


Biochemical applications of chemically induced nuclear polarization in phenols, peptides, catecholamines and related molecules. A photochemical method for producing proton nuclear polarization in phenolic biogenic molecules such as tyrosyl polypeptides and phenolic amines is described. The nuclear polarization is due to the reversible hydrogen-atom transfer from the phenolic hydroxy group to triplet photoexcited xanthene-dye molecules. Typical results obtained with insulin, catecholamines and other phenolic molecules are treated in detail.


The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells. 1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.


Characterization of cholesterol ester hydrolase activities in rabbit and guinea pig aortas. Cholesterol ester hydrolase activity was determined in preparations of rabbit and guinea pig aorta utilizing micellar and glycerol-dispersed cholesterol oleate substrates. Both substrate preparations demonstrated an acid pH optimum of 4--5 for the soluble and particulate rabbit media cholesterol ester hydrolase, suggesting a lysosomal origin for this activity. Approximately one-fifth of the total recovered activity was particulate. Particulate media preparations from guinea pig aorta also demonstrated cholesterol ester hydrolase activity at acid pH values with a definite optimum at pH 5 for the glycerol-dispersed substrate. However, in contrast to the rabbit media enzyme, activity was also observed at neutral pH with another optimum at pH 7. The supernatant enzyme from guinea pig media exhibited only a single pH optimum of 7. Cholesterol ester hydrolase activity from either rabbit or guinea pig media was not influenced by preincubation with cyclic AMP, ATP and protein kinase. The addition of chloroquine resulted in the inhibition of both the rabbit and guinea pig enzyme. Cholesterol ester hydrolase activity from rabbit and guinea pig media was also inhibited by phenyl methane sulfonyl fluoride; activity measured at pH 7 (guinea pig) was more sensitive to inhibition than activity measured at pH 5 (guinea pig and rabbit).


Accuracy of an automated blood-gas analyser operated by untrained staff. Performance of the IL 613 automatic blood-gas analyser has been assessed using a group of 100 "analysts" with no previous training or experience in the use of the instrument. Test material consisted of blood equilibrated to a known PO2 and PCO2 in a tonometer: pH estimations were carried out on thawed aliquots of a large batch of frozen serum which were then equilibrated to a known PCO2. Eighty-six per cent of analyses were within acceptable limits of error. The largest proportion of errors was in the measurement of pH. Satisfactory results were obtained in 98% of the analyses of PO2 and PCO2. Eighty-eight per cent of operators were able to use the analyser after instruction lasting less than 1 min. These results were significantly better than those obtained in a regional survey of 16 blood-gas laboratories, staffed by trained technicians.


Comparative study of two long-acting tranquillizers for oral premedication. Lorazepam 2.5 mg was compared with promethazine 50 mg as oral premedication in a double-blind study in women. The premedication was given at the same time to all patients on each operating list, and both drugs continued to be effective 6 h after ingestion. A similar number of patients considered each drug to have relieved anxiety and the amnesic effect of lorazepam was confirmed. However, the use of lorazepam alone was accompanied by significantly more salivation during and after anaesthesia than the use of promethazine, especially in patients in whom the trachea was intubated. There was also a higher frequency of vomiting during and after operation with lorazepam (seven of 67 patients) than after promethazine (one of 71 patients). Promethazine produced dyskinetic side-effects in six of 71 patients.


A study of naloxone and doxapram as agents for the reversal of neuroleptanalgesic respiratory depression in the conscious rabbit. The effectiveness of naloxone and doxapram in reversing the respiratory depressant actions of fentanyl and droperidol in the rabbit has been examined. Both drugs did not reverse fully the depression of respiratory frequency produced by the neuroleptanalgesic agents. Doxapram also failed to reverse fully the depression of minute volume produced by fentanyl and droperidol, although naloxone was adequate in this respect. However, analysis of arterialized venous blood showed that both naloxone and doxapram not only prevented the increase in PCO2 caused by fentanyl and droperidol, but caused also a significant decrease. A reduction in PCO2 was seen also when either naloxone or doxapram was given to untreated rabbits. With doxapram this appeared to be a result of pure respiratory stimulation. Naloxone also produced a reduction in standard bicarbonate.


Collection, cryopreservation and subsequent viability of haemopoietic stem cells intended for treatment of chronic granulocytic leukaemia in blast-cell transformation. We have stored at -196 degrees C peripheral blood buffy coat (BC) and bone marrow (BM) cells collected from 47 patients with chronic granulocytic leukaemia in the chronic phase. Dimethyl sulphoxide (DMSO) 10% was used as cryoprotective agent. As these cells include CFUc and probably pluripotential stem cells they may be transfused as part of the management of patients who enter blast cell transformation. The mean numbers of nucleated cells collected and stored per procedure was about 9 times greater for BC collections than for BM harvests (106 +/- 49 (SD) X 10(9) versus 11.9 +/- 6.6 X 10(9) respectively). Agar CFUc assay showed that stored cells may remain viable for up to 5 years. Since in vitro studies showed that CFUc proliferation is not inhibited by low concentrations of DMSO the removal of all DMSO during cell reconstitution before transfusion may not be necessary. If autologous BC cells are capable of repopulating the BM of patients treated for CGL in blast cell transformation the routine collection and storage of BC rather than BM cells may be desirable for all newly diagnosed patients.


Continuous fetal tissue pH recordings during labour. A preliminary report. We made continuous fetal pH-measurement in 50 patients and showed a good correlation between tissue pH and umbilical artery, umbilical vein and the fetal scalp blood pH. There were practical problems in the use of the glass pH electrode.


