Systemic and regional blood-flow changes during spinal anesthesia in the rhesus monkey. The radioactive microsphere technique was used to determine the distribution of cardiac output and regional blood flow in rhesus monkeys before and 10,20,40, and 80 minutes after induction of spinal anesthesia. Five monkeys were studied during low spinal anesthesia (sensory level T10) and five other monkeys were studied during high spinal anesthesia (sensory level T1). Each monkey served as its own control. There was no significant change in regional blood flow during T10 spinal anesthesia. During T1 spinal anesthesia, blood flow (per 100 g of tissue) to kidneys was significantly reduced at 20, 40, and 80 minutes, blood flows to liver and carcass were significantly reduced at 20 and 40 minutes and blood flows to miscellaneous organs (lymph nodes, salivary glands, etc.) were significantly reduced throughout anesthesia. Blood flows to heart, brain, and lower extremity during T1 spinal anesthesia showed only non-significant changes. Vascular resistance in the lower extremity was significantly reduced during both levels of spinal anesthesia, indicating arteriolar dilatation. Also, during both levels of anesthesia, the lungs received an increased proportion of the radioactive microspheres, suggesting increased peripheral arteriovenous shunting of microspheres due to the arteriolar dilatation.


Annals of allergy.

Annals of allergy. A review of the world literature regarding the relative efficacy, duration and toxicity of the available beta-adrenergic bronchodilator aerosols for the treatment of asthma is presented. Many of these agents will soon become available in the United States and some familiarity with their actions is necessary for optimal care of asthmatic patients.


In vivo erythrocyte sickling in the Japanese sika deer (Cervus nippon): methodology. The Japanese sika deep (Cervus nippon) proved to be a suitable animal model for the study of acute phases of in vivo erythrocyte sickling. Ophthalmologic studies can be conducted during or after 1 to 6 hours of effective in vivo sickling. Intravenous administration of 1.75 to 3.5% sodium bicarbonate solution at a rate of 500 to 1,000 ml per hour produced a transient state of alkalosis and in vivo erythrocyte sickling in the sika deer. The percentage of sickled erythrocytes increased as the blood pH increased. Concurrently, the packed cell volume decreased. Sickling was enhanced by 100% oxygen ventilation after endotracheal intubation and light anesthetization. After the induction of erythrocyte sickling, a sickling-reversal phenomenon occurred despite continued bicarbonate administration. During the course of this reversal, the percentage of sickled erythrocytes steadily decreased, the venous blood pH decreased, and the packed cell volume slowly increased. Because of the sickling-reversal phenomenon, chronic erythrocyte sickling was not achieved.


Changes in motility and ph in the digestive tract of experimentally overfed sheep. Motility of the digestive tract of 4 sheep was studied with radiotelemetric equipment. After base line records were made, each sheep was overfed with 70 g of grain per kilogram of body weight. The ruminoreticulum did not become static until the ingesta pH was less than 5. The cecum had the same patterns of motility and pH as did the ruminoreticulum, but these returned to normal more quickly in surviving sheep. The motility patterns of the abomasum and the small intestine were more erratic. Results indicated that considerable quantities of grain (substrate for microbial growth) reached the cecum before ruminoreticular motility was inhibited.


Physiologic studies of experimentally grain-engorged cattle and sheep. Seven sheep weighing 34 to 41 kg, each, 3 steers and 1 heifer weighing 230 to 460 kg each, were experimentally, "overfed" (induced grain engorgement). The most significant changes occurred in ruminal ingesta pH, blood pH, packed cell volume (PCV), hemoglobin (Hb), carbon dioxide pressure (PCO2, total CO2 (volume %), blood D-lactic acid, blood HCO3, and base excess. There was no common denominator that was especially pathognomonic.


Erythrocyte metabolism in normal and glutathione-deficient sheep. Glucose utilization and lactate formation in erythrocytes from normal and glutathione (GSH)-deficient sheep were similar. Significant differences were observed, however, between the 2 groups of sheep in the production of 14-CO2 from erythrocytes incubated with ascorbic acid or methylene blue, or both. The greater response of normal erythrocytes compared to erythrocytes deficient in GSH suggests that there are some metabolic differences in the pentose phosphate pathway (ppp) activity of the erythrocytes. The nature and site of these differences are, however, not known. When sheep were kept in a decompression chamber for 2 weeks and subjected to a simulated altitude of 7,000 m for 12 hours/day, the erythrocytes showed a four- to six-fold increase in the activity of the ppp.


The detection of cross-reacting material for alpha-antitrypsin in liver extracts by acid-starch electrophoresis. Purified extracts of alpha-antitrypsin cross-reacting material (CRM) from liver of patients with MM, ZZ, and MZ phenotypes were studied by acid-starch electrophoresis and crossed immunoelectrophoresis. It is believed that CRM-M from liver is derived primarily from postmortem digested serum antitrypsin, whereas CRM-Z is a product of postmortem digested antitrypsin from hepatocytes. All extracts produced small bands of antitrypsin on the cathodal side of the gel: A single M band migrated faster than the single Z band, whereas CRM from an MZ heterozygote produced a combination of 2 mobile bands. The CRM-Z (from a ZZ phenotype) did not migrate toward the anode, in contrast to the presence of a broad plateau of antitrypsin protein with CRM-M (from an MM phenotype) or CRM-MZ. Thus, most of the CRM-Z protein could not be accounted for on routine acid-starch electrophoresis. However, an anodal peak could be seen on starch gels with CRM-Z at less acidic pH, suggesting that the anodal-migrating CRM-Z protein is denatured and loses antigenicity at lower pH.


Determinants of chronic obstructive pulmonary disease in patients with intermediate levels of alpha-antitrypsin. We examined elderly heterozygotes for alpha-antitrypsin deficiency (phenotype MZ) to determine which of several factors might be associated with the development of chronic obstructive pulmonary disease (COPD). Elderly heterozygotes who smoked had a very high incidence of COPD (70 per cent), whereas homozygotes (phenotype MM) who smoked had a significantly lower incidence of COPD (35 per cent). There was no difference in the incidence of COPD between subjects with MM and MZ phenotypes who did not smoke. To determine if leukocyte enzymes were related to the development of COPD in elderly heterozygotes, we measured the activities of monocytic acid cathepsin, neutrophil acid cathepsin, neutral cathepsin, and elastase. The activities of these enzymes were not associated with the development of COPD or with smoking history in the heterozygotes. In conclusion, cigarette smoking seemed to be a significant determinant of the development of COPD in heterozygotes for alpha-antitrypsin deficiency, but the leukocyte enzyme actitivty that we studied was unrelated.


Flurazepam hydrochloride, a benzodiazepine hypnotic. Flurazepam hydrochloride is a benzodiazepine derivative marketed for use as a hypnotic agent. Flurazepam is more effective than placebo and is as effective as other hypnotic drugs in most short-term controlled studies. In long-term dosage studies, flurazepam's efficacy persists while other hypnotics become ineffective. Flurazepam has relatively minor effects upon rapid eye movement (REM) sleep and does not lead to REM rebound; this may reduce the likelihood of drug dependence. Flurazepam does not cause enzyme induction and probably presents little hazard of abuse or overdosage. The rational use of hypnotic agents depends as much upon the underlying cause of the sleep disorder as upon the choice of a particular drug. When hypnotic therapy is indicated, flurazepam appears to have advantages over other drugs currently available in the United States.


Unanswered questions about the periodic health examination. The periodic health examination in its application as a screening procedure in asymptomatic, ostensibly healthy persons is explored with a focus on the following issues: (a) the impact on health, (b) the content of a beneficial health examination, and (c) the effect of the examination on the physician-patient relation. The application discussed is distinct from use of the examination as a tool for diagnosis, prognosis, or therapeutic planning for patients with a specific illness. The discussion also shows a relatively recent change in the goal for the clinical assessment. There has been a shift in emphasis from establishing a diagnosis as the main outcome event of the periodic "checkup" to the identification of an intervention of value to the patient. Evidence from various studies that throw some light on related questions is considered. Special ethical issues surrounding the unsolicited medical assessment are identified. Finally, some ground rules for decisions about the periodic health examination are proposed.


