[Anesthetic considerations in patients with CAD (author's transl)]. The primary aim during anesthetic management of the patient with coronary artery disease is prevention of imbalance between myocardial oxygen supply and demand. Since oxygen supply is limited by restriction of coronary blood flow, prevention of increases in demand plus maintenance of supply, rather than increases in supply, will achieve this aim. The major determinants of myocardial oxygen demand are mechanical, i.e. (a) left ventricular wall tension, dependent in turn upon systolic pressure and ventricular volumn, (b) velocity of contraction, and (c) heart rate. Systolic pressure, pulmonary capillary wedge pressure, and heart rate monitoring will alert the anesthetist to increases associated with an enhanced oxygen demand. Decreased arterial diastolic and increased pulmonary capillary wedge pressure are associated with decreased supply. By appropriate manipulation of these variables and avoidance of episodes of myocardial ischemia, the perioperative morbidity and mortality rates associated with coronary artery disease may be decreased.


[Effect of combined application of halothane and some central depressants (author's transl)]. The combined effects of halothane and various central depressants were studied in mice by means of an isobolographic method which allows the statistical evaluation of interactions. A loss of the righting reflex for at least 2 min was taken as the criterion for the synchronisation of the peak time of drug effects. All interactions turned out to be supra-additive, more pronounced in the case of diazepam and flunitrazepam than in the case of medazepam, pentobarbitone or ethanol. The organic solvent used for the solution of diazepam and flunitrazepam was found to contribute to the observed interactions. The difficulties of estimating and classifying combined drug effects as well as their clinical relevance are discussed.


[Studies of the effects of an injectable combination of a local anaesthetic with a beta-receptor-blocker (author' transl)]. The use of an injectable combination of a local anaesthetic (Xylestesin) and a beta-receptor-blocker (Betadrenol) during maxillo-facial surgery is described in this paper. The statistical analysis of pulse and blood pressure recordings of 30 patients demonstrated no significant blood pressure changes; there was a significant decrease of the cardiac rate, however. It was noted that the negative chronotropic effect was the greater the higher the rate rate was before injection. The local anaesthetic described is of great therapeutic value, in particular for apprehensive patients and patients with tachycardia.


A unitary theory of anesthesia based on lateral phase separations in nerve membranes. This paper relates research on anesthetic effects on lipid membrane systems to mechanisms of neural function. A unitary theory of anesthesia based on anesthetic-induced changes in fluid-solid-phase separations in the lipid region of nerve membranes is presented. It is suggested that anesthetics act by fluidizing nerve membranes to a point where critical lipid regions no longer contain phase separations. As a consequence, the membranes are less able to facilitate the conformational changes in proteins that may be the basis for such membrane events as ion gating, synaptic transmitter release, and transmitter binding to receptors. It is proposed that the anesthetic-modified phase separation behavior of the membrane may alter neural function by a combination of the following effects: inhibition of conformational changes of intrinsic membrane proteins; prevention of the association of protein subunits to form polymeric ion channels; depression of transmitter release by preventing fusion of vesicles containing synaptic transmitter with the membrane of the presynaptic terminal.


[Apneic anesthesia in unventilated patients for various operations and explorations in laryngotracheal surgery]. A technique of anesthesia is considered for laryngoscopy in suspension, and certain acts of surgery of the vocal cords, e.g. myxoma, and of the trachea, e.g. destruction of papillomatosis. The patient is in apnea, unventilated, a catheter brings down to the level of the carena a current of oxygen of 15 liters per minute, the gaseous acidosis is buffered by injections of a solution of molar sodium bicarbonate. After briefly recalling the notions which justify this technique, its repercussion on homeostasis is studied by arterial gasometry. General anaesthesia with apneic oxygenation may offer the ENT surgeon increased possibilities of exploration and operation at the level of the larynx and trachea, but owing to its biological consequences, it should be used only with circumspection and its indications should be totally justified, for acts of limited duration. The technique seems, at present, difficult to adapt to surgery with laser.


[General anesthesia and bronchoscopy].

[General anesthesia and bronchoscopy]. High frequency, positive pressure ventilation during anesthesia for bronchoscopy, uses the intermittent delivery of oxygen and an anesthetic gas mixture thus avoiding the risks of hypoxia, retention of carbon dioxide and concomitant acidosis. This technique may be used with low pressures in the airways (8 to 10 cm of water during insufflation).


[Effects of naloxone on postoperative analgesia]. Two comparable groups of ten patients were studied. After nitrous oxide-oxygen fentanyl-pancuronium anesthesia, half the patients were reversed with a titrated dose of naloxone. Even in titrated doses naloxone rapidly abolished residual post-operative fentanyl analgesia in 80 p. 100 of the patients. In the control group none of the patients complained of pain for an average of six to eight hours. Blood gases in the recovery room were practically the same in reversed and non-reversed patients and were satisfactory.


[Study of pressure and FeCO2 during ventilation by injection (preliminary note)]. This study, carried out in nine patients, attempted to judge the efficacy and possible inocuity of ventilation by injection (jet ventilation), firstly on capnography and. secondly, on recordings of intra-tracheal pressure. The frequency of six jets per minute, is that which, under the conditions of the trial, ensures the best removal of CO2 and the maintenance of stable CO2 levels in the trachea. Although pressures recorded in the airways varied between 15 and 40 torrs, the finding of very high peaks of pressure in certain cases, makes the authors somewhat reserved concerning the security of the method when the free reflux of gas is liable to be inhibited.


[Value of jet ventilation during otorhinolaryngologic endoscopies practiced under general anesthesia]. A technique of jet ventilation under general anesthesia (Alfaterinse suxamethonium) was used in 67 patients, in 55 cases for multiple endoscopic examinations in a single stage, usually laryngoscopy in suspension followed by oesophagoscopy, and in 12 cases, for an operation on the larynx. Arterial gasometry (PaO2 and PaCO2) proved the efficacy of the method, the major risk remaining, in the opinion of the authors, the possibility of excessive pressure which may be avoided by strict supervision.


[Seeking an ideal protocol for the stability of blood gases during general anesthesia for bronchosopies]. During general anesthesia for bronchoscopy, hypoxemia is a major risk, especially in patients with a severe intrapulmonary shunt. With the technique of apnea in pure oxygen, after one hour denitrogentation, and with an intake of 50 liters of oxygen per minute through the bronchoscope, the PaO2 was greater than 400 mm of Hg, but hypercapnia and acidosis occurred. To compensate the latter, five minute sessions of apnea, alternating with two minutes of jet hyperventilation, nevertheless, have the disadvantage of producing a Ventrui phenomena at the proximal end of the bronchoscope, hence a fall in FiO2 which was dangerous in these high risk patients. The authors propose a method so that the Venturi phenomenon, which cannot be prevented, occurs in pure oxygen.


[General anesthesia for bronchoscopy and lipidol bronchography in children and infants]. In 160 cases of children and infants, a technique of general anesthesia is described (penthiobarbital-suxamethonium, then halothane-oxygen with a mask in spontaneous ventilation if the bronchoscopy is followed by lipiodol bronchography). This technique is simple and efficacious and makes the child comfortable, especially in cases of repeated operation, and for the operator whose gestures are more rapid and less traumatising and this permits rapid awakening. No incidents or severe complications were noted.


[Salvage of a patient with respiratory distress caused by tracheal obstruction, by use of a Fogarty catheter]. The author observed a state of acute asphyxia due to a mucosal plug in a patient undergoing total laryngectomy. After failure of classical methods of removal and the plug and in view of the critical state of the patient, a heroic procedure was used: extraction of the mucosal plug using a Fogarty catheter, as used in arterial surgery.


[Choice of anesthesia technic for suspension larygoscopy]. Anesthesia, during suspension laryngoscopy should permit the ENT surgeon to work in a field without the hindrance of an intra-tracheal tube, and with a calm larynx. After rapid review of anesthesias of brief duration, which are eliminated, several techniques of anesthesia  of variable duration are discussed. Neuroleptanalgesia is nevertheless exclused owing to the environmental conditions necessary for its use. Gamma OH was reserved for patients in poor general health, and gave satisfaction. Alfatisine was used here and procured anesthesia of good quality, with minimal respiratory depression, provided a precise protocol is respected, but one cannot hope for success of suspension laryngoscopy without local anesthesia in addition.