Low pH dimerization of chymotrypsin in solution. The mechanism of the acid dimerization of alpha-chymotrypsin in solution was reexamined using a number of chemical derivatives. Blocking of the carboxyl of Tyr-146, while that of ASP-64 remained free, eliminated completely the ability of alpha-chymotrypsin to dimerize, as did methylation of His-57. O-Acetylation of Tyr-146 reduced the dimerization constant to that of gamma-chymotrypsin, and deacetylation of the other accessible tyrosines did not affect the dimerization. It is concluded that the mechanism proposed by Aune and Timasheff [Aune, K.C., and Timasheff, S.N.(1971) Biochemistry 10, 1609-1617] for the solution dimerization which involves the electrostatic interaction between the His-57 imidazolium ring and the terminal carboxyl of Tyr-146 is still most consistent with all the experimental observations. The interactions in dilute solution may differ somewhat from those observed in crystals. In particular, the two intermolecular bridges formed by sulfate ions in crystals cannot be present in solution.


Phosphorus-31 nuclear magnetic resonance studies of active proton translocation in chromaffin granules. ATP hydrolysis and proton translocation in chromaffin granules were followed using 31P nuclear magnetic resonance. The intragranular pH affects the resonance frequency of the gamma-phosphate of granular ATP. By measuring frequency vs. pH in solutions which simulate the intragranular matrix, this may be calibrated to give quantitative pH measurements. The pH in the resting granule is 5.65 +/- 0.15. This drops by 0.4 to 0.5 pH unit when ATP is added externally and protons are actively pumped into the granules. Because of differences in the composition and pH of the internal and external solutions, the resonances of internal and external nucleotides and Pi can be distinguished. Consequently, ATP hydrolysis and changes in internal pH may be observed simultaneously and continuously in a single sample of chromaffin granules. From the measured buffering capacity of a reconstituted intragranular solution, pH changes were converted into an absolute number of protons translocated. The net proton flux (protons translocated/ATP hydrolyzed) was about 1.0 immediately after external ATP addition but fell toward zero as the pH gradient increased to a new steady state. These 31P NMR results agree with intragranular pH measurements determined from methylamine distribution and with H+/ATP stoichiometries calculated from pH changes observed in the external medium.


Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness. ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels. From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.


Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition. We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out ability to generate this species to study reactions of a3+(3) with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O2 and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a3+(3) reacts within less than 10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a3+(3) species responds to O2 and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a3+(3) registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a3+(3) in either case. In all these experiments, in which EPR detectable a3+(3) was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a3+(3) to other 'activated' species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.


Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles. The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.


Surface charges on chloroplast membranes as studied by particle electrophoresis. 1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts. 2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge. 3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface. 4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues. 5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting chlorophyll a/chlorophyll b pigment . protein complex. 6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts. 7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory. 8. Calculations of the zeta-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500--2000 A2. 9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.


Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes. Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.


Role of beta-carotene in the reaction centres of photosystems I and II of spinach chloroplasts prepared in non-polar solvents. Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl4, n-hepatane) and the beta-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b6 in the ratio 1 : 2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP+ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn2+ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of beta-carotene in photochemistry separate from its roles in energy transfer and photoprotection. Removal of the fraction of beta-carotene closely associated with the Photosystem I reaction centre caused the rate of NADP+ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of beta-carotene, photochemistry can still be observed, however the specific association of beta-carotene with the reaction centre is required for maximal rates. We propose that beta-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state beta-carotene and chlorophyll in the first excited state.


Erythrocyte membrane potentials determined by hydrogen ion distribution. If the extracellular fluid is left unbuffered, dynamic membrane potential changes in the red blood cell may be determined from external pH readings. For some types of experiments it is necessary to accelerate H+ equilibration by adding minute amounts of hydrogen carriers. The method is independent of hematocrit over a wide range of membrane potential changes. Membrane potential jumps produced by permeability changes or by changes in ionic composition may be measured. The method provides a convenient means of measuring parameters of both the conductive and non-conductive anion pathways in the red cell.


The hyperpolarizing and depolarizing effects of 2,4-dinitrophenol on Ehrlich cells. The ability of glucose to reverse the effects of dinitrophenol on amino acid uptake in Ehrlich cells is a function of pH. At pH 6.0, the presence of glucose does not reverse the inhibitory action of the uncoupler. Nearly complete restoration occurs with glucose at pH 7.4. At pH 8, the presence of glucose may cause a modest increase in amino acid uptake in presence of dinitrophenol. At all pH values, glucose restores ATP and cellular K+ to the control levels at the same pH. Although the cytoplasmic pH changes with changes in the external pH, the cell interior is more alkaline than the medium near pH 6.0 and more acid than the medium at pH 7.8 even after 45 min incubation at 37 degrees C. With dinitrophenol and in presence of glucose the difference in pH between the medium and the cell is minimal at both pH 6.0 and 7.8.


The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes. In the absence of albumin, v/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the beta-ketostearoyl-coA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentration-dependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some beta-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of beta-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of 2-trans-octa-decenoyl-CoA was 18 nmol . min(-1) . mg(-1) in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2-trans-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.


Parvalbumins from coelacanth muscle. II. Amino acid sequence of the two less acidic components. The primary structure of the two less acidic parvalbumins (pI = 5.44 and pI = 4.95) from coelacanth muscle (Latimeria chalumnae) has been determined. They differ only by the presence or absence of a N-terminal blocking group. By the use of the automatic degradation, 69 amino acids could be placed unambiguously in the N-terminal part and 24 amino acids following the single arginine 75. Tryptic peptides were used to establish the sequence and the position of the remaining residues. The two parvalbumins examined belong to the alpha-lineage, and the rate of their molecular evolution is comparable to that found in other vertebrates.