[Malabsorption of carbohydrates in children (author's transl)]. Physiological bases of digestion and absorption of carbohydrates are reviewed, as a preliminary step, in order to draw a general scheme of its patholophysiology. Clasification of different types of carbohydrate malabsorption is presented. Various exploration methods are discussed in terms of autors' own experiences. Relationship between a sugar screening test, faecal lactic acid contents and a simplified lactose tolerance test, is described in detail. Systematic diagnoses of these diseases are established. Different clinical pictures are reviewed. It is not yet well defined if a starch malabsorption can be caused by either a primary or secondary duodenal amylase deficiency. The clinical forms of congenital sucrose-isomaltose intolerance may be more attenuated than its classical form; incertain cases, secondary sucrose intolerance may also be present due to mucosa anatomic lesions. Maltose malabsorption has no clinical implications. As compared to other alpha-glycosidades, the trehalase activity has been not more affected by not using trehalose in feeding. Primary congenital lactase deficiency is not frequent, whereas secondary forms as much more usual and appear, in primary malabsorption syndromes and in the coeliac disease, very often along with clinical tolerance to lactose. In Spain, lactose nonabsorbers in 16.5% for adults 11.2% for adolescents and 18.3% for children, meaning, that it is being favored by environmental factors in the latter. The unspecified sugar malabsorption during the child's first year is still the most frequent cause of carbohydrate intolerance in children and, although certain progress has been achieved in its diagnosis and therapy, its pathogenic mechanism is not satisfactorily known yet.


A military surgical team in Belfast.

A military surgical team in Belfast. This paper details the experiences of a military surgical team in Belfast from 1972 to early 1974. The overall picture of the problem is given and the current management of 'war' injuries discussed. Up to February 1974 over 1000 servicemen have been injured in Northern Ireland as a result of the vivil disturbance. Over 200 have died. Because of the close proximity of the hospital to many battle areas, casualties may arrive with massive injuries, requiring major resuscitation. Limb wounds have predominated. There is no short cut to adequate wound debridement, especially in the surgery of high-velocity missile injury. Missile wounds of the large bowel require a colostomy. Formal thoracotomy is increasingly used for the through-and-through gunshot wounds of the chest. Controlled ventilation is playing an increasingly important role in the management of some missile wounds of the head. Mine and bomb explosions frequently cause multiple injuries, requiring extensive surgery on any one patient.


Partial 4q trisomy. Apropos of 3 cases.

Partial 4q trisomy. Apropos of 3 cases. Three observations of partial trisomy 4q are reported: the first due to a familial translocation the second to a de nove translocation, the third to a "mirror" duplication. The very characteristic phenotype is compared to that of 4 other patients already reported in the literature. The most evocatory symptoms include: absent or poorly indicated nose bridge; pursed lips; shortness of the philtrum; and constant existence of a fold on the antitragus continuing the anthelix reachinghe insertion of the pinna.


Hyperglycemia, polyol metabolism, and complications of diabetes mellitus. This brief review of the sorbitol pathway has attempted to present our current knowledge of this accessory pathway of glucose metabolism in the development of some diabetic complications. Clearly hyperglycemia in the diabetic patient is an important factor controlling the activity of the sorbitol pathway. Hyperglycemia in both diabetic patients and experimental animals results in significant accumulations of the products of this pathway in some tissues, and these diabetic manifestations. The development of inhibitors of the aldose reductase enzyme affords new means for preventing and treating some of these complications. Nevertheless, we are still hampered by the lack of knowledge of the normal role of this pathway in tissue metabolism. Many technical problems still exist concerning sensitive and specific assays for the products of the sorbitol pathway in tissue studies, as well as with valid techniques for the measuremtn of the activity of this pathway in clinical situations. It is hoped that clinical studies with aldose reductase inhibitors in the future will further clarify the importance of this accesory pathway of glucose metabolism in diabetes.


Pharmacology of the esophageal motor funciton. In the striated muscle of the upper esophageal sphincter, tonic maintenance of closure is probably mediated via tonic central excitation of the extrinsic motor innervation; relaxation represents central inhibition of this mechanism. The motor nerves are probably cholinergic and act through nicotinic receptors like those of somatic striated muscle. In the striated muscle of the esophageal body, swallowing-induced contraction is also probably a cholinergic and nicotinic response. In the smooth muscle of the esophageal body, the control of contractions is cholinergic and muscarinic in part, but there is evidence for a nonadrenergic and noncholinergic component as well. The muscarinic component may arise from the cholinergic innervation of the longitudinal muscle layer. The other component may lie in the cryptic innervation of the circular muscular layer. In the smooth-muscled lower esophageal sphincter, resting closure tension appears to reflect a variety of possible control mechanisms. No single control system predominates. The evidence for muscarinic excitation is equivocal. An excitatory adrenergic alpha mechanism and inhibitory adrenergic beta receptors may contribute. A role for the polypeptide hormones from the gastrointestinal tract seems unlikely. Relaxation of the lower sphincter with swallowing seems not to involve any of these mechanisms, but is apparently accomplished by nonadrenergic noncholinergic inhibitory nerves like those present elsewhere in the gut (87). The possibility that the transmitter of these nerves is an adenine nucleotide has been raised from studies of other parts of the gut, but that hypothesis has not yet been examined critically in the lower esophageal sphincter.


In vitro models in the study of structure-activity relationships of narcotic analgesics. Evidence has been adduced for the view that the morphiness receptor or receptors in the myenteric plexus of guinea pig ileum are very similar to the brain receptors that mediate the analgesic action of the narcotic alagesics.  Such an in vitro model is very suitable for the study of structure-activity relationships because the angonist and antagonist activities of compounds can readily be assessed. One example is the investigation of the effects of changes at the N atom. Another point of interest is the role of substitutions at the C14 atom in morphine and morphinans. Such alterations in structure may have a profound effect on the relative agonist and antagonist activities and may convert a drug with dual agonist and antagonist actions into an antagonist devoid of agonist action.


Effect of pH and human serum on the susceptibility of group D streptococci (Enterococci) to ampicillin in vitro. The in vitro susceptibility of group D streptococci (enterococci) to ampicillin was studied comparing the results obtained in Mueller-Hinton broth (MHB) with those obtained in normal human serum (NHS). The rate of enterococcal killing was consistently faster in NHS than in MHB at equivalent ampicillin concentrations. Whereas an increasing media pH appeared to decrease the susceptibility of enterococci to ampicillin by determinations of the minimum bactericidal concentration (MBC) of ampicillin, an opposite increase in susceptibility was observed when the rate of bactericidal activity was studied. This difference may be explainable by the instability of ampicillin at higher pH values. In both MHB and NHS a paradoxical decrease in the rate and extent of enterococcal killing occurred as the ampicillin concentration was increased above the minimally effective concentration. These results demonstrate the inadequacies of the MBC test system and the need for standardizing test media used for determining the susceptibility of enterococci to ampicillin.


Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide. The antibacterial effects of 24-h exposures to high-pressure oxygen in relation to environmental CO(2) were studied at 3 atm absolute (ata) and at 1 ata. Eight gram-negative, aerobic and facultatively aerobic, pathogenic enteric bacteria (Salmonella typhosa, Salmonella paratyphi, Salmonella schottmuelleri, Shigella dysenteriae, Shigella flexneri, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli) were exposed as shallow-broth cultures and agar surface cultures. Although broths supplemented with 0.2% glucose permitted some growth of Salmonella typhosa, Salmonella schottmuelleri, Shigella dysenteriae, and Shigella flexneri during exposure to high-pressure oxygen in the presence of CO(2), the other species grew only after the exposure, indicating a bacteriostatic effect. Both bacteriostatic and bactericidal effects were demonstrated on the surface of Trypticase soy agar, where killing of Salmonellea typhosa, Proteus vulgaris, and Pseudomonas aeruginosa was significantly greater after exposure to pure O(2) at 3 ata than at 1 ata. At 3 ata, significantly more killing occurred upon exposure of all species (except Shigella dysenteriae and S. flexneri) on an agar surface to 100% O(2) as compared with exposure to a mixture of 95% O(2) + 5% CO(2). Thus, deprivation of CO(2) during exposure to pure O(2) enhanced the bactericidal effect of high-pressure oxygen.


Inactivation of enterovirus by glutaraldehyde. A study on the rate of inactivation by glutaraldehyde of coxsackievirus was conducted using different concentrations, temperatures, and pH values. It was found, that 2 percent glutaraldehyde at pH 7.4 and 25 C, as recommended for a sporicide, reduced the titer of infectious virus by 2 log10U in 1 min or less. The reduction was not negatively affected by high concentrations of organic matter (serum and animal spillings) in the reaction mixtures.