[Bronchoscopies under Alfatesine].

[Bronchoscopies under Alfatesine]. Alfatesine in perfusion, associated with laryngo-tracheal local anesthesia, was used to carry out 131 bronchoscopies. The comfort of the patient and of the bronchoscopist were then assessed. A few cardio-vascular side-effects, e.g. severe hypotension in two cases, hypertensive effects and tachycardia during introduction of the bornchoscope, were noted.


[Severe asphyxiant hemoptysis. Bronchoscopy under extracorporeal circulation]. The authors report a case of bronchoscopy made necessary by severe hemoptysis in a young man with very severe respiratory distress. Bronchoscopy was carried out under excellent conditions thanks to respiratory assistance supplied by partial femoro-femoral extra-corporeal circulation. The interest and limits of this method are discussed.


[Anesthesia during resections-anastomoses of the trachea and tracheal bifurcation. Apropos of 4 cases]. The author reports 4 cases of surgery of the lower trachea and of tracheal bifurcation, and consider: -that it is preferable only to intervene after improvement by medical treatment of the phase of acute asphyxia; -that a technique of general anesthesia, with deep curarisation and oro-tracheal catheter placed above the stenosis, permits correct ventilation; -that in cases of impossibility of ventilation, one should use extra-corporeal circulation, which should always be provided for.


[Prevention of tracheal lesions by rhythmic insufflation of the intubation catheter ballone]. The prevention of tracheal lesions due to the ballons of the intubation catheters depends on the correction of several factors concerning: -the material used (polyvinyl is better tolerated than rubber); -the shape (the sleeve-shape balloons are less traumatic); -the pressure of use, which should remain moderate; -the permanence of the compression (one should use intermittent filling of the ballon). A device designed by the authors and which is described here, permits intermittent insufflation of the ballons of the endotracheal catheters, by adaptation of certain apparatus of artificial ventilation, type LOGIC and SF4, has given excellent results.


[A rational naso-tracheal catheter].

[A rational naso-tracheal catheter]. For the last 4 years, the author has been using, especially for oral surgery, a naso-tracheal catheter (Rosso catheter), the intra-facial part of which has an S-shape which follows the curves of the airway and the outer part of which, in reinforced latex, permits attachment to the face and easy, reliable connection to the anesthetic apparatus. This catheter is easy to introduce, keeps well in place and frees perfectly the operative field and gives the anesthetist satisfactory mastery of the patient's ventilation.


[Difficult intubations in anesthesia].

[Difficult intubations in anesthesia]. The authors  present six cases of difficult intubation and review the literature on this subject; The circumstances during which difficult intubation may be feared are recalled; some are due to the morphology of the subject, the others to his previous pathology which may be rheumatological or dermatological. The authors then discuss the method of anaesthesia for this unusual intubation and the various technical means proposed to carry out safely the intubation itself.


[Difficult intubations and proposed solutions. Intubation by fiberscopy]. After a review of the various techniques used in difficult intubations, the authors emphasize recent progress in the field of fiber endoscopy. The advantages of this technique, e.g. absence of trauma, increased field of exploration and reduction in dose of anaesthetic drugs, exceed the present disadvantages, e.g. high price of this material, relatively fragile apparatus, too long and diameter too great for use in children.


[Use of closed circuit television for teaching tracheal intubation to medical students]. There are many occassions when patients could benefit from early intubation. This skill should be acquired by all doctors and not only by resuscitators and trainee anaesthetists. In Liverpool, all undergraduates in their fifth year of medicine are expected to intubate a minimum of ten patients lnder supervision of the anaesthetist. The use of closed circuit television has been explored and its value assessed in the teaching of intubation to undergraduates. The availability of hardware that is inexpensive, relatively simple to use and requiring minimal staff, such as Phikips VCR N1500 has made this approach to all. Videotapes are superior to film for this, since they are cheap, reusable and do not require chemical processing. The medium is versatile and replay immediate. Student performance can be recorded and replayed to allow positive identification of an imperfect technique. The videotape permits discussion and demonstration of methods of improvement of technique. A pre-recorded tape can be used to illustrate the anatomy and to demonstrate intubation in the absence of a patient. The fact that this is in black and white at the moment may be a disadvantage, but colour is not essential for recognition of the anatomy of the larynx. It must not be forgotten that in Britain, written permission is required before recording patients on "videograms". This can be obtained by modification of the consent forms signed by the patients prior to surgery.


[Problems posed by difficult intubations]. The difficulties of tracheal intubation are mainly encountered in facial surgery and E.N.T.  surgery, but they may nevertheless, occur in any form of surgery. The indications for nasal intubation, without visible glottis, the problems raised and the means of solving them, whilst respecting safety precautions, are briefly recalled.


[Difficult intubations].

[Difficult intubations]. The author reports twelve cases of difficult tracheal intubation, sometimes in acrobatic conditions, in victims of railway accidents. The position of the patient, the circumstances of the accident and the importance of the ventilatory distress, make tracheal intubation essential "in situ". The success of this act depends on the qualification of the medical and para-medical personnel, the technique proposed and the choice of material used.


[Sterilization of intratracheal tubes].

[Sterilization of intratracheal tubes]. After a brief review of classical and modern methods of sterilisation of endo-tracheal catheters, where they emphasize the dangers of the use of ethylene oxide, the authors recall the common procedure which they used: washing with water and soap, and soaking for 6 to 7 hours in formaldehyde or in a solution of quaternary ammonium salts, and all manipulations should be made using gloves.


[Laryngo-tracheal stenoses after intubation during resuscitation (notes apropos of a series of complications)]. The authors observed an abnormal frequency of laryngo-tracheal stenosis over a period of three months, corresponding to the use of a defective lot of supple catheters for single use and made of polyvinyl chloride. They emphasize the extreme difficulty to demonstrate the precise etiology of these severe complications.


[Complications during use of the Carlens catheter]. Two complications occurring during anesthesia with the use of Carlens catheter are discribed: -in the first case, perforation of the carena by the tip of the catheter and a tear along that left main bronchus, owing to invasion of the area by carcinoma, and death on the second post-operative day in spite of sutures that were carried out. -in the second case, poor indications for selective intubation of the left main bronchus by left upper lobectomy initially foreseen, whereas pneumonectomy was necessary, hypoventilation, anoxia, cardiac inefficacy.


[Glycosyltranferases. Biological and pathological importance]. Catalysing the transfer of oses and ose derivatives on various accepters, glucosyltransferases are of increasing importance in biology and pathology. Their sub-cellular localisation is now known together with the physico-chemical parameters of their activity and the importance of polyprenic intermediates. The first phenomenological studies concerning them are in favour of a mechanism of Bi-Bi type. For example, they may play a role in mucoviscidosis, virus diseases, vaccination and arthrosis.


Effect of beta-blockade during bowling competitions. Seventeen amateur bowlers taking part in local bowling competitions were given either 40 mg of oxprenolol or placebo before the competitions, so that each subject played two competitions on oxprenolol and two on placebo in random order. All the bowlers had a relative tachycardia 15 min before the competition which was abolished by oxprenolol. A similar reduction in the heart rate during and after the compeptition was likewise seen after oxprenolol. The drug caused only minor changes in the systolic blood pressure in comparision with placebo. On the whole, the total scores of placebo and oxprenolol competitions did not differ significantly from each other, nor was there any difference between the scores of the trial competitions and the average results during previous season. However, when the bowlers were divided into subgroups according to the effect exerted by oxprenolol on performance, about one third of the bowlers did benefit from the oxprenolol treatment while 50% had worse results. The bowlers whose bowling performance was improved by oxprenolol exhibited significantly higher heart rates before, during and after the competition as compared with the subgroups not responding beneficially to the active drug. The effect of oxprenolol was most pronounced during the very first series and the harmful effect of the drug was mainly seen in the last series of the competition. One reason for the impairment of the general performance could conceivably be an "overrelaxed" state as indicated by the greater number of misses made by the bowlers when treated with oxprenolol.