The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process. Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.


Structural interpretation of lanthanide binding to the basic pancreatic trypsin inhibitor by 1H NMR at 360 MHz. The weak binding of lanthanides to the five carboxyl groups of the basic pancreatic trypsin inhibitor (hereafter termed "the inhibitor"), has been investigated in detail using high resolution 1H NMR at 360 MHz. Lanthanides bind to the C-terminus with an apparent binding constant of 30 M-1, and thus competitively inhibit the formation of a salt-bridge between the C-terminus and the N-terminus, Lanthanides bind also to the side chain carboxyl groups of Asp 3, Glu 7, Glu 49 and Asp 50, with binding constants of 10--30 M-1. With the use of lanthanides individual resonance assignments for Phe 4 and Phe 45 were obtained in the 1H NMR spectrum of the inhibitor, and for several spin systems previous  identifications were independently confirmed. The present experiments also provide a nice illustration for the use of shift reagents to improve the resolution in 1H NMR spectra of proteins. The exchange broadening for Tyr 35 and Phe 45 over the temperature range 4--72 degrees C could thus be observed for almost all the components of these aromatic spin systems and new details on the dynamic properties were obtained also for other aromatic residues.


The effect of acid pH and citrate on the release and exchange of iron on rat transferrin. The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1.10(-3) M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.


Origin of the ribityl side-chain of riboflavin from the ribose moiety of guanosine triphosphate in Pichia guilliermondii yeast. In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.


Characterization of an agglutinin from human serum. After exposure to serum, an agglutination of mitochondria from yeast, liver, heart and kidney was observed. The degree of agglutination was dependent on the ratio between the amount of serum proteins and mitochondrial protein. The serum protein which induced agglutination was bound irreversibly to the mitochondria, was heat stable and partly resistant to acidification. Maximal agglutination was observed at an ionic strength equal to 40 mM Tris, at pH 6.0-7.5. Preincubation of mitochondria with calcium ions at slightly acidic pH prevented the agglutination. Neuraminidase treatment of either serum or mitochondria had no effect upon the agglutination.


The relation between carbon monoxide binding and the conformational change of hemoglobin. The spectral difference between normal and rapidly reacting deoxyhemoglobin (Sawicki and Gibson (1976), J. Biol Chem. 251:1533-1542) is used to study the relationship between CO binding to hemoglobin and the conformational changes to the rapidly reacting form in a combined flow-laser flash experiment. In both pH 7 phosphate buffer and pH 7 bis(2-hydroxy-ethyl)imino-tris (hydroxymethyl)methane buffer (bis-Tris) with 500 muM 2,3-diphosphoglycerate (DPG), the conformational change lags far behind CO binding; rapidly  reacting hemoglobin is not observed until more than 10% of the hemoglobin is liganded. In pH 9 borate buffer the formation of rapidly reacting hemoglobin leads CO binding by a significant amount. A simple two-state allosteric model (Monod et. al. (1965), J. Mol. Biol. 12:88-118) which assumed equivalence of the hemoglobin subunits in their reaction with CO was used to simulate the experimental results. In terms of the model, the conformational change lead observed at pH 9 suggests that significant conformational change has occurred after binding of only one CO molecule per tetramer. In the presence of phosphates good agreement between experimental results and simulations is obtained using parameter values suggested by previous experimental studies. The simulations suggest that the conformational change occurs after binding of three CO molecules.


Bicarbonate-chloride exchange in erythrocyte suspensions. Stopped-flow pH electrode measurements. A pH-sensitive glass electrode was used in a temperature-controlled stopped-flow rapid reaction apparatus to determine rates of pH equilibration in red cell suspensions. The apparatus requires less than 2 ml of reactants. The electrode is insensitive to pressure and flow variations, and has a response time of < 5 ms. A 20% suspension of washed fresh human erythrocytes in saline at pH 7.7 containing NaHCO(3) and extracellular carbonic anhydrase is mixed with an equal volume of 30 mM phosphate buffer at pH 6.7. Within a few milliseconds after mixing, extracellular HCO(3) (-) reacts with H(+) to form CO(2), which enters the red cells and rehydrates to form HCO(3) (-), producing an electrochemical potential gradient for HCO(3) (-) from inside to outside the cells. HCO(3) (-) then leaves the cells in exchange for Cl(-), and extracellular pH increases as the HCO(3) (-) flowing out of the cells reacts with H(+). Flux of HCO(3) (-) is calculated from the dpH/dt during HCO(3) (-)-Cl(-) exchange, and a velocity constant is computed from the flux and the calculated intracellular and extracellular [HCO(3) (-)]. The activation energy for the exchange process is 18.6 kcal/mol between 5 degrees C and 17 degrees C (transition temperature), and 11.4 kcal/mol from 17 degrees C to 40 degrees C. The activation energies and transition temperature are not significantly altered in the presence of a potent anion exchange inhibitor (SITS), although the fluxes are markedly decreased. These findings suggest that the rate-limiting step in red cell anion exchange changes at 17 degrees C, either because of an alteration in the nature of the transport site or because of a transition in the physical state of membrane lipids affecting protein-lipid interactions.