Common mesophilic anaerobes, including Clostridium botulinum and Clostridium tetani, in 21 soil specimens. A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 X 10-2 to 3.3 X 10-6 per g. The organisms most frequently isolated were Clostridium subterminate, C. sordelii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridium. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content.


Extracellular lipids of Cladosporium (Amorphotheca) resinae grown on glucose or on n-alkanes. Cladosporium (Amorphotheca) resinae was grown in shake culture on glucose, n-dodecane, or n-hexadecane. Growth was most rapid on glucose, and more acid accumulated in the medium than in n-alkane-grown cultures. Neutral lipid was the major lipid fraction and triglycerides were the only extracellular neutral lipids detected. Dodecanoic (lauir) acid was the predominant fatty acid (greater than 60%) in neutral lipids from all three media, with lesser amounts of tetradecanoic, hexadecanoic, and octadecanoic acids. Extracellular phospholipids identified were phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin or a cardiolipin-like compound. Phospholipids from all three media contained dodecanoic acid as their principle fatty acid. Dodecanoic acid was the only extracellular free fatty acid detected. Glucose medium contained acetic, glyoxylic, and glycolic acids and an unidentified organic acid which may contribute to the lower pH in cultures after growth on glucose. In all classes of extracellular lipids the fatty acids do not correspond to the fatty acids previously determined to be associated with cellular lipids. Moreover, the fatty acids of extracellular lipids do not reflect the chain length of the n-alkane growth substrate.


Effect of pH on sporulation of Bacillus stearothermophilus. An improved broth medium was developed for high growth yields of Bacillus subtilis var. niger NCIB 8649, Bacillus cereus NCIB 9373, and Bacillus stearothermophilus NCIB 8919 and ATCC 7953. Sporulation was abundant (1.1 times 10-8 B. subtilis var. niger and 9.2 times 10-7 B. cereus per ml) at an initial pH of 7.0. Sporulation of both strains of B. stearothermophilus took place (1.9 times 10-7 and 2.4 times 10-7/ml, respectively) in this medium when initial pH values of 7.7 to 8.7 were used.


Effects of trace metals on the production of aflatoxins by Aspergillus parasiticus. Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low micrograms-per-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 mug/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 mug of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 mug or less of the metal per ml, salts of iron, manganese, cooper, cadmium, trivalent chromium, silver, and mercury partly or completelyinhibited aflatoxin production, without influencing mat weight.


Metabolism of deoxycorticosterone by human fecal flora. 21-Dehydroxylation, a feature of metabolism of corticoids in humans, was observed in mixed cultures of fecal flora of normal individuals on a Western diet. The model substrate, 11-deoxycorticosterone (DOC), was metabolized to 3alpha-21-dihydroxy-5beta-pregnan-20-one (THDOC), 3alpha-hydroxy-5beta-pregnan20-one (pregnanolone), and to two unidentified structures, metabolites X and Y. DOC was not metabolized in all media supporting growth of fecal flora. Conversion required an initial pH between 6.0 and 8.0. 21-Dehydroxylation occurred within 4 days of incubation in media inoculated with 10-minus 1 to 10-minus 7 fecal suspensions. In higher dilutions, containing obligatory anaerobes only, DOC was converted to metabolite X and sometimes also to metabolite Y. The yield of pregnanolone was related to the promptness with which the specimen was processed, to the presence of cysteine in the medium, and to the concentrationof substrate (optimum, 16 to 64 mug of DOC per ml). The yield of THDOC was related to the delay in the processing of the specimen, the concentration of substrate (maximum at 256 mug/ml), and aeration of the culture. Pure cultures of aerobic organism of fecal origin either failed to metabolize DOC or converted it to metabolite Y. Pure cultures of fecal anaerobes converted DOC to metabolite X and sometimes also to metabolite Y. Neither THDOC nor pregnanolone was produced by pure cultures.


Necrotizing vasculitis. Etiologic aspects of immunology and coagulopathy. Thirty-one patients with cutaneous necrotizing vascultis were studied for immunological and coagulation disturbances. Serum immunoglobulin levels did not correlate with tissue deposition of the corresponding immunoglobulins in the lesions of cutaneous necrotizing vasculitis. In all instances, localization of immunoglobulins, complement, and fibrinogen, when present in the lesions of necrotizing vasculitis, was limited to the vascular wall or perivascular space. Soluble fibrinogen-fibrin complexes (cryoprofibrin) were detected in the blood of four of 17 patients with cutaneous necrotizing vasculitis. Since it represents a product of the limited action of thrombin on fibrinogen, its presence in the blood in some patients with necrotizing vasculitis suggests that intravascular coagulation may play a part in the pathogenesis of the disease. In 12 of the 31 patients studied, a cause of the vasculitis was found or presumed.


Familial glucocorticoid deficiency. Studies of diagnosis and pathogenesis. The clinical and biochemical findings are described in 2 brothers who had intermittent hypoglycaemia generally precipitated by the "stress" of infection. Both were tall and pigmented. Both boys showed a failure of adrenocortical response to ACTH which was progressive in the eldest boy. The diagnosis of familial glucocorticoid deficiency (hereditary adrenocortical unresponsiveness) was confirmed by the absence of electrolyte imbalance even on a low sodium diet, and by very high levels of ACTH in plasma. High levels of deoxycorticosterone (DOC) were found in both children with normal levels of other plasma corticosteroids. It is suggested that the high levels of DOC may be in some way related to the apparent persistence of a "fetal" type of adrenocortical steroid biosynthesis for 18 months or more in these boys. After the diagnosis, established by relatively simple methods, treatment with cortisone acetate has 0een highly effective.


Plasma gastrin in congenitial hypertrophic pyloric stenosis. A hypothesis disproved. Fasting plasma gastrin levels were measured in babies with pyloric stenosis and in normal babies of similar age. There was no difference in gastrin levels either before or after operation between the babies with pyloric stenosis and normal babies. Similarly, neither the fasting blood glucose nor the fasting gastric pH of the babies with pyloric stenosis differed significantly from the values obtained in normal babies. Our findings do not support the hypothesis that gastrin in the fasting baby has an aetiological role in the development of hypertrophic pyloric stenosis of infancy. An alternative hypothesis is suggested.


Separation and molecular weight estimation of phosphatases in psoriatic scales by disc-electrophoresis. Disc-electrophoretic investigations of psoriatic scale homogenates (15000 x g supernatant) revealed several different phosphatase activities. At pH 5 either five different phosphatase active bands (substrates: p-nitrophenylphosphate, naphthylphosphate) or three different bands (substrate: glycerophosphate) could be obtained. At pH 7 only one band (substrate: p-nitrophenylphosphate) showed phosphatase activity. At pH 9.9 either two bands (substrate: p-nitrophenylphosphate) or three bands (substrate: glycerophosphate) could be demonstrated. the acid p-nitrophenylphosphatase activity of the psoriatic specific bands "9" and "10" could be distinguished by their different fluoride and tartrate susceptibility. Also the alkaline glycerophosphatase activities could be differentiated by distinct Ca and Mg susceptibility.


Abolition of reactive hyperemia in the dog heart following alpha adrenergic blockade and its reversal by vasopressin. The local intracoronary application of the alpha adrenergic blocking agents phenoxybenzamine or dibenamine, in open chest anesthetized dogs, regularly resulted in maximal coronary vasodilatation thus abolishing reactive hyperemia. Infusion of vasopression or angiotensin returned coronary flow to near control levels and restored reactive hyperemia. It is thus concluded that although alpha blocking agents can relax coronary vessel smooth muscle, reactive hyperemia does not appear to be mediated through the inhibition of alpha adrenergic constrictor tone.


Timolol maleate, a new beta-adrenergic receptor blocking agent. Some pharmacodynamic properties of an oxypropanolamine substituted novel heterocyclic compound are described. Initial studies involve comparison of the racemic mixture, dl-timolol maleate, with the beta-adrenergic receptor blocking agent propranolol in the rat, dog and cat. dl-Timolol maleate is shown to be a potent inhibitor of cardiovascular beta-adrenergic receptors activated by isoproterenol or adrenergic nerve stimulation. Blockade of alpha-adrenergic receptors is not observed even after extremely high doses. The compound is approximately 3 times more potent than propranolol in suppressing isoproterenol-induced cardioacceleration by the i.v. route of administration. dl-Timolol maleate is also extremely well absorbed when given orally, being then about 10 times more active than propranolol. Unlike propranolol, neither dl-timolol maleate nor its optical isomers possess demonstrable local anesthetic activity. Similar to propranolol and other beta-adrenergic receptor blocking agents, activity of the compound resides predominantly in the l-isomer (timolol maleate.