The role of microsomal cytochrome b5 in the metabolism of ethanol, drugs and the desaturation of fatty acids. Liver endoplasmic membrane contains two hemoproteins, cyt. P-450 and cyt. b5. Cytochrome P-450 catalyzes the hydroxylation of lipid-soluble compounds, while the cyt. b5 system is involved in desaturation of fatty acids. NAD(P)H and oxygen are essential components for both systems. Oxidation of ethanol to acetate in the liver, via alcohol and acetaldehyde dehydrogenases, leads to an elevated cellular NADH content. It has been proposed that oxidation of the cytosolic NADH occurs predominantly in the mitochondria via the substrate oxidation-reduction shuttle. In order to investigate the effects of elevated levels of cytosolic NADH on the state of the endoplasmic hemoprotein system, microsomes from a fatty human liver (post-ethanol intake) were isolated and studied. Microsomal cyt. b5 reductase was found to reoxidize cytoplasmic NADH directly and transfer the reducing equivalents readily to the microsomal oxidases. Addition of catalytic amounts of alcohol dehydrogenase, NAD, and ethanol to microsomes resulted in a rapid reduction of microsomal cyt. b5. These results are consistent with the proposal that the catalytic moiety of cyt. b5 reductase is exposed to the aqueous phase of the membrane and directly accepts reducing equivalents from the cytoplasm. Microsomes from fatty human liver showed an increased rate of cyt. b5 dependent desaturation of fatty acids. These findings suggest that ethanol metabolism may selectively affect the activity of one or the other microsomal hemoprotein. Thus, when the desaturase activity is low, drug metabolism by the cyt. P-450 pathway may predominate. Conversely, an increase in the desaturase level may lead to a decreased drug metabolism. This mechanism may underlie the clinical observations of drug intolerance reactions associated with alcohol intake.


Kinetics of the conformational changes of hemopexin in acid media. Under the action of acid media the hemopexin molecule unfolds with resulting heme expulsion from the binding site, followed by heme dimerization and reassociation of dimeric heme with the unfolded protein molecule. The rate of the reaction is pH dependent and the whole process is fully reversible for a certain time interval. Prolonged treatment of hemopexin at acidic conditions, however, leads to the irreversible denaturation of this protein.


Comparative in vitro studies of cinoxacin, nalidixic acid, and oxolinic acid. Cinoxacin and nalidixic acid were found to be similar in in vitro activity against 138 Shigella isolates and somewhat less active than oxolinic acid on a weight basis. Cross-resistance developed when 10 shigellae were transferred on increasing amounts of the respective agent contained in Mueller-Hinton agar. Plate dilution studies of the effect of changes in agar pH on the minimum inhibitory concentration revealed that the antibacterial activity increased with decreasing pH. Protein binding investigations revealed a high degree of binding, with nalidixic acid > oxolinic acid > cinoxacin.


Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035. Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.


Virus inactivation by grapes and wines.

Virus inactivation by grapes and wines. Infusions and extracts of different grapes inactivated poliovirus; agents responsible for this property resided in the skin of the grape. Commercial grape juice at both natural and neutral pH inactivate various enteric viruses and herpes simplex virus; a 1,000-fold reduction in poliovirus infectivity occurred after incubation with grape juice, pH 7.0, for 24 h at 4 degrees C. A variety of wines were antiviral but to a lesser extent than grape juice; red wines were more antiviral than white. Antiviral activity was demonstrable in fractions of grape juice varying in molecular weight from less than 1,000 to greater than 30,000 as determined by membrane filtration. Some restoration of poliovirus infectivity from virus-grape juice complexes was achieved with 1% gelatin, 0.1% Tween 80, 0.5% polyvinyl pyrrolidone, and 0.5% polyethylene glycol.


Kinetics and biochemical properties of haemopoietic stem cells during chicken development. Haemopoietic stem cells from donors of different ages (embryos, adults) have been grafted into irradiated hosts. Cell multiplication kinetics, foetal haemoglobin production, age-specific antigen production were examined during two weeks after grafting. The results obtained tend to show that the erythrocyte characteristics are, for a large part, already determined in a stem cell type endowed with a large proliferative capacity.


Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein. A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.


Growth of Aureobasidium pullulans on straw hydrolysate. Growth characteristics and cell properties of Aureobasidium (Pullularia) pullulans were studied. The organism grew well on an acid hydrolysate of ryegrass straw over a wide range of pH and temperature. The optimum temperature and pH for the growth of the organism were 32 degrees C and 5.5, respectively. A cell yield of 1.5 g/liter of straw hydrolysate was obtained. The dried cell mass contained 42.6% crude protein, 0.4% crude fat, and 6.4% nucleic acids. The essential amino acid profile of the microbial protein was comparable to that of Candida utilis. A rat feeding study indicated that the A. pullulans cells were not toxic and that the feed intake, weight gain, and protein efficiency ratio values were superior to those obtained with C. utilis. Once the question of mathogenicity is resolved, A. pullulans could be useful for production of single-cell protein from cellulosic wastes.


[Effect of neonatal injections of estradiol, testosterone and cryproterone acetate on plasma and testicular testosterone and on the genital system in adult male mice]. On day old male mice received a single injection of oestradiol benzoate, testosterone propionate or cyproterone acetate in order to study their action on testicular development, particularly testosterone secretion. Oestrogenization of newborn males leads, when the animals mature, to a high proportion or cryptorchidism, to atrophy of testes and seminal vesicles, and inhibition of spermatogenesis. Testosterone levels were reduced in the plasma. Testosterone propionate produced moderate reduction of testicular weight but spermatogenesis was not impaired. Plasma testosterone level was reduced. Cyproterone acetate increased significantly testicular testosterone level.


[Problems posed by anesthesia in the hypertensive treated with beta-blockaders]. The use of beta-blocking agents against hypertension and their rapid extension in daily practice we have to consider the problem of anesthesia of a patient undergoing such treatment. Continuation of the treatment increases the operative risk, discontinuation long before the operation leaves the hypertension to reappear and discontinuation 48 hours before may lead to a coronary or arrhythmic accident. Therefore, except in case of emergency, treatment may be maintained until the day before the operation, and in case or emergency extensive atropinisation is desirable. Finally prescription of selective beta-blocking agents decreases the risks.


[The effect of various pH values on in vitro peptic digestion of proteins in the presence of Cu2 ions]. Studies were carried out to investigate the effect of different pH values on the peptic digestion of soya protein in the presence and in the absence of Cu2+ ions. The studies were performed in vitro at a pH of 2.2 and 3.2, with 2.96 X 10(-5) mole of Cu2+ ions present in 11 of the reaction mixture. The reaction was carried out in a digestion apparatus permitting dialysis of the cleavage products. Different parameters were used as criteria of digestion, viz. the quantity of N contained in the reaction vessel (residue) and in the resulting dialysis products as determined by Kjehldahl microanalysis and automatic amino acid analysis, the proportions of digestion products found in the different molecular ranges after partition of Sephadex G 75 and the composition of amino acids in the cleavage products. From the distribution of the reaction products on the residue and dialysis products and on the different molecular ranges it was found that additions of Cu2+ ions at pH 2.2 produced a considerable inhibition of digestion. With a rise in pH to 3.2 peptic digestion decreased even without the addition of Cu2+. Supplementation of Cu2+ ions produced only a slight additional effect in the molecular range termed "exclusion limit". In the case of the amino acids tyrosine and phenylalanine it was found that an increase in pH changed the composition of peptides within the different molecular ranges. Additions of Cu2+ had no influence on the amino acid composition.


Aging and extrapyramidal function.

Aging and extrapyramidal function. Measurements on human brain samples of some enzymes concerned with neurotransmitter synthesis suggest serious losses with age. The most severe loss found was that in striatal tyrosine hydroxylase activity, the rate-controlling enzyme in the synthesis of dopamine. Cell counts in the substantia nigra where this dopaminergic tract originates suggest that the decrease in enzyme activity is partly due to cell loss, but must largely reflect decreased activity of residual cells. It is possible that this loss may account for some of the difficulties in movement seen in aged individuals and that it might be less if pigment formation in these cells could be inhibited.