[Change in the concentration of nicotinamide nucleotides in rat brain and myocardium under the influence of factors inducing neurogenic dystrophy]. The content of nicotinamide dinucleotides in the brain and myocardium induced by electrical stimulation of rats and by injection of toxic noradrenaline doses was investigated. The total level of pyridine nucleotides and of their oxidized forms proved to be decreased, and the content of triphosphopyridine nucleotides and their synthesis activity rose. The data obtained point to disturbances of the redox processes and to increase of hexose monophosphate shunt activity in the tissues by neurogenic dystrophies induced by excessive irritation.


beta-Adrenoceptor antagonists inhibit the behavioural responses of rats to increased brain 5-hydroxytryptamine. 1 The effect of various beta-adrenoceptor blocking agents on the 5-hydroxytryptamine (5-HT)-induced hyperactivity response produced in rats by administration of tranylcypromine (10 mg/kg i.p.) followed by L-tryptophan (50 mg/kg i.p.) has been investigated. 2 (+/-)-Alprenolol, (+/-)-timolol, (+/-)-sotalol, (+/-)-pindolol (all at 40 mg/kg) all inhibited the hyperactivity response to some degree when given 45 min before the tranylcypromine, as did (+/-)-oxprenolol when given after the L-tryptophan. 3 beta-Adrenoceptor antagonists that are not found in the brain appreciable amount after peripheral injection, (+/-)-atenolol, (+/-)-practolol, (+/-)-labetalol and (+/-)-acebutalol, did not inhibit the 5-HT-mediated behaviour. 4 Neither the beta1-selective drug (+/-)-metoprolol, nor the beta2-selective drug (+/-)-butoxamine inhibited the behavioral response. 5 The drugs that blocked the 5-HT-mediated behaviour did not alter brain 5-HT concentrations, synthesis rate or the accumulation of 5-HT following tranylcypromine/L-tryptophan. However, they did inhibit the hyperactivity produced by the suggested 5-HT agonist, 5-methoxy N,N-dimethyltryptamine, indicating that the beta-adrenoceptor blocking drugs were inhibiting the post-synaptic 5-HT-mediated response. 6 Circling produced by methamphetamine (3 mg/kg) in unilateral nigro-striatal lesioned rats was not altered by alprenolol, sotalol, pindolol or metaprolol, indicating that these drugs do not alter dopamine-mediated behaviour. 7 It is concluded that non-selective (beta1 and beta2) adrenoceptor antagonists which have a high brain/blood ratio following their peripheral injection, block 5-HT-mediated behavioural responses in the rat.


Occurrence of H1- and H2-histamine receptors in the guinea-pig gall bladder in situ. 1 Histamine has a dual action on the in situ gall bladder of the guinea-pig: a spasmogenic and a relaxant effect mediated through H1- and H2-receptor stimulation respectively. 2 The contracturant action, mimicked by 2-(2-aminoethyl) thiazole (a specific H1-receptor agonist), is blocked by mepyramine and the relaxation, mimicked by dimaprit (a specific H2-receptor agonist), is inhibited by cimetidine.


Release of 3H-purines from [3H]-adenine labelled rabbit kidney following sympathetic nerve stimulation, and its inhibition by alpha-adrenoceptor blockage. 1 Rabbit kidneys were isolated and perfused with Tyrode solution. Release of 3H-purines was studied after labeling of the adenine-nucleotide stores with [3H]adenine (more than 60% uptake during a single passage). 2 One hour after labelling the spontaneous 3H-outflow amounted to 0.1 to 0.2% of the total tissue content per minute. The release rate was enhanced following nerve stimulation (3 to 10 Hz), or brief infusion of noradrenaline (0.1 to 2.4 microgram i.a.). Release of radioactivity was also enhanced by angiotensin II, by interruption of perfusion flow for 0.5 to 2 min and by hypoxia (5 to 25% O2). 3 The release of tracer induced by nerve stimulation or noradrenaline was markedly reduced or abolished by phenoxybenzamine, which also inhibited the vasoconstrictor response. The release following angiotensin II, ischaemia and hypoxia could not be antagonized by this alpha-adrenoceptor antagonist. 4 the radioactivity in the kidney was predominantly in nucleotide form, while that released was composed mainly of nucleosides, of which adenosine predominated. 5 The results indicate that in the rabbit kidney vasocontriction, arterial clamping or reduced perfusion oxygen tension, cause release of adenosine and related compounds. In view of the reported actions of adenosine on noradrenaline effects and release in the kidney a possible physiological role is discussed.


Differences between presynaptic and postsynaptic alpha-adrenoceptors in the isolated nictitating membrane of the cat: effects of metanephrine and tolazoline. The alpha-adrenoceptor blocking agent, tolazoline, and the O-methylated metabolite of adrenaline, metanephrine, produced a concentration-dependent increase of tension in the smooth muscle of the cat isolated nictitating membrane. These effects were considered to be due to the activation of postsynaptic alpha-adrenoceptors. 2 The responses to nerve stimulation of this muscle were neither potentiated nor blocked by tolazoline (0.1 to 10 micrometer) or metanephrine (1 to 10 micrometer). 3 3H-transmitter overflow evoked by electrical stimulation was not modified by tolazoline or metanephrine in concentrations in which these drugs stimulated the postsynaptic alpha-adrenoceptors. 4 Since tolazoline and metanephrine failed to activate the presynaptic alpha-adrenoceptors of the cat nictitating membrane under experimental conditions in which they stimulated the postsynaptic alpha-adrenoceptors, these results further support the view that the presynaptic (alpha2) adrenoceptors differ from the postsynaptic (alpha1) adrenoceptors.