Predictive value of various parameters for the antihypertensive effect of the beta blocker ICI 66,082 (1). The antihypertensive effect of the new beta-blocker, ICI 66,082 was studied in 37 patients with renal or essential hypertension. A daily dose of 75 mg produced a significant antihypertensive effect and a further blood pressure decrease was obtained by increasing the daily dose up to 300 mg; at this treatment level, the antihypertensive effect averaged 29.4/24.0 mm Hg. Increasing the dose from 300 to 600 mg daily did not produce a significant additional blood pressure decrease. Six of the 37 patients retained fluid, producing a loss in the antihypertensive effect. No significant correlation was found between the percentage systolic blood pressure fall and the following parameters determined before therapy: systolic blood pressure and its variability, heart rate (at rest and after stimulation by exercise or isoproterenol), cardiac output and total peripheral resistance, renin concentration (at rest and after stimulation) and urinary excretion of catecholamines and derivates.


The effect of clonidine and penbutolol, respectively on catecholamines in blood and urine, plasma renin activity and urinary aldosterone in hypertensive patients. In various kinds of hypertension clonidine induced a decrease in urinary catecholamines, plasma renin activity and urinary aldosterone, concommitant with a fall in blood pressure and pulse rate in both short term and chronic studies. Furthermore, clonidine lowered the plasma levels of noradrenaline and adrenaline but a postural increase in upright position still occurred. The capacity to increase renin during salt restriction seemed mainatined. When clonidine was withdrawn all parameters returned to pretreatment levels but in some cases a marked rebound increase in catecholamine production was seen. --During clonidine the increase in catecholamines and renin after insulin induced hypoglycemia was largely abolished. Under basal conditions oral penbutolol induced a decrease of pule rate and blood pressure but no change in plasma or urinary catecholamines. During treatment plasma renin was suppressed at rest and after exercise. A work load, which led to only minor changes in blood catecholamines before treatment, was associated with a marked increase during penbutolol. Medication with penbutolol reduced the response in plasma catecholamines after hypoglycemia and renin activity remained low. Clonidine seems to act mainly by central inhibtion of symapthetic tone. Penbutolol probably acts mainly peripherally but may also have a central effect.


Centrally induced hypotension unreleated to alpha-adrenergic stimulation. Central hypotension is induced by erythro-1-(1-[2-(1,4 - benzodioxan - 2 - yl) - 20H - ET] - 4 - piperidyl) - 2 - benzimidazolinone (R28935), as evidenced by local injections into the cisterna magna of anaesthetized dogs. This hypotensive effect is not inhibited by piperoxan, 100 mug/kg s.o. R28935, contrary to clonidine or norepinephrine, does not provoke vasoconstriction in the perfused isolated artery of the rabbit ear. Very low doses of R28935 antagonize vasoconstriction induced by KC1-depolarization. The centrally-induced hypotension is not due to alpha-adrenergic stimulation.


[Cardiopulmonary effects after i.v. administration of maprotiline in the dog]. The cardiopulmonary effects of the antidepressive agent maprotiline were investigated in the dog. In ventilation experiments on dogs anaesthetized with chloralose, respiratory minute volume decreased by 5-10% shortly after intravenous injection of 1 and 3 mg/kg; acid-base-balance variables remained unchanged. After an intravenous dose of 10 mg/kg ineffective hyperventilation was observed, associated with a transient decrease of P-O2 and a slight increase in P-CO2 in the arterial blood. In cardiac-catheterization experiments on dogs anaesthetized with chloralose and morphine and receiving artificial respiration, an intravenous dose of 3 mg/kg maprotiline had no appreciable effect on the cardiovascular system, apart from a transient and slight, but statistically significant increase in pulmonary capillary pressure immediately after the injection. Anaesthesia was slightly deeper and more prolonged after the intravenous administration of maprotiline.


Rheumatoid arthritis associated with hyperviscosity syndrome and intermediate complex formation. A patient with long-standing rheumatoid arthritis developed hyperviscosity syndrome that correlated with the presence of abundant intermediate complexes in plasma. The intermediate complexes were isolated, and evidence is presented that these complexes are composed predominantly of igG-rheumatoid factor.


Rapid method for eluting IgG antibodies from erythrocytes coated with anti-Rh immune antibodies. A method for eluting IgG antibodies from erythrocytes coated with anti-Rh immune antibodies has been developed, using temperature 56 degrees C as decomplexing factor and ion-exchange on DEAE Sephadex A-50 as antigen-binding factor.


Studies on stimulation and properties of antiglobulin antibodies. II. Antiglobulin antibodies against homologous globulins and antigens of thermally denatured autologous sera in the sera of rabbits immunized with various antigens. Sera of 48 rabbits after long-term immunization with suspensions of Diplococcus pneumoniae, Klebsiella friedländeri, Proteus OX19 and solution of bovine albumin were examined for antibodies reacting with homologous gamma-globulins and antigens of thermally denatured autologous sera. Appearance of antibodies of this type was related to duration of immunization, but not related to the type of antigen used. The experimental results were explained as a result of altered antigenic properties of antibodies following reaction with homologous antigen.


Rat interferons. III. Antiviral activity and effect of "priming" on interferon production in homologous and heterologous cell lines. Studies on interferon from rat serum sensitive to pH 2-0 showed the following: 1) Its action is stronger in cultures of heterologous L cells than in rat fibroblast cultures. 2) Resistance acquired under the influence of interferon lasts longer in a heterologous than in a homologous system. 3) Actinomycin D added to cultures after 4 hours of contact with interferon inhibits its activity in L cells, and potentiates its activity in RE cells. 4) In rat embryonic cells treated with interferon, interferon production following induction with ND virus is blocked. Similar blocking was observed in cultures of L cells after 24-hour priming; after 6 hours of exposure of L cells to rat interferon, on the other hand, production of interferon was enhanced.


Proteins in mouse embryonic cell (MEC) culture fluids following interferon induction. A study was made on the proteins and interferon activity in supernatant culture fluids from mouse fibroblast cultures induced with Newcastle disease virus, and in controls. The studied materials showed low protein content, the concentration of proteins depending on the length of time from induction to their collection. Proteins from induced and control cultures were separated on CM-Sephadex C-50 and by electrophoresis in polyacrylamide gel. No difference was found in the character of proteins from control materials and from materials induced 24 hours after infection; the elution profile was the same, and the distribution of electrophoretic fractions was similar. Interferon activity was not accompanied by the appearance of a new protein fraction. However, 5--10 hours after induction differences were noted in electrophoretic mobility and a marked reduction in the number of protein fractions occurred in control and stimulated culture fluids.


Some biochemical indices of posttransfusion shock induced under protection by selective hypothermia of the brain. Biochemical disorders accompanying posttransfusion shock in cats under protection by selective cerebral hypothermia, by our own method was studied in cats. In 11 of 22 cats, selective cerebral hypothermia was performed before transfusion of human group A blood. In 11 control cats, transfusions were made without prior selective cerebral hypothermia. The following parameters were determined in venous blood: pH, pCO2, HCO3, content and excess of buffer bases. Selective cerebral hypothermia diminished mortality in cats in posttransfusion shock and reduced the intensity of the biochemical changes accompanying shock.


Staged orchiorrhaphy. Therapeutic procedure in cryptorchic testicle with a short spermatic cord. In 62 planned staged orchiorrhaphies for a "short" spermatic cord, satisfactory location of the testicle following the second operation was achieved in 90% of cases. The testicle was atrophic and had to be excised in only 3.2%. After a follow-up period of two to ten years, the testicle remained satisfactorily located in 77% of cases; adequate size and configuration of the testicle (compared to the contralateral side) was found in 64.5%. In 17%, a partial or complete atrophy developed. No correlation was found between the initial size of the testicle and the late results. It is advocated, therefore, that excision be avoided unless definite atrophy exists. Planned staged orchiorrhaphy is recommended for testicles in high locations that are difficult to bring down. The late preschool period (5 to 6 years) is suggested as the optimal age for the first attempt at orchiorrhaphy and the second stage two years later, if required.