Mannosidosis. New clinical presentation, enzyme studied, and carbohydrate analysis. Mannosidosis is a rare inborn error of metabolism characterized by deficiency of the lysosomal enzyme alpha-mannosidase and widespread storage of complex carbohydrate, which is enriched in mannose. Two affected unrelated males, aged 6 and 26 years, are reported. Both had a nonprogressive encephalopathy with moderately severe mental retardation. The older patient showed several unique features, including massive gingival hyperplasia associated with histiocytes containing large amounts of a material with the staining characteristics of glycoprotein. The best determinant of mannose storage proved to be the ratio of mannose to other carbohydrates in urinary polysaccharides. The enzyme deficiency in this disease is most convincingly demonstrated at pH values below 4.0. The ability of zinc to activate the mutant enzyme in vitro offers a possible mode of therapy for this disease. Retarded individuals with a Hurler-like appearance and gum hyperplasia of unknown cause should be screened for alpha-mannosidase deficiency.


[The measurement of the partial pressure of oxygen and carbon dioxide and pH of the aqueous humor in man. A preliminary study on 32 normal eyes]. Using a micromethod the authors have measured the pH, and the partial pressure of oxygen and carbon dioxide in the aqueous humour. The results are presented in 32 normal eyes. The authors have studied the variation of these levels in the aqueous in comparison with the same levels measured at the same time under the same conditions in the arterial blood.


Electrophoretic demonstration and inital characterization of human red cell NAD(P)+ase. A method for electrophoretic detection of NAD(P)+ase solubilized from red cell membranes is described. The method reveals both NAD+ase and NADP+ase and can be applied to a screening procedure for the detection of electrophoretic variants. The initial chracterization of the solubilized enzyme is also described.


The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme. D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.


A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase. Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.


Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase. After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate.


Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles. 1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 X 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.


The transport and oxidation of succinate by Ehrlich ascites-tumour cells. The transport and oxidation of succinate by functionally intact Ehrlich ascites-tumour cells was investigated. On the basis of pH dependence and inhibitor sensitivity it was concluded that succinate may be transported across the cell membrane by the organic anion carrier system. Thus the ability of isolated Ehrlich cells to oxidize succinate is real, and is not necessarily a result of damage to cell integrity.


The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase. The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.


Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism. 1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.


Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization. The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.


Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity. 1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.


Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts. 1. The tRNA methyltransferase activity in vitro of leaves, cotyledons and roots of 85-day-old tea seedlings was studied. 2. The activity of extracts prepared from tea leaves with Polycar AT (insoluble polyvinylpyrrolidine) had optimum pH7.7 and was greatly influenced by thiol compounds, but only slightly by metal ions and ammonium acetate. 3. The activities of extracts, expressed per mg of protein, were as follows: roots greater than leaves greater than cotyledons. The only methylated base isolated after incubation with these preparations was 1-methyladenine. 4. The results did not support the view of involvement of methylation of nucleic acids in caffeine biosynthesis in tea plants. In contrast, it is suggested that theophylline is synthesized from the specific methylated precursor in nucleic acids, namely 1-methyladenylic acid, via 1-methylxanthine.


Effect of phenylalanine metabolites on the activities of enzymes of ketone-body utilization in brain of suckling rats. 1. The effects of phenylalanine and its metabolites (phenylacetate, phenethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) 3-oxo acid CoA-transferase (EC 2.8.3.5) and acetoacetyl-CoA thiolase (EC 2.3.1.9) in brain of suckling rats were investigated. 2. The 3-hydroxybutyrate dehydrogenase from the brain of suckling rats had a Km for 3-hydroxybutyrate of 1.2 mM. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the enzyme activity with Ki values of 0.5, 1.3 and 4.7 mM respectively. 3. The suckling-rat brain 3-oxo acid CoA-transferase activity had a Km for acetoacetate of 0.665 mM and for succinyl (3-carboxypropionyl)-CoA of 0.038 mM. The enzyme was inhibited with respect to acetoacetate by phenylpyruvate (Ki equals 1.3 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). The reaction in the direction of acetoacetate was also inhibited by phenylpyruvate (Ki equals 1.6 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). 4. Phenylpyruvate inhibited with respect to acetoacetyl-CoA both the mitochondrial (Ki equals 3.2 mM) and cytoplasmic (Ki equals 5.2 mM) acetoacetyl-CoA thiolase activities. 5. The results suggest that inhibition of 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase activities may impair ketone-body utilization and hence lipid synthesis in the developing brain. This suggestion is discussed with reference to the pathogenesis of mental retardation in phenylketonuria.


Nicotinamide-adenine dinucleotide-linked "malic" enzyme in flight muscle of the tse-tse fly (Glossina) and other insects. 1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.


The electrophoretic properties and aggregation of mouse lymphoma cells, chinese-hamster fibroblasts and a somatic-cell hybrid. 1. The electrophoretic mobilities of a mouse lymphoma cell, a Chinese-hamster fibroblast and a somatic-cell hybrid (also fibroblastic), produced by fusion of the hamster cell and a mouse lymphoma cell, were measured at 25 degrees C over a range of pH, concentration of Ca2+ ions and concentration of La3+ ions. 2. All the cells have pI at pH3.5. 3. Ca2+ ions decrease the mobilities and zeta potentials of the cells to zero in the range 1-100mM. 4. La3+ ions lower the mobilities and zeta potentials in the range 10 muM-1 mM, and the cells become positively charged above 1 mM. 5. The data are consistent with specific adsorption of La3+ ions on approx. 2 X 10(14) sites/m2 of cell surface with a free energy of approx. -37kJ/mol. 6. The effects of Ca2+, La3+ and ionic strength on the extent of aggregation of the cells and of neuraminidase-treated cells were studied. 7. Ca2+ ions do not markedly increase aggregation, whereas La3+ ions gave rise to extensive aggregation in the range 10 muM-1 mM, corresponding to the region of La3+ adsorption. 8. Both fibroblastic cell lines are aggregated at high ionic strength. 9. The fibroblastic cells have larger amounts of trypsin-sensitive carbohydrate than does the lymphoma cell; the possible role of this material in cellular aggregation is discussed.


Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression. 1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN'-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN'-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.


Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions. 1. In the lung and liver of tocopherol-deficient rats, the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase were increased substantially, suggesting an important role for both enzymes in protecting the organ against the deleterious effects of lipid peroxides. 2. Facilitation of the glutathione peroxidase reaction by infusing t-butyl hydroperoxide caused the oxidation of nicotinamide nucleotides and glutathione, resulting in a concomitant increase in the rate of release of oxidized glutathione into the perfusate. Thus the rate of production of lipid peroxide and H2O2 in the perfused organ could be compared by simultaneous measurement of the rate of glutathione release and the turnover number of the catalase reaction. 3. On hyperbaric oxygenation at 4 X 10(5)Pa, H2O2 production, estimated from the turnover of the catalase reaction, was increased slightly in the liver, and glutathione release was increased slightly, in both lung and liver. 4. Tocopherol deficiency caused a marked increase in lipid-peroxide formation as indicated by a corresponding increase in glutathione release under hyperbaric oxygenation, with a further enhancement when the tocopherol-deficient rats were also starved. 5. The study demonstrates that the primary response to hyperbaric oxygenation is an elevation of the rate of lipid peroxidation rather than of the rate of formation of H2O2 or superoxide.


Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats. Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.