Pharmacological characterization of the presynaptic alpha-adrenoceptors regulating cholinergic activity in the guinea-pig ileum. 1 The presynaptic alpha-adrenoceptors located on the terminals of the cholinergic nerves of the guineapig myenteric plexus have been characterized according to their sensitivities to alpha-adrenoceptor agonists and antagonists.2 Electrical stimulation of the cholinergic nerves supplying the longitudinal muscle of the guinea-pig ileum caused a twitch response. Clonidine caused a concentration-dependent inhibition of the twitch response; the maximum inhibition obtained was 80 to 95% of the twitch response. Oxymetazoline and xylazine were qualitatively similar to clonidine but were about 5 times less potent. Phenylephrine and methoxamine also inhibited the twitch response but were at least 10,000 times less potent than clonidine.3 The twitch-inhibitory effects of clonidine, oxymetazoline and xylazine, but not those of phenylephrine or methoxamine, were reversed by piperoxan (0.3 to 1.0 mug/ml).4 Lysergic acid diethylamide (LSD) inhibited the twitch response, but also increased the basal tone of the ileum. Mepyramine prevented the increase in tone but did not affect the inhibitory action of LSD. Piperoxan or phentolamine only partially antagonized the inhibitory effect of LSD.5 Phentolamine, yohimbine, piperoxan and tolazoline were potent, competitive antagonists of the inhibitory effect of clonidine with pA(2) values of 8.51, 7.78, 7.64 and 6.57 respectively.6 Thymoxamine was a weak antagonist of clonidine; it also antagonized the twitch-inhibitory effect of morphine. Thus, its effect against clonidine is probably not mediated specifically at presynaptic alpha-adrenoceptors.7 Labetalol, itself, depressed the twitch response but did not antagonize the inhibitory effect of clonidine on the residual twitch.8 The results demonstrate that the presynaptic alpha-adrenoceptors in the guinea-pig ileum are of the same type as those located presynaptically in sympathetically innervated tissues. They are alpha(2)-adrenoceptors and are different from those located postsynaptically.


Radiologic manifestations of epidemic haemorrhagic fever with renal syndrome. Epidemic haemorrhagic fever is a viral disease characterized by acute febrile onset followed by profound renal dysfunction. The endemic areas are widespread throughout the Eurasian continent and the Japanese islands. The patient with EHF usually seeks medical care as an emergency for an "acute abdomen". Consequently the X-ray examination becomes an essential part of the clinical study. We analysed the plain abdomen and chest films of 62 patients with this disease. The radiologic manifestations represented diffuse capillary damage with resultant oedema and haemorrhage, particularly in the retro-peritoneal space. Abnormal findings were seen in 94% of the patients on the plain abdomen, and in 63% on the chest film. The most significant finding was obliteration and blurring of anatomical detail in both intra- and retro-peritoneal cavities and renal enlargement. We believe that simultaneous presence of oedema and effusion in both intra- and retro-peritoneal cavities along with renal enlargement is in practice pathognomic of EHF. Obliteration of the renal outline and cardiomegaly with overt lung oedema was associated with the most severe renal dysfunction.


Glutamate secretion and NAD(P)H levels during calcium-dependent depolarization of slices of the dentate gyrus. Evidence from studies involving release, postsynaptic responses, inactivation, storage and synthesis etc. support the contention that glutamate may be the transmitter of the perforant input to the granule cells in the dentate gyrus of the hippocampus. In the present report the release of endogenous glutamate and the levels of reduced pyridine nucleotides (NAD(P)H) has been measured in parallel experiments on slices from the dentate gyrus of the hippocampus. A Ca-dependent release of glutamate is evoked by tissue depolarization caused either with electrical field stimulation or with elevated KC1. Electrical stimulation induced a transient increase in tissue NAD(P)H levels, the increase being inhibited by approximately 50% during Ca-free conditions. KC1 stimulation, on the other hand, produced a long-lasting decrease in NAD(P)H, the decrease being halved in the absense of Ca. A metabolic relation between stimulus secretion and energy utilization is discussed.


A thermodynamic analysis of the secondary transition in the spontaneous precipitation of calcium phosphate. A thermodynamic analysis has been made of the secondary transition stage in the spontaneous precipitation of calcium phosphate following the amorphous-crystalline transformation. The first formed crystalline material has a solubility similar to that of octacalcium phosphate (OCP) and the computed thermodynamic solubility product remains invariant in the pH range 7.00--8.60. The duration of the secondary stage is sensitive to pH and the transition appears to occur by hydrolysis of the first formed OCP-like phase to a more basic apatitic phase with a tricalcium phosphate (TCP) stoichiometry. The crystalline material at the end of this transition has an invariant solubility product, in the pH range 7.00 to 8.60, when the TCP-like molecular formula is assumed. Changes in the solution chemistry which accompany the solid-to-solid transitions are consistent with the above conclusions. The results of this study are also consistent with those of a previous study which suggest that the stability of the amorphous calcium phosphate phase is dependent upon the instability of the solution phase with respect to OCP formation.


The role of renal nerves in the response to dipsogenic stimuli in the rat. The effect of bilateral renal denervation on water intake and urine volume during specific thirst challenges was studied in rats. Renal denervation attenuated significantly the drinking response elicited by the administration of 30% polyethylene glycol (PG, extracellular challenge) but had no effect on the drinking response after an intracellular challenge (2.5 M NaCl) or after a 24-h water deprivation period. Furthermore, during a PG challenge total water intake was the same in two groups of rats, one with denervated kidneys and the other with beta-adrenergic neural activity in efferent renal nerves eliminated by blocking agents. Urine volumes were not affected by PG administration or water deprivation in denervated rats but were increased significantly after administration of 2.5 M NaCl. These results indicate that renal nerves play an important role in the physiological processes controlling extracellular thirst, and suggest that this role may be related to the neural control of release of renin.