Effect of alkaline maintenance medium upon the growth of rabies virus in chick embryo cells. HEP Flury strain of rabies virus was propagated in chick embryo cells under maintenance media of different pH. It was found that viral growth was better and reached a markedly higher maximum titer when the initial pH of maintenance medium was 8.2 to 9.0 than when it was 7.4. The enhancement of viral growth was not ascribable to mere neutralization of acids produced from infected cells, because the different media became almost equally neutral within an early phase of growth curve. Serial passage of the virus in chick embryo cells using pH 8.2 maintenance medium resulted in altered growth characteristics of the progeny virus; first, the virus so passaged could now grow equally well under alkaline and neutral maintenance media, and, secondly, autointerference observable with the parent virus eventually lowered virus yield when neutral maintenance medium was used, but this effect of undiluted passage was eliminated by the use of pH 8.2 maintenance medium.


[Isolation of surface antigens of influenza virus A/Hong Kong 1/68 (H3N2) by density gradient fractionation with a new carboxypolypeptidase (author's transl)]. Isolation of pure neuraminidase and hemagglutinin from influenza virus A/Hong Kong 1/68 (H3N2) can be achieved using the coupled action of a new carboxypolypeptidase from Streptomyces fradiae and sodium dodecyl sulfate (SDS). Biochemical, physical and physico-chemical studies showed that the two molecules are in pure form and of glycoproteic nature.


Graft-versus-host reaction (GVHR). A case report suggesting GVHR occurred as a result of maternofetal cell transfer. A female infant with combined immune deficiency syndrome exhibited graft-versus-host reaction (GVHR) clinically during the nine weeks of her life. Pathologically, the first clue for GVHR was the lymphohistiocytic dermal and epidermal infiltrate with single cell necrobiosis seen on a skin biopsy specimen at 6 weeks of age. Two days before death, she received irradiated (3,500 rads) whole blood and plasma with specific antibodies against mother's lymphocytes, which was the first introduction of an allogenic hematopoietic substance. This suggests that the GVHR, which was confirmed at autopsy, occurred as the result of maternofetal cell transfer in utero or at time of delivery.


A new analysis of allogeneic interactions. Allogeneic reactions have conventionally been considered as typical immune responses by one population of cells to antigens present on the other. This view is inadequate, since it does not explain many features of these reactions, among which are: (1) reactivity is much higher between different strains within a species than between species, in spite of the much greater antigenic disparity in the second case; (2) a very high proportion of cells may respond to allogeneic stimuli; (3) major histocompatibility differences are not essential for vigorous allogeneic reactions; (4) the responding population need not be immunologically competent to respond to antigens of the stimulating population; (5) the stimulating population must be both metabolically active and immunocompetent. We have tried to produce a model of cell interaction which will account for these and other anomalies, which at the same time explaining both normal antigenic stimulation (through cell-cell cooperation) and allogeneic interactions as examples of the same basic mechanisms. The model is based on the Bretscher-Cohn scheme of cell interaction. An allogeneic reaction is seen as having two stages: (1) Cells come together when antibody receptors on cells of one population combine with antigens on cells of the other. To this extent, our model is the same as the conventional one. It need not be the responding population which has the receptors, however. (2) A species-specific proliferation signal passes between the cells. This is the same signal as is involved in normal antibody induction. Even antigen-receptor bonds which are very weak may result in effective stimulation of one or both partners because of enhancing effect of this signal, and because the antigens involved are probably repeated over the cell surface, enabling multipoint binding. This explains the very proportions of cells which proliferate. The exact outcome of any allogeneic interaction will depend on which of the two populations have antibody receptors for antigens on the other, which can produce the proliferative stimulus, and which can respond to either the proliferative signal alone or to this stimulus plus antigen.


Purification, properties and kinetics of sheep and human renin substrates. Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.


Cardiopulmonary changes following 24-36 hours of hyperoxia. Cardiopulmonary variables were studied in rabbits breathing room air following 24-36 h of 100 percent 0-2 exposure. Initially, arterial pH and P-co-2 remained within normal limits while arterial P-0-2 decreased significantly. Cardiac output and oxygen consumption increased significantly. Static lung compliance was decreased, and histologic examination showed pulmonary hemorrhage, atelectasis, and adema. Myocardial function under these conditions was restored, and the myocardium was able to produce a compensatory increase in cardiac output. Therefore, changes in myocardial function, as related to oxygen toxicity, are reversable phenomena.


Polymorphism at the alpha-glycerophosphate dehydrogenase locus in Drosophila melanogaster. I. Properties of adult allozymes. A biochemical comparison was made between alpha-glycerophosphate dehydrogenase allozymes from Drosophila melanogaster. Enzymes extracted from the three major genotypes were indistinguishable in terms of their pH optima and thermal stabilities. Distinctive differences were observed for three parameters; temperature dependence of specific activity, temperature dependence of K-m, and reaction rate constancy over a physiological temperature range. These results are discussed in terms of a model of balancing selection and the existence of spatial and temporal allele frequency clines in natural populations.


Genetic expression of beta-mannosidase activity in different tissues of mice. Beta-Mannosidase activity of liver, kidney, and spleen of two inbred strains of mice and their crosses has been assayed with the synthetic aubstrate p-nitrophenyl-beta-d-mannoside. Activity is low in C57BL/Kl mice and high in DBA/2/Kl mice. Hybrid animals have intermediate levels of beta-mannosidase activity. Segregation of enzyme activities occurs in the F-2 and backcross generations, and there are good correlations between acitities in the three tissuses of F-2 and backcross animals. Some evidence points to a single gene difference in crosses between C57BL and DBA with respect to this mannosidase variation. Curves for enzyme activities at different substrate concentrations and pHs obtained with preparations from DBA and C57BL mice show some differences. These are interpreted as a possible strain variation in a structural gene for this enzyme.


The identification and biosynthesis of siderochromes formed by Micrococcus denitrificans. 1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.


Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine. 1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.


The stoicheiometry of the absorption of protons with phosphate and L-glutamate by yeasts of the genus Saccharomyces. 1. A study was made of the pH changes occurring when 0.1-4 mumol of glutamate, phosphate and certain phosphate esters was added at about pH 4.8 to washed cell preparations (50 mg dry wt.) of strains of Saccharomyces. The system also contained deoxyglucose and antimycin to inhibit energy metabolism and so prevent proton ejection from the yeast. 2. A strain of Sacc. carlsbergensis was grown in a chemostat with a limiting supply of phosphate in order to enhance the subsequent rate of phosphate transfer into the yeast. These preparations absorbed 0.2 mumol of phosphate with about 3 equiv. of protons/mol of phosphate. The charge balance was maintained by the efflux of 2 equiv. of K-+ from the yeast. 3. Larger amounts of phosphate were absorbed with fewer proton equivalents. 4. Arsenate and phosphate caused similar pH changes. 5. Glucose 6-phosphate, ATP and certain order phosphate esters each initiated a rise in pH, possibly because hydrolytic extracellular enzymes released phosphate that was subsequently absorbed. 6. Four strains of yeast were grown with glutamate as principal source of nitrogen. Each absorbed extra protons in the presence of L-glutamate. 7. One of them, a strain of Sacc. cerevisiae, absorbed 0.2 mumol of glutamate with 3equiv. of protons/mol of glutamate, and in these circumstances 1-2 equiv. of K-+ left the yeast cells. 8. The role of ionic gradients in the transport of these anions is discussed.


The effects of diet, lipolysis and limb ischaemia on the distribution of plasma tryptophan in the rat. A non-linear relationship between the plasma non-esterified fatty acid concentration and the percentage of free plasma tryptophan was found in rats in different nutritional states, although non-esterified fatty acids are not the only factors determining the percentage of free tryptophan. This relationship was not seen in rats injured by limb ischaemia. The effect of drugs causing rapid increases in the plasma non-esterified fatty acid concentration was also studied. Isoprenaline decreased the total plasma tryptophan concentration. Dichloroisoprenaline caused a sustained increase in the plasma non-esterified fatty acid concentration which was accompanied by an increase in the concentration of free plasma tryptophan and followed by a fall in the concentration of total tryptophan. The loss of tryptophan from the plasma was attributed to an altered distribution of tryptophan in the extracellular space rather than to increased metabolism. This interpretation was supported by determinations of the irreversible disposal rate of plasma tryptophan which in uninjured rats was unaffected by the concentration of free plasma tryptophan. In the injured rats this rate was unaltered during limb ischaemia but was decreased after removal of the tourniquets; increased competition for tissue entry by other neutral amino acids and the fall in body temperature could be factors in this fall.