[Investigations on the Stability of Proscillaridine, Proscillaridine-3'-methylether and Proscillaridine-4'-methylether in Artificial Gastric and Intestinal Fluids (author's transl)]. The stability of bufadienolides in artificial gastric and intestinal fluids was investigated at different pH-values as a function of incubation time using proscillaridine, proscillaridine-3'-methylether and proscillaridine-4'-methylether. These glycosides were completely stable on pH 3.0--8.5 during the investigated time interval of 1 h. At lower pH-values cleavage of the glycosidic bond occurred. At pH 1.0 73% of proscillaridine, 33% of proscillaridine-3'-methylether and 41% of proscillaridine-4'-methylether were decomposed. At pH 2.0 only about 10% decomposition of these compounds could be detected. These experiments show that the stability of proscillaridine in gastric juice is comparable with the stability of digitalis glycosides and digoxin derivatives. The stability of methylated proscillaridine derivatives in artificial digestion fluids, however, proved to be superior to any orally administered cardiac glycoside.


Piroxicam, a novel anti-inflammatory agent. Piroxicam (CP-16 171) is a potent acidic anti-inflammatory agent structurally distinct from the current agents such as indometacin, phenylbutazone or naproxen. Pharmacokinetic studies indicate a longer plasma half-ife for piroxicam than for these agents. Potency in the range of indometacin is observed when piroxicam is tested in the carrageenan rat paw edema model. This activity is not dependent on an intact adrenocorticoid system. The high potency, long half-life and absence of cardiovascular or cental nervous system effects have encouraged clinical trial of piroxicam.


Cardiorespiratory activitirs of 3-formylamino-4-hydroxy-alpha-(n-1-methyl-2-p-methoxyphenethylaminomethyl)-benzylalcohol-hemifumarate(BD 40A) and some other beta-adrenoceptor stimulants in conscious guinea pigs. The beta-stimulant activity of 3-formylamino-4-hydroxy-alpha-(N-1-methoxyphenethylaminomethyl)-benzylalcohol-hemifumarate (BD 40A) was compared with those of isoproterenol, orciprenaline, trimetoquinol and salbutamol in conscious guinea pigs. Bronchodilator effects of BD 40A were most potent among five agonists by s.c., oral and aerosol administration, and were lasting by oral and aerosol administration. BD 40A was similar to isoproterenol, more potent than trimetoquinol, orciprenaline and salbutamol in increasing heart rate by s.c. route. The cardiostimulating effect of BD 40A was more potent than that of isoproterenol, trimetoquinol and salbutamol orally. However, the order of bronchoselectivity (ratio between ED30 beats/min vs. ED50) in conscious guinea pigs was salbutamol greater than BD 40A = trimetoquinol greater than orciprenaline greater than isoproterenol by s.c. administration, and by oral administration the order was BD 40A = salbutamol greater than trimetoquinol = isoproterenol. Orciprenaline showed no beta-stimulating effect in guinea pigs orally. In guinea pigs, BD 40A, like salbutamol, seems to be a beta2-adrenoceptor stimulant.


[The influence of pentagastrin on the ratios of Na+,K+H+/Amino acids in gastric juice. Short communication (author's transl)]. Pentagastrin has a distinct effect on the ratios of Na+,K+,H+/amino acid in the gastric juice in the different states of acidity. Contrary to the ratios of normacidity the values of all quotients in patients with hyper-, hypo- and anacidity are significantly reduced.


[Profile of pharmacological actions of NAB 365 (clenbuterol), a novel broncholytic agent with selective activity on adrenergic beta2-receptors (author's transl)]. Effects of 4-amino-alpha-[(tert.-butylamino)methyl]-3,5-dichlorobenzyl alcohol hydrochloride (clenbuterol, NAB 365) on the adrenergic beta-receptors were investigated and compared with those of isoproterenol and salbutamol. The beta2-mimetic activity of clenbuterol on the smooth muscle of bronchi, uterus and vessels after i.v. injection corresponds to that of salbutamol in all laboratory animals. When given subcutaneously or as an aerosol clenbuterol is even somewhat more effective than isoproterenol. Clenbuterol differs from the known beta-mimetic drugs in its much longer duration of action. Therefore the integral of activity of single doses of clenbuterol, which are equally effective in the period of their maximal action, is remarkably greater than that of the other beta-mimetic substances. Clenbuterol differs from known beta-mimetic drugs used as bronchodilators in its efficacy after oral administration and in its mode of action on the heart. In the isolated auricle of the rabbit it has proved to be a weak partial agonist. In conscious rabbits, anesthetised guinea-pigs, dogs and cats the maximum of tachycardia obtainable by clenbuterol is lower than that of salbutamol. The higher degree of tachycardia in conscious dogs provoked by clenbuterol is a result of a reflex reaction to the vasodilation analogous to that of salbutamol. In higher doses clenbuterol shows beta1-blocking properties. Like other beta-blocking agents it owns qualities of a local-anesthetic and prolongs refractory period of the heart of guinea-pigs. In contrast to other beta-mimetic substances, clenbuterol causes only slight mobilization of heart muscle glycogen by doses higher than those which have broncholytic effects. The lipolytic and lactacidemia inducing activity of clenbuterol in rabbits corresponds to that of isoproterenol. The blood sugar is only slightly increased by clenbuterol as well as by other beta-mimetic agents. Degree and duration of action of clenbuterol and the other sympathomimetic amines on skeletal muscle of the cat shows parallelism with that of the broncholytic effect. In the rat clenbuterol inhibits the gastric secretion more than does isoproterenol. In contrast to other broncholytic substances, a very small dosage of clenbuterol is sufficient to protect rats against the liberation of histamine and serotonin caused by the anaphylactic reaction.


Synthetic analgesics: N-(1-[2-arylethyl]-4-substituted 4-piperidinyl) N-arylalkanamides. The synthesis of several 4-arylamino-4-piperdinecarboxylic acids is reported. These acids were starting materials for the preparation of alpha-amino esthers, ethers and ketones. Different synthetic approaches are described. Suitable substitution on both nitrogen atoms afforded extremely potent analgesics. Thus, methyl 4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylate (22),N-(4-(methoxymethyl)-2-[2-(2-thienyl)ethyl]-4-piperidinyl)-N-phenylpropranamide (67) and N-[4-acetyl-1-(2-phenylethyl)-4-piperidinyl]-N-phenylpropanamide (82) were found to be respectively 7682, 3987 and 4921 times as potent as morfine. Both cis- and trans-3-methyl homologs of 22 have been prepared. As expected, analgesic activity resides mainly in the cis-isomer.


In vitro effects of bencyclan on coagulation, fibrinolysis and platelet function. The in vitro effects of N-3-(1-benzyl-cycloheptyloxy)-propyl-N,N-dimethylammonium-hydrogenfumarate (bencyclan) on clotting, fibrinolytic and platelet function test were investigated by adding the drug to normal human plasma. An anticoagulant activity, mainly of an antithromboplastin nature (directed against later stages of intrinsic thromboplastin formation and against tissue thromboplastin), was observed, while thrombin phase was unaffected. No effect was found in the fibrinolytic system tested (euglobulin lysis, UK-activated fibrinolysis, "hanging clot" method). The drug, although capable of aggregating platelets by itself at very high concentrations, showed a striking inhibitory effect, over a wide range of concentrations, both on platelet aggregation induced by ADP, epinephrine or collagen and on platelet adhesiveness to glass or collagen. Clot retraction was also clearly inhibited. PF3 availability was influenced with a peculiar two-phase behaviour dose-dependently. High concentrations showed a promoting action, while the lower were obviously inhibitory. It is suggested that the effects on platelet function may be due to an influence of the drug on cell membrane.


N-4-Substituted 1-(2-arylethyl)-4-piperidinyl-N-phenylpropanamides, a novel series of extremely potent analgesics with unusually high safety margin. The intravenous analgesic activity and toxicity of a novel series of N-[4-substituted 1-(2-arylethyl)-4-piperidinyl]-N-phenylpropanamides was studied in rats. Onset, potency and duration of analgesic action were assessed in the tail withdrawal test and compared with the activity of fentanyl, (+)-cis-3-methylfentanyl (R 26 800), morphine, and pethidine. All compounds studied were found to be extremely potent analgesics characterized by an unusually high safety margin. Methyl 4-[N-(1-oxopropyl)-N-phenyl-amino]-1-(2-phenylethyl)-4-piperidinecarboxylate (R 31 833; lowest ED50 = 0.00032 mg/kg) is the most potent compound (10 031 times morphine). cis-Methyl 3-methyl-4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidine carboxylate (R 32 792) is the longest acting compound (more than 8 h at 4 times the lowest ED50) and N-[4-(1-oxopropyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 33 352) is the shortest acting compound (0.74 h at 4 times the lowest ED50) of the 4-substituted fentanyl derivatives. N-[4-(Methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 30 730) was selected for further investigation. R 30 730 has a rapid onset of action and is 4521 times more potent than morphine at the time of peak effect; it has a relatively short duration of action comparable to that of fentanyl and its safety margin (LD50/lowest ED50 = 25 211) is unusually high.