Pharmacological studies on the pulmonary vein of the horse. I. Effects of selected spasmogens. Horses suffer from a respiratory condition, similar to human allergic asthma, that is characterized by severe dyspnea, wheezing, coughing, and mucus production. Mediator substances released during the allergic reaction may contract airways and pulmonary vasculature. Nothing is known of the effects of autacoids and other vasoactive substances on equine pulmonary vessels. Therefore, spiral strips of equine pulmonary vein were prepared in vitro and the effects of histamine (H), 5-hydroxytryptamine (5HT), bradykinin (BK), carbachol (Carb), and phenylephrine (phen) were studied. The order of contractile effectiveness for the agonists on the vein was found to be 5HT greater than H greater than Bk greater than Phen greater than Carb, although H consistently produced the greatest maximal effects. H1-receptors appeared to mediate H contractions while H2-receptors had no measurable effect. 5HT responses were mediated directly by 'D-type' smooth muscle receptors. Bk produced contractions but of a lesser magnitude than either H or 5HT. Varying degrees of tachyphylaxis were observed for each agent. alpha-Adrenergic receptor stimulation by Phen initiated low-magnitude contractions whereas Carb exhibited virtually no activity on the pulmonary vein. Contractile responses of pulmonary veins to various spasmogens may contribute to the equine asthmatic response by raising vascular hydrostatic pressure, thereby enhancing edema formation.


The ganglionic blocking properties of the cholinesterase reactivator, HS-6. Following intravenous administration of the cholinesterase reactivator HS-6 (30 mg/kg), blood pressure fell (up to 50 mmHg) and maximal blood levels of HS-6 reached 242 microgram/ml. HS-6 attenuated the pressor response resulting from carotid occlusion and the depressor effect of vagal stimulation. Doses of HS-6 below those used to protect against soman in different animal species (10--30 mumol/kg) progressively blocked the ganglion-stimulating effects of nicotine and dimethylphenylpiperazinium but not the pressor effect following adrenaline, a pattern similar to that produced by hexamethonium but only 1/84 as potent. HS-6, like hexamethonium and mecamylamine, progressively blocked the contraction of the nictitating membrane of the cat resulting from preganglionic stimulation. The results indicate that HS-6 possesses ganglion-blocking properties at doses likely to be used in the protection against soman poisoning. The ganglion-blocking properties of the drug may be a factor in the beneficial effects of HS-6.


A presynaptic component of the action of iontophoretically applied flurazepam on feline cortical neurones. The effect of iontophoretically applied flurazepam on the spike activity of pericruciate cortical neurones of the cat was studied. Flurazepam increased cortical inhibition produced either by local electrical stimulation (which is known to release gamma-aminobutyric acid (GABA) or by iontophoretically applied GABA. Following intravenous treatment with thiosemicarbazide (a GABA-synthesis inhibitor), flurazepam still augmented the action of GABA but was much less effective on electrically evoked cortical inhibition. These findings suggest that part of the action of flurazepam on inhibitory cortical transmission might be at the presynaptic level.


Intracellular actions of monoamine transmitters. Intracellular injections of noradrenaline or dopamine in spinal motoneurones of cats have a clear depolarizing action associated with particularly marked depression of spike potentials and their after-hyperpolarization, but with little slowing-down of the falling phase of the action potential. These effects are associated with an increase in input resistance, and they are reversible and reproducible in the same neurone. Intracellular injections of 5-hydroxytryptamine have some depolarizing action and increased input resistance, but they produced no comparable depression of the action potential and tended to enhance the after-potentials and increase excitability. It is concluded that changes in intracellular levels of monoamines, whether physiological or drug induced, may be of significance for central neuronal function.


Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp. A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.


Effects of antianxiety agents on relaxation training: a preliminary investigation. The purpose of this preliminary study was to investigate the effects of certain antianxiety agents, that is, minor tranquilizers, sedatives, and hypnotics, on relaxation training. Twelve inpatients and two outpatients were referred to group relaxation training because of tension-anxiety complaints and underwent three training sessions. Results for those patients who were not having any psychotropic medication (Relaxation Only group) were compared with results for those who were receiving anxiolytic drugs (Medication-Relaxation group). Patients in the second group were on antianxiety medication prior to and during relaxation training. Self-report measures indicate that the Relaxation Only group benefited more from relaxation training and showed more treatment generalization effects. The results are interpreted as lending some support to the view that antianxiety drugs may have an interfering effect on relaxation training. Implications and limitations of the study are discussed.


Therapeutic trials in tardive dyskinesia. The search for a treatment of tardive dyskinesia has generally been guided by the putative biochemical mechanisms underlying the extrapyramidal disorders, but no markedly effective treatment has yet been found. The currently postulated mechanism in tardive dyskinesia involves namely an imbalance between the central dopamine-acetylcholine systems whose balance may also be influenced by neuroendocrine factors. The agents reported having some clinical efficacy in the management of this neurological complication act on these systems. The clinical investigation for the treatment of tardive dyskinesia is laborious and raises several problems that could account for the unpredictability and the discrepancies in results. These problems can be divided into three broad categories: patient variables, experimental treatment variables and methodological variables. These variables are discussed and some suggestions made.