[The Heidelberg capsule, an antacid of improved galenics]. By means of the Heidelberg capsule the influence of two different antacids on the acidity of gastric juice was measured in 16 normal test subjects. The antacidic combination drugs were Roha salt tablets (RSTB) containing herba absinthii 1.9 mg; fruct. anisi 35.5 mg; rhiz. calami 22.2 mg; bolus alba 14.0 mg; calcium phosphoricum 53.3 mg; magnesium sulfuricum 41.1 mg; calcium carbonicum 70.0 mg; magnesium carbonicum 42.0 mg; sodium bicarbonicum 1120 mg, and Roha gastric tablets (RMTB) with retarded release and containing fruct. anisi 40 mg; herba absinthii 3.2 mg; calcium carbonicum 688 mg; sodium bicarbonicum 480 mg; magnesium carbonicum 40 mg. Compared to the simple antacid RSTB, the duration of the neutralizing effect of the retarded RMTB was prolonged significantly by 300%.


[Changes in imipramine induced noradrenaline potentiation by varying activity of gastric juice under oral medication]. From the serial studies on imipramine dependent noradrenaline (NA) rise at different values of gastric acid with 336 registrations of blood pressure, it follows that a lawful relation exists between imipramine absorption and gastric juice acidity. At pH values of 3.5--6.0 a distinct decreased absorption of orally supplied imipramine was stated. After changing the pH value by acid substitution, a full absorption is reached. This state seems to be of special practical importance in the treatment of depression with thymoleptics.


Modification of the cardiostimulatory and coronary dilator effect of isopreenaline caused by etafenone. Effect of Etafenone on the Cardisotimulatory (1) In the isolated  guinea-pig heart (Langedorff), 2'-[2-(diethylamino)-ethoxy]-3 -phenyl-propioheonoe  HCI (etafenone, Baxacor) produced a parital and non-competitive inhibition of the stiumulant actions of isoprenaline on glycogen phosphorylase, rate and amplitude of contraction, but did not reduce the increase in coronary flow. (2) In isolated cirular strips from bovine coronary arteries, etafeonoe reduced tension (ED 50 = 2.8 x 10-5M), and increased the relaxing effect of isoprenaline. (3) Etafeonoe inhbitied the acitivty of phosphodiesterase in vitro; the Ki value with the enzyme frombovine coronary arteries was 9.2x10-4M, which was 160-fold higher than the Ki for papaverine.(4) The results suggest that etafenone probably affects the myocardial as well as the coronary smoth muscle cell by a direct inhibitory effect on Caiinflux, which could accountfor the depressant effect and the non-competitive antagonism aginst isoprenaline  in  the heartas well as the relaxing action on coronary smooth  muscle. The inhibitory effect on phospodiesterase is likely to contribute to the relaxing action of etafenone by accumulation ofcyclic AMP.


A potentially antianginal benzo [b]thiophene with an amiodarone-like haemodynamic profile. L 9146 or 2-methyl-3(3,5 dimethyl-4-gamma-di-n-butylaminopropoxy-benzoyl)-benzo [b] thiophene is a substance belonging to the amiodarone series which induces in the anaesthetized dog a decrease of myocardial oxygen consumption which is mainly due to slowing of the heart rate and reduction in systemic blood pressure. L 9146 also enhances coronary blood flow. L 9146 has also antiadrenergic properties since catecholamine-induced hypertension, tachycardia and increase of myocardial oxygen consumption are markedly antagonized; these antiacrenergic effects are not due to a competitive blockade of the beta-adrenoceptors. L 9146 does not decrease cardiac output, but increases it appreciably in the initial phase of its action. Several findings indicate that when the intensity of certain properties is considered, l9146 is more active than aniodarone since only half the dose used with aniodarone is required to achieve a given level of action. The overall haemodynamic properties of L 9146, which are similar to those of amiodarone, are considered to be potentially valuable for the long-term treatment of angina pectoris.


A study of some beta-adrenoceptor blocking agents on skeletal muscle. The effect of three recently introduced beta-adrenoceptor blockers practolol, USVC 6524 and Inpea was studied on various skeletal muscle preparations. Practoloo, USVC 6524 and Inpea produced a dose related inhibition of acetylcholine induced contractions of rectus abdominis muscle of frog. These drugs also blocked neuromuscular transmission when tested on in vitro rat phrenic nerve diaphragm preparation; the blockade was partially reversed by physotigmine, KCl and adrenaline and was potentiated by d-tubocurarine. In gastrocnemius sciatic muscle-nerve preparation only Inpea exhibited neuromuscular blocking activity, while practolol and USVC 6524 did not show any effect up to a dose of 10 mg/kg (intraarterially). The apparent discrepancies between the results of in vitro and in vivo experiments could not be adequately explained. It has been discussed that the neuromuscular blockade caused by presently investigated beta-adrenoceptor blocking agents is essentially due to curare-like activity and to a small extent may be due to local anaesthetic activity.


Nuclear magnetic resonance studies of anaesthetic interactions with haemoglobin. The use of 270 MHz Fourier Transform nuclear magnetic resonance (NMR) spectrometer, combined with signal processing techniques to improve resolution, enabled proton resonances from the individual aromatic residues of haemoglobin to be distinguished. In the presence of clinical concentrations of the general anaesthetic drugs halothane and methoxyflurane, specific changes in the NMR spectrum can be distinguished which probably reflect local changes of conformation. When higher concentrations of anaesthetic are used, extensive changes in the NMR spectrum occur which are consistent with non-specific binding of the anaesthetic to the hydrophobic parts of the haemoglobin molecule.


Ventilation and blood-gas studies during experimentally produced Mendelson's syndrome in the dog. Hydrochloric acid was instilled into the bronchial tree in anaesthetized dogs. A severe respiratory acidosis resulted. The metabolic component of acid-base balance was little affected.


Hypertension in the immediate postoperative period. Sixty patients out of 1,844 recovery room admissions had significant postoperative arterial hypertension. Nearly 60% of them had a history of hypertension. The postoperative hypertension usually began within 30 min from the end of operation and lasted about 2 hours. In 20% of the patients it lasted 3 hours or longer. Complications attributable to hypertension were confined to this latter group. The principal factors possibly contributing to the pressure elevations were pain (35%), hypercarbia (15%) and emergence excitement (16%). Ten of the patients (17%) had no demonstrable cause for hypertension. The hypertension in this group appeared to have a shorter and more benign course.


The effect of intravenous premedication with lorazepam (ativan), pentobarbitone or diazepam on recall. Sixty-nine patients undergoing regional anaesthesia were premedicated with either lorazepam (1, 2, 3 or 5 mg), diazepam (5 or 10 mg), pentobarbitone (50 mg) or placebo given i.v., on a double-blind, randomized basis. A simple yet rigorous protocol was used to test the effect of these drugs on anterograde recall. Lorazepam 3 and 5 mg was found to affect anterograde recall significantly. None of the other drugs tested affected recall.


Calcium uptake into muscle of pigs susceptible to malignant hyperthermia: in vitro and in vivo studies with and without halothane. MHS and normal pigs were anaesthetized with nitrous oxide and diazepam. Halothane 1% was then administered for 1 hr. Immediately before and immediately after the halothane inhalation, 10 g of vastus lateralis muscle was excised. SR was isolated from this muscle. Calcium accumulation by the MHS sarcoplasmic reticulum in the absence of halothane was greater than normal. Halothane in vitro produced a similar increase in calcium uptake into both the MHS and the normal SR. Halothane in vivo significantly reduced calcium accumulation by hyperthermic SR but had no significant effect on calcium uptake into the normal SR. Thus the action of halothane on the MHS sarcoplasmic reticulum is indirect, occurring only in the intact cell. It is possibly a result of the deleterious effect which lack of ATP, a low pH or a high temperature is likely to have on the calcium accumulating mechanism of the SR membrane. Our results suggest that the primary defects of porcine and human malignant hyperthermia are not entirely synonymous.


Procaine in porcine malignant hyperthermia. Serum creatine phosphokinase, rectal temperature and muscle pH at death were measured in three halothane-susceptible Landrace pigs. Two of the pigs were treated with procaine. Although the course of the syndrome was different in the treated pigs, procaine did not reverse the established malignant hyperthermia syndrome.