Sufentanil, a very potent and extremely safe intravenous morphine-like compound in mice, rats and dogs. Sufentanil (R 30 730), N-[4-methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide, is a chemically novel, highly potent and extremely safe intravenous morphine-like agent in laboratory animals. In mice R 30 730 i.v. is 2304 times more potent than morphine (hot plate ED50's: 0.0028 and 6.45 mg/kg, respectively). The i.v. safety margin of R 30 730 in mice is 1 : 6 679 (LD50 = 18.7 mg/kg). Under the same experimental conditions the safety margin of pethidine is 1 : 7.97, of morphine 1 : 34.9 and of fentanyl 1 : 454. In rats R 30 730 i.v. is 4521 times more potent than morphine (tail withdrawal test ED50's: 0.00071 and 3.21 mg/kg, respectively). The i.v. safety margin of R 30 730 in rats is 1 : 25 211 (LD50 : 17.9 mg/kg). Under the latter experimental conditions the safety margin of pethidine is 1 : 4.80, of morphine 1 : 69.5 and of fentanyl 1 : 277. In dogs R 30 730 i.v. is 2429 times more potent than morphine (apomorphine antagonism test ED50's: 0.00028 and 0.68 mg/kg, respectively). The i.v. safety margin in dogs is approximately 1 : 50 000, the LD50 being +/- 14.0 mg/kg. All morphine-like effects of R 30 730 are immediately antagonized by nalorphine. These pharmacological findings are relevant in connection to the increasing interest for use of morphinomimetics in anesthesia.


[Blood gas analytical study on cerebral circulation under the influence of dihydroergotoxin (author's transl)]. The demand for sufficient blood supply and oxygenation of cerebral tissue after brain surgery or severe head injuries requires the clinical investigation of drugs which are supposed to improve cerebral blood flow and metabolism. We have therefore used the hydrogenated derivatives of the ergotoxin components of the secale alcaloids in the post-operative and posttraumatic period of neurosurgical patients, respectively, and studied their effect on cerebral metabolism by successive analysis of blood gases in the arterial and venous blood. A significant increase of cerebral venous CO2-concentration following the administration of dihydroergotoxin in these patients in all probability is attributable to an improved O2-utilisation in the cerebral tissue.


Failure of benzoctamine to influence the activity of rat striatum tyrosine-hydroxylase. The influence of benzoctamine (Tacitin) on rat striatum tyrosine-hydroxylase was analized. Injection of 100 mg benzoctamine/kg body weight caused no alteration in the tyrosine-hydroxylase activity whilst a decrease of about 60% in the activity was recorded after treatment with a-methyl-p-tyrosine a known inhibitor of tyrosine-hydroxylase. The present results differ from those of Maitre et al. which indicated that benzoctamine inhibited tyrosine-hydroxylase activity.


Pharmacological basis for antihypertensive effects of intravenous labetalol. Labetalol 1-5 mg/kg administered intravenously to normal subjects in the supine position produced an immediate mean fall in systolic (16%) and diastolic (25%) blood pressure with a concomitant increase in heart rate (12%). After graded exercise, intravenous labetalol inhibited increases in heart rate and blood pressure. Isoprenaline log dose response curves of increase in heart rate and reduction in diastolic pressure after intravenous labetalol shifted to the right in a parallel manner compared with pre-labetalol response curves suggestive of competitive antagonism at beta-adrenoceptor sites. Similarly, phenylephrine dose response curves of increase in systolic pressure before and after intravenous labetalol were suggestive of competitive antagonism at alpha-adrenoceptor sites. The ratio of relative potency alpha: beta adrenoceptor antagonism after intravenous labetalol was approximately 1:7, whereas in the same subjects after oral labetalol the ratio was approximately 1:3 as previously reported. Using the inhibition of isoprenaline tachycardia to estimate the potency of the beta-adrenoceptor antagonism of labetalol relative to that of propranolol the potency ratio was 1:6. However, using inhibition of Valsalva tachycardia as the index, the estimated ratio was approximately 1:3. Estimates of relative potency using inhibition of tilt tachycardia were complicated by the additional effects upon blood pressure after labetalol not seen after propranolol. Labetalol produced adrenoceptor blockade at both alpha and beta sites in man sufficient to explain its therapeutic antihypertensive effect.


Diffusion coefficients for protein molecules in blood serum. A new technique is described for the measurement of the self-diffusion coefficients of protein macromolecules in solution. The method makes use of the phenomenon of Taylor dispersion of a solute introduced into a solvent flowing in the laminar regime through a tube of circular section. Results are reported for the self-diffusion coefficient of cholesterol associated with lipoprotein molecules in dogs' serum at pH 7.4 in the temperature range 18-37 degrees C. The diffusivity of bovine serum albumin in serum has also been studied as a function of temperature at pH 7.4 and 4.7. In the more basic solution, measurements of the diffusivity as a function of protein concentration substantially agree with earlier work. For all the systems studied the diffusivity varies rapidly with temperature. The pH of the solution, in the case of bovine serum albumin, also has a significant effect on the diffusivity of the macromolecule. The latter observation is related to the amount of water bound to the protein molecule in solution.


[Acid-base equilibrium in healthy newborns. Pattern of normality in Mexico City]. During their spontaneous recovery after birth, the acid-base balance was studied in 32 healthy newborns. The observation period lasted for 4 hours. Determinations were carried out, both in umbilical vessels as in central arterial blood. The most outstanding phenomenon was an increase in acidosis at 30 minutes of life. The metabolic component predominating over the respiratory was a definitely evident fact. At the end of four hours, the acid-base balance reached figures considered normal for the adult.


The effects of Bordetella pertussis vaccine on cerebral vascular permeability. The effect of Bordetella pertussis vaccine on the cerebral vascular permeability in the mouse was studied by a radio-isotope method (131I-labelled HSA). Intravenous injection of 4 x 1010 heat-killed pertussis organisms caused a measurable increase in permeability in normal mice. Cryoinjury to the cerebral hemispheres resulted in a striking increase in vascular permeability at 24 h. This declined within 48 h and stabilized at a level fractionally higher than normal at 7 days ("healed lesion"). When pertussis organisms were injected into mice bearing ("healed lesion"). When pertussis organisms were injected into mice bearing "healed lesions" the increase in permeability was similar in magnitude to that in uninjured brain. The effect was increased by a second administration of pertussis 24 h after the first. The action of pertussis on a newly inflicted cryoinjury was protective. It is suggested that permeability changes in the cerebral vessels may be involved in the evolution of the encephalopathy attributed to the use of Bordetella pertussis vaccine in man.


Inhibition of the liver and plasma protein acute-phase response in mice by D-galactosamine. Local inflammation evoked in Swiss albino mice by subcutaneous injection of Celite resulted in a rise of liver tyrosine aminotransferase activity and plasma level of fibrinogen and seromucoid, while liver alanine aminotransferase activity and plasma level of fibrinogen and seromucoid, while liver alanine aminotransferase activity and the plasma level of albumin and total protein remained unaltered. By measuring the incorporation of [14C] leucine, stimulation of liver and plasms protein synthesis by Celite injection was demonstrated. Administration of D-galactosamine (2-5 mg/10 g body weight) inhibited the enhanced synthesis of liver proteins, and especially of trauma-induced synthesis of plasma fibrinogen and seromucoid. The inhibitory effect of galactosamine was most pronounced when the amino sugar was injected simultaneously with Celite and then protein synthesis was measured 6 h later. The results obtained support the idea that high doses of galactosamine inhibit transcription of trauma-inducible mRNA in the liver and thus block the acute-phase response.