Carcinoembryonic antigen (CEA) and other tumor markers in ovarian and cervical cancer. Combinations of carcinoembryonic antigen (CEA), gamma glutamyl transpeptidase (GGT), pregnancy-associated macroglobulin (PAM) and placenta-like alkaline phosphatase (PLAP) were studied in groups of patients with ovarian and cervical cancer. In ovarian cancer, only CEA and PLAP levels appeared to reflect tumor burden and were complementary in detecting active disease. In cervical cancer, CEA and GGT reflected tumor burden, while PLAP showed just the reverse--the highest degree of positivity being present in minimal disease. PLAP positivity was even more pronounced in patients with cervical dysplasia and carcinoma in situ while CEA and GGT were negative. The data indicate that the use of marker combinations can improve our capacity to detect minimal disease and provide information regarding tumor biology that may not be available by studying individual markers or by other means. It remains to be determined whether the use of tumor markers can influence existing therapy sufficiently to alter the outcome in cancers which are notoriously difficult to treat.


Seminoma in a 12-year-old male with 46 XY/45 XO karyotype. A pure seminoma in a 12-year-old boy with an undescended testis and 46 XY/45 XO karyotype is presented. The relationships between an undescended testis, an abnormal karyotype, dysgenetic gonads and cancer are discussed.


Adenosine triphosphate induced change in light scattering of isolated basal bodies. Suspensions of isolated basal bodies undergo a characteristic decrease in turbidity following their exposure to 10(-3) M ATP. Typically, turbidity changes range from 8% to 20%, depending on the preparation, with an average change of 12%. Nucleotides other than ATP did not cause a turbidity decrease. The reaction has a pH optimum of pH 8.5 and is inhibited by concentrations of divalent cations greater than 2 X 10(-3) M. These results indicate that ATP induces a conformational change in the basal body that may be related to its activity in the cell.


Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro. Following the simultaneous subcutaneous administration of CdCl2 and Na2SeO3 to rats, evidence of a Cd-Se complex was detected in plasma by gel filtration chromatography. A similar complex was found in plasma after incubation of selenite, Cd, rat erythrocytes, and plasma in vitro, and after incubation of H2Se, Cd, and plasma in vitro. No interaction of selenite, selanete, or selenodiglutathione with Cd and plasma in the absence of erythrocytes in vitro was noted. Characterization by gel filtration, ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation showed that these Cd-Se complexes are similar. The results support the hypothesis that H2Se or a similarly reduced selenide is the product of selenite metabolism by rat erythrocytes. Hydrogen selenide also altered the distribution of inorganic mercury in rat plasma in vitro in such a way that the apparent molecular weights of the Se-Hg and Cd-Se complexes associated with protein were similar. Hydrogen selenide had no effect upon the distribution of methylmercury in plasma. The stability of the Cd-Se complex in plasma depended upon the integrity of the native protein components, as shown by incubation with Proteinase K. The properties of the complex suggested that it existed in a single form associated with different plasma components under various conditions.


Lymphocyte surface poisons: disulfides and thiolsulfonates. Eight disulfides (I-VIII) and a thiolsulfonate (IX) were promising blocking agents of lymphocytes in graft-versus-host reactions (GvHR) without comensurate intracellular effects. The blocking effects were assayed through inhibition of the local GvHR after parental lymphocytes had been incubated with agents at suitable concentrations and then inoculated into F1 hybrid offspring. The intracellular effects were assessed beforehand by measuring the inhibition of [6-3H]thymidine incorporation by lymphocytes in the presence of a wide range of concentrations of agents. Concentration levels which induced no greater than approx. 50% inhibition of the [6-3H] thymidine incorporation were considered to reflect sufficiently small intracellular effects and were used for the subsequent GvHR comparisons. Cellular survival always was 90% or more for the GvHR tests (unless stated otherwise), even when inhibition of thymidine incorporation was as high as 50%; hence the thymidine data are useful not only as guides for dose levels in the GvHR but also as leads to new agents that may show immunosuppressive or anti-leukemic activity through intracellular effects. Structural specificity of the active compounds as cell-surface poisons is evidenced by little or no activity (less than 30% inhibition of GvHR) of 28 other disulfides, 2 trisulfides, 2 Bunte salts, and 8 other thiolsulfonates. Active agents may owe this function to replacement of the H of SH in cell-surface thiol receptors by an SR group. Glutathione did not significantly inactivate agents, probably because the products of reaction also are active disulfides. When two agents (III, IX) were given orally or intraperitoneally to F1 hybrid recipients of untreated parental cells, doses of 10--15 mg/kg produced a GvHR inhibition of 17--53%.


Correlation between tissue pH, cellular transmembrane potentials, and cellular energy metabolism during shock and during ischemia. The relevance of two direct techniques for monitoring of cellular function during tissue hypoxia has been evaluated. Tissue pH and cellular transmembrane potentials were registered in canine skeletal muscle during intestinal exteriorization shock and during prolonged local tourniquet ischemia. The obtained pH and transmembrane potential changes were correlated to simultaneous changes in high-energy phosphagen (ATP + CP) and lactate levels in skeletal muscle. In control dogs no significant changes in either of the studied variables occurred. Intestinal exteriorization shock as well as local tourniquet ischemia resulted in a gradual increase in tissue lactate and a concomitant decrease in tissue pH and transmembrane potentials. In both experimental situations there was a close correlation between the transmembrane potential reduction and the tissue lactate increase. Tissue pH registrations, on the other hand, did not similarly reveal the full extent of the tissue lactate increase under the two experimental conditions. Possible reasons for this discrepancy are discussed. On the basis of the present results it may therefore be concluded that the transmembrane potential seems to be a better variable for revealing the full extent of cellular metabolic deterioration during various situations with tissue hypoxia.