Pulmonary oxygen transfer during IPPV in man. The efficiency of pulmonary oxygen transfer, measured as the difference between pulmonary end-capillary and arterial oxygen content, was assessed at several different inspired oxygen concentrations in 20 patients during intermittent positive pressure ventilation (IPPV) with oxygen/nitrogen mixtures. In only three of the patients did the patterns of response correspond with those predicted from iso-shunt and ventilation/perfusion mismatch models of lung function, in which it is assumed that oxygen exerts only a passive physical effect within the lungs. It is suggested that the pattern of response obtained during IPPV in the remaining 17 patients is explicable on the basis of an active physiological effect of oxygen such that an increasing alveolar oxygen partial pressure is associated with a progressive increase in the proportion of the pulmonary blood flow which bypasses ventilated alveoli. This "hyperoxic shunt", which is readily reversible, may be the result of redistribution of either ventilation or blood flow within the lung.


A clinical comparison of AH8165 and pancuronium as muscle relaxants in patients undergoing cardiac surgery. The non-depolarizing muscle relaxant AH8165 has been compared at two doses (0.5 and 1.0 mg/kg) with pancuronium (0.1 mg/kg) during induction of anaesthesia for patients having major cardiac surgery. After barbiturate-opiate premedication and thiopentone induction, administration of pancuronium was followed by no significant alteration in heart rate or arterial pressure. Both doses of AH8165 werr followed by significant tachycardia, and the higher dose by arterial hypotension. The lower dose of AH8165 was unsatisfactory for tracheal intubation, but the AH8165 1 mg/kg gave intubating conditions similar to those with pancuronium 0.1 mg/kg.


Mechanism of action of local anaesthetic agents and biotoxins. Four current theories for the mechanism of action of local anaesthetics are that these drugs: (1) interfere with some chemical, such as acetylcholine, that is involved in nervous conduction; (2) alter the density of fixed charges on the surface of the membrane; (3) cause an expansion of some volume of membrane that is critical for conduction; and (4) react with some specific receptor in the nerve membrane. The first theory fails for lack of evidence that acetylcholine is involved physiologically in conduction. The other three theories, however, all seem to apply.


Plasma concentration of local anaesthetic agents in regard to absorption, distribution and elimination, with special reference to bupivacaine. Absorption from the injection site, tissue distribution, metabolism and urinary excretion are the factors determining the plasma concentration of local anaesthetic agents. Other factors such as physico-chemical properties of these drugs may have important influences on these processes. An example of this is the difference in tissue distribution and plasma/erythrocyte ratio as well as an increase in plasma concentration of bupivacaine seen in acidosis. The importance for the plasma concentration, of protein binding, vasoactivity and the effect of depressed circulation are discussed.


Central nervous system effects of local anaesthetic agents. A review is given of an experimental study on cats where the influence of acid-base changes on central nervous system toxicity of local anaesthetic agents was studied. The conclusion of this study was that a respiratory acidosis increased the central nervous system toxicity of local anaesthetics and that the underlying metabolic conditions modified this increase. Thus a respiratory acidosis increased this toxicity more if it was based on a metabolic acidosis than on a metabolic alkalosis (Englesson, 1974; Englesson and Grevsten, 1974). An extended analysis is presented where automatic frequency analysis was performed on the e.e.g. recordings performed during the i.v. infusion of lignocaine, bupivacaine, L 134, HS 37 and its optical isomers. The preliminary results show that the electrical changes appearing in the e.e.g. from the start of the i.v. infusion until seizure activity were the same if this time interval was as short as 1 min or as long as 8 min. It also revealed remarkable individual differences between agents, for instance lignocaine displaying marked electrical changes already in the first third of this time period where bupivacaine showed no changes until shortly before seizures.


The activation of concanavalin A by lanthanide ions. Divalent cadmium and lead and the trivalent lanthanides bind in the trasition metal site (S1) of concamavanlin A and induce saccharide binding to the protein in the presence of calcium. Partial activation of the protein in the presence of lanthanides alone indicates these ions bind into both transition metal (S1) and calcium sites (S2). The activity of a lanthanide-protein derivative may be increased by the addition of either calcium or a transition metal ion. The saccharide binding activity decreases in the order Zn2+ is greater than Ni2+ is greater than Co2+ is greater than Mn2+ is greater than Cd2+ reflecting the order of binding constants for these ions in the transition metal site. Like the lanthanides, divalent cadmium substitutes for both the transition metal ion and calcium ion to partially activate the protein. Divalent lead substitutes only for the transition metal ion and partially activates the protein upon addingcalcium. The data are consistent with a model in which saccharide binding activity is independent of the metal size in S1 but critically dependent upon metal size in S2.


The mechanism of the bond forming events in pyridine nucleotide linked oxidoreductases. Studies with epoxide inhibitors of lactic dehydrogenase and beta-hydroxybutyrate dehydrogenase. 2,3-Epoxybutyrate and 2,3-epoxypropionate act as effective competitive inhibitors of pig heart lactic dehydrogenase. KIapp for both inhibitors was pH dependent and varied according to the general equation KIapp = KI(1 +Ka/H+) which may be predicted if the binding of the epoxide to the E-NADH complex involves a compulsory protonation step. Values of KI(epoxybutyrate), KI(epoxypropionate) and pKa were estimated as 150 muM, 860 muM, and 6.8, respectively. The formation of an E-NADH epoxide inhibitor complex was followed directly by fluorescence measurements. Both epoxybutyrate and epoxypropionate enhanced fluorescence of the E-NADH complex and caused a 20-nm blue shift in the maximum emission wavelenght. The dissociation constants measured by fluorescence titration for both epoxides increased as the pH was raised reflecting a decreased affinity for the E-NADH complex. 2,3-Epoxybutyrate was also shown to inhibit beta-hydroxybutyrate dehydrogenase by a mechanism which is consistent with compulsory protonation prior to addition of the epoxide. These results are discussed in terms of a general mechanism for the bond forming events in pyridine nucleotide linked oxidore-ductases.


Zinc and magnesium content of alkaline phosphatase from Escherichia coli. Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared.


Structural and catalytic properties of hydrogenase from Chromatium. The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.


The interaction of an epoxide with yeast alcohol dehydrogenase: evidence for binding and the modification of two active site cysteines by styrene oxide. Yeast alcohol dehydrogenase is inactivated and alkylated by styrene oxide in a single exponential kinetic process. The concentration dependence of half-times for inactivation indicates the formation of an enzyme inhibitor complex, KI = 2.5 times 10(-2) M at pH 8.0. Reduced nicotinamide adenine dinucleotide (NADH), at a concentration of 3 times 10(-4) M where Kd congruent to 1 times 10(-5) M, has a small effect on kinetic parameters for inactivation. Although benzyl alcohol and acetamide-NADH increase the KI for styrene oxide in a manner consistent with their dissociation constants, substrate also increases the rate of inactivation at high styrene oxide concentrations. The reciprocal of half-times for inactivation, extrapolated to infinite styrene oxide concentration, increases with pH between 7.6 and 9.0, pK congruent to 8.5. The stoichiometry of alkylation by [3H]styrene oxide is 2.2 mol of reagent incorporated/mol of subunit, and is accompanied by the loss of 1.9 mol of sulfhydryl/mol of subunit; prior alkylation with iodoacetamide reduces the stoichiometry to 0.88:1, and increases the rate of labeling. Tryptic digests of enzyme modified with [14C]iodoacetamide or [3H]styrene oxide produce two major peptides which cochromatograph, indicating that styrene oxide and iodoacetamide modify the same cysteine residues. Previous investigators have reported that iodoacetate, iodoacetamide, and butyl isocyanate alkylate either of two reactive cysteines of yeast alcohol dehydrogenase; both cysteines cannot be modified simultaneously [Belke et al. (1974), Biochemistry 13, 3418]. The inactivation of enzyme by p-chloromercuribenzoate (PCMB) is reported here to be accompanied by the incorporation of 2.3 mol of PCMB/mol of enzyme subunits, in analogy with styrene oxide; the planarity of the alkylating agent appears to be an important factor in determining the stoichiometry of labeling.