Atenolol versus propranolol. A comparison of ocular hypotensive effect of an oral dose. In a controlled double-blind cross-over trial in 10 patients comprising six with open-angle glaucoma, three with closed-angle glaucoma, and one with ocular hypertension, a single oral dose of atenolol (50 mg) was significantly more effective than propranolol (40 mg) in reducing ocular tension.


Milk-substitute diet composition and abomasal secretion in the calf. 1. The effect of different protein sources in milk-substitute diets on abomasal acidity and proteolytic activity was studied in Friesian calves, aged 20-58 d (Expt 1). The diets contained 'mildly' preheated, spray-dried skim-milk powder (MHM), severely preheated, spray-dried skim-milk powder (SHM), fish-protein concentrate (FPC) or solvent-extracted soya-bean flour (SF) as the main protein source. 2. Gastric juice was collected from abomasal pouches before feeding and at 15 min intervals for 8 h after the morning feed. Samples of digesta were obtained from the abomasum at 1 h intervals during the same period. 3. Digesta pH was lower and titratable acidity higher 0-3 after giving the diet containing MHM than when any of the other three diets was given. 3. Acid secretion from the pouches for the different diets was in the order: FPC greater than MHM greater than SHM greater than or equal to SF. 5. Protease secretion from the pouches, assayed at pH 2-1, was in the order: MHM greater than SHM = FPC greater than SF. 6. The effect of dry matter (DM) intake and concentration on abomasal acidity was also studied in calves given diets which contained MHM (Expt 2). This diet was reconstituted at either 100 or 149 g DM/kg liquid diet and fed at either 32-5 or 49-0 g DM/kg live weight 0-75 per d. Samples of abomasal digesta were collected as in Expt 1. 7. A high intake of DM at a low DM concentration resulted in low acidity of the digesta in the first 3 h after feeding, which suggested a dilution effect. Comparison of two diets of different DM concentration, which were fed in the same volume of liquid, indicated that the greater the DM intake, the greater was the amount of acid secreted. 8. It is concluded that the protein sources varied in their ability to stimulate abomasal acid and protease secretion and it is suggested that this may relate to calf performance.


Studies on lipid digestion in the preruminant calf. The source of lipolytic activity in the abomasum. 1. Lipolytic and proteolytic activities and pH values were determined in secretions collected from innervated abomasal pouches and in abomasal contents from preruminant calves given liquid diets. 2. No lipolytic activity was detected in pouch secretions collected during 1 h after feeding, though lipolytic activity was present in abomasal contents; pepsin (EC 3.4.23.1) and renin (EC 3.4.23.4) were present in both pouch secretions and abomasal contents. The pH values of pouch secretions ranged from 1-2 to 1-8 and those of abomasal contents from 4-2 to 5-9. 3. When diet was placed directly into the abomasal pouch soon after feeding, the pH values of pouch and abomasal contents decreased similarly (i.e. from 6-3 to approximately 5). Protease activity (U/ml) of pouch contents ranged from 0-1 to 0-8 and that of abomasal contents from 0-1 to 0-2. No lipolytic activity was detected in pouch contents, though abomasal contents contained 0-6 to 1-2 U/ml and when the diet contained milk-fat as the dietary fat source considerable lipolysis of triglycerides containing shorter-chain fatty acids was found. 4. It is concluded that there is no significant secretion of lipolytic enzymes by the fundal mucosa and that the lipolysis of triglycerides in the abomasum of the preruminant calf is due predominantly to a lipolytic enzyme in saliva.


Studies on digestion and absorption in the intestines of growing pigs. Measurements of the flow of digesta and pH. 1. Thirty-five pigs were fitted with single re-entrant cannulas in either the duodenum, jejunum or ileum. A further twenty-four pigs were used in a conventional digestibility trial. 2. Methods for collecting, sampling and returning digesta were developed. 3. A 'practical-type' diet and two purfied diets were used, fed twice daily. 4. Flow and pH of digesta were measured hourly in the duodenum and jejunum, and every 6 h in the ileum. 5. In the duodenum and jejunum there were clear flow responses to feeding, while such an effect was not found in the ileum where the flow-rate was much lower and more uniform than in the former sites. 6. In the duodenum and jejunum, and within 6 h periods in the ileum, there was considerable variation in the flow-rate between different pigs within each hour but there was less variation in pH. 7. The pattern of flow in the duodenum and jejunum was similar for each of the diets but the total flow and the average pH in 24 h differed significantly between diets. There were more digesta of a lower pH from the 'practical-type' diet than the purified diets. 8. The pH in the duodenum was highest after feeding and decreased with increasing time after feeding. In the jejunum and ileum the pH-varied over a much smaller range than in the duodenum. 9. Collections for 6 h periods appeared to be insufficiently long to predict the values obtained in 24 h collections with reasonable accuracy.


Proton-dependent dissociation equilibrium of hemoglobin. 1. A 700-nanometer light-scattering study on horse methemoglobin in the pH range 4.8 to 7.2. The effect of proton concentration upon the subunit dissociation of horse methemoglobin has been investigated at two ionic strengths by light scattering photometry at 700 nm. Differential refractometry revealed a slight but systematic decrease of the specific refractive index increment with decreasing protein concentration for solutions in dialytic equilibrium with the solvent. In the pH range 4.8-7.2 the dissociation can be described by a simple equilibrium between tetramers and dimers. The dissociation constant Kd of the met derivative is found to be very similar to those of the O2- and CO-ligated states. From the slope of a plot of log Kd vs. pH, the number of protons bound is n = 1.3 +/- 0.1 resulting from an increase in the pK values of two groups upon dissociation. These two groups must be identical because the dissociation is symmetrical.


Proton-dependent dissociation equilibrium of hemoglobin. 2. Surface pressure measurements in monolayers of horse hemoglobin (III). The molecular weight of hemoglobin (III) in monolayers on aqueous subsolutions has been determined by measuring the surface pressure as a function of the protein surface concentration. The dissociation equilibrium between tetrameric and dimeric hemoglobin (III) was determined for spread as well as adsorbed monolayers. The results were compared with analogous measurements in solution. It was found that the numerical value and the pH dependence of the dissociation constant were similar both in the bulk and in the surface phase of the solution. From these findings it was concluded that the native conformation of hemoglobin (III) is retained after adsorption at aqueous surface.


Conformational characteristics of luliberin. Circular dichroism and fluorescence studies. By circular dichroism and fluorescence spectroscopy, the conformation of luliberin (luteinizing hormone-releasing hormone) has been investigated under various conditions of pH and solvents. Several structural parameters have been defined which seem predominant for the maintenance of the hormone in some privileged conformation(s). Formation of an intramolecular hydrogen bond between CO (His) and NH (Ser) seems likely when dissolving the hormone in organic solvent such as dioxane. Energy transfer has been demonstrated between Tyr and Trp residues. Calculation of the energy-transfer efficiency at different pH's allowed us to estimate in the range of 10 A the distance which separates these residues. Evidence is also provided for a charge-transfer interaction between protonated histidine and tryptophan. These data suggest that, when luliberin has organized structure (under appropriate surrounding conditions), its conformational pattern would resemble that of beta-turn structure in which a beta bend would exist at the level of the aromatic residues.


Conformational characteristics of luliberin. Luminescence properties at liquid-nitrogen temperature. The luminescence properties (fluorescence and phosphorescence) of luliberin have been investigated at liquid-nitrogen temperature (77 K) in 50% ethylene glycolaqueous buffer at various pH's. Calculation of the energy-transfer efficiency between Trp and Tyr residues leads to an evaluation of the distance separating these residues. In alkaline medium, when tyrosine is ionized, a 100% transfer efficiency occurs from Trp to Tyr- at the singlet level and also from Tyr- to Trp at the triplet level indicating that the TRP-Tyr distance is less than about 5A. When luliberin is at pH 7.8 (or 4.5), transfer efficiency from Tyr to Trp at the singlet level is 85-90% which should correspond to a distance between Trp and Tyr of about 10-12 A. These results are discussed with respect to the role played by aromatic amino acids both in the hormone conformation and in the hormone biological potency. Moreover, comparison with the data obtained from fluorescence or circular dichroism studies at room temperature allows us to deduce some characteristics of luliberin conformation. Structure-activity relationships in the aromatic region of luliberin are also discussed.


Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system. An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.


Intermediate states of actomyosin adenosine triphosphatase. The early kinetic steps of actomyosin subfragment 1 (acto-S1) adenosine triphosphatase have been investigated by simultaneous monitoring of fluorescence and light scattering and also by observation of the time course of the production of phosphate. The results show that fluorescence enhancement occurs after the dissociation of actomyosin and that the rate of enhancement is similar to the maximum rate of enhancement for S1 alone, under similar conditions of pH and temperature. The maximum rate of the phosphate burst for acto S1 is also approximately the same as that for S1 alone. The maximum rates for fluorescence enhancement or phosphate formation are reached at much lower adenosine triphosphate concentrations for acto-S1 than for S1. An extension of the actomyosin scheme is presented which accounts for these results.


Energetics and mechanism of actomyosin adenosine triphosphatase. Rate constants were determined for the reaction of actin with subfragment 1 (S1), S1-product complex, heavy  meromyosin (HMM), and HMM-products complex for a range of temperatures, pH's, and ionic strengths. For actin concentrations up to 10 muM, the rate of reassociation of the product intermediate was equal to the rate of actomyosin subfragment 1 (acto-S1) or acto-HMM adenosine triphosphatase (ATPase). Therefore, under these conditions, the only  important pathway for adenosine triphosphate hydrolysis is through the dissociation and recombination of S1 or HMM. The apparent rate constants for the association of S1 and S1-product with actin showed a similar large ionic strength dependence. The S1-product reaction had a large temperature dependence paralleling the rate of acto-S1 ATPase, while the reaction with S1 had a much smaller variation with temperature. The low value of the rate constant for the S1-product reaction and its relationship to the s1 areaction suggests that the apparent rate constant does not measure a simple second-order reaction. A plausible mechanism is a rapid equilibrium for the binding step, followed by a transition (product release) which  increases the association constant. A refractory state could also reduce the apparent rate constant of recombination. An approximate assignment of equilibrium constants for the acto-S1 ATPase reaction was made based on the interpretation of the present evidence and equilibrium constnats for the S1 ATPase.


Relaxation spectra of yeast hexokinases. Isomerization of the enzyme. Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.


Effect of alcohols on the structure and function of D-amino-acid oxidase. The absorption spectrum of D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was significantly perturbed by various alcohols; typical fine structures were observed in the visible absorption bands, accompanied by blue shifts of the peaks. Both fluorescence intensity and fluorescence polarization were increased upon the addition of alcohols, indicating that the coenzyme is not liberated from the apoenzyme but the hydrophobicity of the environment of the enzyme-bound flavin is increased. Upon the addition of alcohols, the circular dichroism of the enzyme was markedly modified in the visible and near-ultraviolet regions, while that of the apoenzyme in the near- and far-ultraviolet regions was scarcely modified, indicating a change in the interaction between the flavin coenzyme and protein. Both the apparent maximal velocity and the apparent Michaelis constant of the enzyme were increased by the addition of alcohols. The presence of alcohols tends to dissociate the dimer of this enzyme into the monomer, but the dissociation does not fully explain the increase in the maximal velocity of the enzyme by alcohols, because the increase in the maximal velocity caused by alcohols is larger than that expected from the dissociation. Since the rate of formation of the purple intermediate was decreased by alcohols in both the dimer and the monomer, the increase in the maximal velocity could be ascribed to an increase in the rate of dissociation of the enzyme-product complex. This increase could be ascribed to the protein conformational change, which is probably provoked by combination of alcohols with the enzyme at a locus other than that for substrate binding.


S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme. Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.


Purification and properties of Renilla reniformis luciferase. Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.


Oxidation of spermidine and spermine in rat liver: purification and properties of polyamine oxidase. A novel enzyme responsible for the oxidation of spermidine and spermine has been found in rat liver. Spermidine is shown to be degraded to putrescine and 3-aminopropionaldehyde, and spermine to be cleaved to spermidine and 3-aminopropionaldehyde. A single enzyme catalyzing both reactions and designated as polyamine oxidase has been purified 4000-fold to electrophoretic homogeneity. Polyamine oxidase appears to be a flavoprotein, containing flavin adenine dinucleotide (FAD) as a prosthetic group. Hydrogen peroxide is evolved in the reaction and no other electron acceptors except molecular oxygen have been found. The molecular weight of the enzyme was approximately 60 000 and the sedimentation coefficient 4.5 S. The enzyme appears to be a single polypeptide chain since no evidence for structural subunits was obtained. Polyamine oxidase was sensitive to sulfhydryl and carbonyl group reagents. The optimum pH value for the oxidation of polyamines was close to 10. The reaction velocities were enhanced by various aldehydes, especially certain aromatic aldehydes. Polyamine oxidase appears to be localized in peroxisomes of liver cells, although the existence of an isoenzyme in the cytosolic fraction was not definitively ruled out. No marked changes were observed in the activity of polyamine oxidase in rat liver after partial hepatectomy, carbon tetrachloride poisoning, and after treatment with growth hormone or thioacetamide, conditions which are known to alter profoundly the metabolism and accumulation of polyamines.


Hydrolysis of chyle cholesterol esters with cell-free preparations of rat liver. 1. The effect of pH on the hydrolysis of chylomicron and chylomicron remnant cholesterol ester with rat liver homogenate was examined. The hydrolysis had three pH optima, at pH 4.5, at pH 6.0-6.5 and at pH 8.5. At the two upper pH optima extensive cholesterol ester hydrolysis occurred without simultaneous degradation of the triacylglycerol portion. 2. Similarly, microsomes (at pH 6.5-8.0) and 100 000 X g supernatant (at pH 7.5-8.5) efficiently hydrolyzed the cholesterol ester but not the triacylglycerol of chylomicron remnants. 3. With the same substrate no enrichment of neutral cholesterol esterase activity was seen in isolated plasma membranes. 4. At pH 4.5 lysosomes efficiently hydrolyzed both the cholesterol ester and the triacylglycerol portion of chylomicron remnants. 5. Three conclusions are drawn: (a) the study provides evidence against the existence of a plasma membrane-bound enzyme-hydrolyzing chylomicron cholesterol ester before or during its penetration into the cell; (b) enzymes of the cell sap and possibly of the endoplasmic reticulum can degrade cholesterol ester of chylomicron remnants without preceeding hydrolysis of the triacylglycerol core; and (c) lysosomal enzymes can degrade both the cholesterol ester and the triacylglycerol portion of chylomicron remnants if these are taken up as whole particles by endocytosis.


Characterization of the acetyl-CoA synthetase of Acetobacter aceti. The acetate activating system of Acetobacter aceti has been studied. The enzyme responsible, acetyl-CoA synthetase, has been purified about 500-fold from crude cell extracts and was approximately 85% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The purified enzyme showed optimal activity at pH 7.6 in both Tris-HCL and potassium phosphate buffers. In its purest form, the enzyme was stable at 4 degrees-C but denatured upon freezing. The Km values for CoA, ATP and acetate were found to be 0.104 mM, 0.36 mM and 0.25 mM respectively; propionate and acrylate were also activated by the enzyme but not butyrate, isobutyrate or valerate. GTP, UTP, CTP and ADP could not replace ATP in the reaction, and cysteine or pantetheine failed to replace CoA. The cationic requirements were studied and of the divalent cations tested, only Mn2+ could significantly replace Mg2+ in the reaction; K+ and NH4+ stimulated enzyme activity but inhibited at high concentrations; Na+ was a poor activator, but did not inhibit at higher concentrations. The effect of a number of glucose and other metabolites on enzyme activity has been tested.