Chemical characteristics of a high molecular weight renin from the renal cortex of the dog. We found an acid extract of normal dog kidneys to contain two distinct molecular weight forms of renin-like activity. Gel filtration chromatography showed peaks of activity as estimated molecular weights of 65,000 and 41,000. The high molecular weight fraction (HMW) comprised only 1% of the total activity of the extract. Both HMW and low molecular weight (LMW) fractions were inhibited by anti-human renin antibody and had similar broad pH-dependent activity optima between pH 6.0 and 7.5 in homologous substrate. The Michaelis constant (Km) of HMW was 3.6 times the Km of LMW. Both renins bound reversibly to concanavalin A-Sepharose with comparable affinities. HMW and LMW eluted from DEAE-Sephadex at similar salt concentrations without conversion of HMW to LMW. Transient acidification effected partial conversion of HMW to LMW without changing the total activity. Preincubation of HMW with trypsin increased the activity 40% and effected complete conversion of HMW to LMW. The apparent molecular weight difference between HMW and LMW is probably due to a covalently bound fragment(s) and not to a noncovalently bound moiety such as has been described in the rabbit and the hog. Both HMW and LMW are glycoproteins whose terminal sugar constituents possibly are similar. HMW dog renin is a new molecular form of renin that is convertible to a more active lower molecular weight renin with tryptic proteolysis.


Treatment of chronic idiopathic urticaria with combined H1 and H2 blockers. Combined H1 (cyproheptadine) and H2 antihistamines (cimetidine) were tried in eight patients with chronic urticaria who were previously unresponsive to conventional anti-urticarial regimens, including type I antihistamines, hydroxyzine, ephedrine and corticosteroids. 50% of the patients responded with 90% or greater improvement within 10 days. The remaining 50% showed no benefit. Side effects of the medications were minimal. These results indicate that a 10 day trial of both H1 and H2 antihistamines may be useful in patients with chronic urticaria resistant to all other standard treatment modalities.


Specific method for serum creatinine determination based on ion exchange chromatography and an automated alkaline picrate reaction -- a proposed reference method. A proposed reference method for serum creatinine has been developed under the auspices of the Committee on Reference Methods and Reference Materials of the Canadian Society of Clinical Chemists. A serum ultrafiltrate at pH 2.0 is applied through a closed sample loop injection system to a short column containing cationic resin of high resolving power. Elution with sodium citrate buffer by means of minipump at constant rate passes the eluate into alkaline picrate reagents in a continuous flow system (Technicon AAIII pump, AAII colorimeter 50 mm x 1.5 mm flow cell, narrow band width filter). The colour reaction peak is monitored visually to verify specificity and the area is calculated electronically. Specificity has been demonstrated by use of Jaffé-reactive substances such as glucose, sodium acetoacetate, L-ascorbic acid, pyruvic acid, L-dopa and glycocyamidine and also by use of an alternate colour reaction, sodium 3,5-dinitrobenzoate in place of alkaline picrate in the analysis of serum pools. Routine methods in common use, i.e., manual and automated alkaline picrate procedures, demonstrated a statistically significant high bias in interlaboratory studies in which this procedure was used for reference.


Multiple molecular forms of certain lysosomal enzymes separated by isoelectric focusing on cellulose acetate membranes. Analytical isoelectric focusing on cellulose acetate membranes offers greater resolution of the enzyme pattern than does standard electrophoresis and offers numerous advantages over other support media in isoelectric focusing. A series of lysosomal enzymes, deficiencies of which cause lethal storage diseases in infants and children, can be shown to exist as multiple molecular forms by analytical isoelectrical focusing on cellulose acetate membranes. This analytical technique should be a valuable tool in the investigation of enzyme polymorphism and genetically determined enzyme deficiency diseases.


Measurement of serum thyroxine-binding capacity. A new method for free thyroxine-binding sites on thyroxine-binding globulin has been described. This method for thyroxine-binding capacity has the virtues of reasonable specificity and convenience for routine use. When used with serum thyroxine levels it should be valuable in the calculation of free thyroxine and total thyroxine-binding globulin. A more directly estimated TBC as reported here should have more value than the various and more empirical T3 uptake methods.


A new automated method for the determination of true creatinine in serum by means of the centrifichem centrifugal analyzer, based on Slot's principle; with special reference to low substrate concentrations. A new automated method is proposed for the accurate determination of the true creatinine concentration in 20 microliter serum, based on the measurement of the initial rate of the decomposition reaction of the creatinine picrate complex caused by decreasing the pH. The results of this method, performed with a Centrifichem centrifugal analyzer system (type 300 F), are compared with the results obtained by two other methods: a manual method based upon the adsorption on Fuller's earth, and the Auto Analyzer method. In these comparative studies special attention is paid to the analysis of sera containing low (normal) substrate concentrations. Calculation of the orthogonal regression between the results obtained by the adsorption method (x) and the proposed method (y) gave: y=0.97x +/- 5 mumol/l (x=91.3 mumol/l, y-93.5 mumol/l), while for the correlation coefficient (r) 0.9721 was found. All sera (n=113) had creatinine concentrations between 40 and 180 mumol/l.


Angiotensin I-converting enzyme in human urine. It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.