The two-step reversible denaturation of lactate dehydrogenase at low pH. Upon exposure to conditions of low pH, beef B4 lactate dehydrogenase rapidly loses enzymatic activity, but this process can be completely reversed yielding 100% of the original activity if the enzyme is immediately returned to neutral conditions. As the time of exposure to low pH is increased, the fraction of activity recovered declines to a values of 50--60% and remains nearly constant over a period of many hours. Correlated with this behavior is a change in the kinetics of the recovery of activity. Recovery of activity has been shown to be a second-order process for enzyme exposed to low pH for brief periods of time (Anderson, S., and Weber, G. (1966), Arch. Biochem. Biophys. 116, 207). After several minutes at low pH recovery of activity is found to become first order and to occur at a considerably slower rate. Gel filtration chromatography at low pH separates the protein into two fractions. The lower molecular weight fraction is found to be primarily monomeric, as indicated by equilibrium ultracentrifugation, and is capable of recovering enzymatic activity. The higher molecular weight fraction is generated from the lower molecular weight fraction, and is incapable of recovering activity. These results are interpreted to indicate that the enzyme exists sequentially in three denatured forms at low pH, the first two capable of being restored to the native state, and the third irreversibly denatured.


Substrate activity of structural analogs of isocitrate for isocitrate dehydrogenases from bovine heart. D-Garcinia acid (D-threo-1,2-dihydroxy-1,2,3-propanetricarboxylate), like D-isocitrate, has an alpha-DS-hydroxyl group and a beta-LS configuration of the second carboxyl group. The maximal velocity of pyridine nucleotide reduction with D-garcinia acid is 8 and 21% of D-threo-isocitrate with the DPN-linked and TPN-linked isocitrate dehydrogenase from bovine heart, respectively. The other stereoisomers of hydroxycitrate [L-garcinia acid, D- and L-hibiscus acid (D- and L-erythro-1,2-dihydroxy-1,2,3-propanetricarboxylate)] are inactive. DL-threo-Homoisocitrate (DL-threo-1-hydroxy-1,2,4-butanetricarboxylate) supports DPN+ reduction at 10-15% of the rate observed for isocitrate with the DPN-specific enzyme, but is not a substrate for TPN-linked isocitrate dehydrogenase. The values of apparent S0.5 for total isocitrate and total garcinia acid are similar with both enzymes; the apparent S0.5 of total homoisocitrate is two- to threefold higher than that of total isocitrate with the DPN-linked enzyme. Enzymatic oxidative decarboxylation of garcinia acid and homoisocitrate leads to formation of alpha-keto-beta-hydroxyglutarate and alpha-ketoadipate, respectively. DL-Methylmalate (DL-1-hydroxy-2-methylsuccinate) is inactive as a substrate for either dehydrogenase as are the newly synthesized compounds: DL-threo-gamma-isocitrate amide (DL-threo-1-hydroxy-3-carbamy01,2-propanedicarboxylate), beta-methyl-DL-isocitrate (DL-1-hydroxy-2-methyl-1,2,3-propanetricarboxylate), beta-methyl-DL-garcinia acid (DL-threo-1-hydroxyl-2-methoxy-1,2,3-propanetricarboxylate), DL-1-hydroxyl-1,2,2-ethanetricarboxylate, and DL-1,4-dihydroxy-1,2-butanedicarboxylate.


Purification and characterization of a major endonuclease from rat liver nuclei. A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.


Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate. The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form  of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.


The pH dependence of the thermodynamics of the interaction of 3'-cytidine monophosphate with ribonuclease A. The apparent free energy (deltaGapp) and enthalpy changes (deltaHB) associated with the interaction of 3'-cytosine monophosphate (3'-CMP) and ribonuclease A (RNase) are reported for the pH range 4--9, T = 25 degrees, mu = 0.05. The pH dependence of deltaGapp and deltaHB has been interpreted in terms of coupled ionization of histidine residues 12, 48, and 119, assuming that only the dianionic form of the inhibitor is bound. The results of this analysis are consistent with the calorimetric and potentiometric titration results for the free enzyme and its 3'-CMP complex reported in the previous paper (M. Flogel and R. L. Biltonen ((1975), Biochemistry, preceding paper in this issue). This analysis allows the calculation of the thermodynamic quantities associated with hypothetical but clearly defined reactions (e.g., the reaction of the dianionic inhibitor with the completely protonated enzyme). It is concluded that the primary thermodynamic driving forces for the reaction are van der Waals interactions between the riboside moiety and the protein fabric and electrostatic interaction between the negatively charged phosphate group of the inhibitor and the positively charged histidine residues at the binding locus. It is also suggested that the binding reaction is weakly coupled (approximately to 0.5 kcal/mol) with a conformational change of the protein associated with protonation of residue 48. These results are consistent with the model originally proposed by G. G. Hammes ((1968), Adv. Protein Chem. 23, 1) and lend additional quantitative detail to the nature of the reaction.


The magnitude of electrostatic interactions in inhibitor binding and during catalysis by ribonuclease A. It is demonstrated that a model of nucleotide binding to ribonuclease A similar to that proposed by Hammes and coworkers (G. G. Hammes (1968), Adv. Protein Chem. 23, 1) is at least, approximately applicable for both cyclic nucleotide substrates and mononucleotide inhibitors at pH values less than or equal to 6.5 and as a function of ionic strength. Calorimetric data on various inhibitors show that the binding reaction can be thermodynamically dissected into a contribution arising from van der Waal's interaction of the nucleoside moiety, characterized by a large negative enthalpy change, and a contribution arising from electrostatic interactions between the negatively charged phosphate group of the inhibitor and the positively charged protein fabric, characterized by a large positive unitary entropy change. Assuming a catalytic mechanism involving the formation of a dianionic pentacoordinated phosphate transition state intermediate, the magnitude of the effect of electrostatic interactions on the overall rate enhancement by the enzyme is estimated to be 2 times 10(2) to 10(6). It is suggested that this effect, along with substrate approximation effects, is sufficient to "explain" the catalytic behavior of the enzyme.


Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution. The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpApT have been monitored at low ionic strength and in the presence of Mg ions at neutral pH in aqueous solution to determine the thermodynamic parameters associated with fraying (D. J. Patel (1974), Biochemistry 13, 2396) at the terminal and internal base pairs as a function of temperature and pH. From studies in H2O-MeOH (3:2), the fraying process persists down to approximately -20 degrees for the internal TA base pair and down to and probably beyond -30 degrees for the terminal AT base pair. The observed average chemical shift at each of these base pairs as a function of temperature suggests rapid exchange on the nuclear magnetic resonance (NMR) time scale between helix and coil (chemical shift separation of 3.2 ppm) and have been utilized to determine the dissociation constant at the terminal and internal base pairs. Comparison of the reaction enthalpies elucidated from the chemical shift parameters with those reported from optical studies suggests that the symmetry related internal TA base pairs break in a coupled manner at low ionic strength, with the coupling removed in the presence of Mg ions and high salt. By contrast, the symmetry related terminal AT base pairs break independently of each other in the absence and presence of Mg ions and high salt. The terminal base pair exhibits a Tm of 10-15 degrees lower than that of the internal base pair in the hexanucleotide, with divalent Mg ions and high salt stabilizing the double helix as reflected in the Tm values of these base pairs. The observed line width changes as a function of temperature provide an estimate of the exchange rate of the proton from the coil form with water. The exchange reaction from the coil state is base catalyzed with rate constants in the diffusion limit.


Proton nuclear magnetic resonance investigations of the nucleation and propagation reactions associated with the helix-coil transition of d-ApTpGpCpApT in H2O solution. The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpCpApT have been monitored in the presence of 0.1 M phosphate at neutral pH in aqueous solution. While the resonance positions of the terminal and internal AT base pairs shift upfield and broaden as average resonances with increasing temperature (helix and coil exchange several times prior to exchange with water from the coil form), those of the central GC base pairs broaden in the absence of upfield shifts (exchange with water occurs each time helix converts to coil). The line-width changes at the AT base pairs monitor the lifetime of the coil state at these positions prior to exchange with water while the line-width changes at the GC base pairs monitor the lifetime of the helix prior to dissociation to strands. This permits the separation of the propagation reaction at the AT base pairs from the nucleation reaction at the GC base pairs during helix formation. The experimental data have been quantitatively analyzed to yield (at 20 degrees) a nucleation formation rate of approximately 10(3) sec-1 for the GC base pairs (bimolecular rate constant of approximately 6 times 10(6) l. mol-1 sec-1) and a dissociation rate of 6 times 10(2) sec-1 at these same base pairs (unimolecular dissociation to strands). The unimolecular propagation reactions at the terminal and terminal base pairs are associated with reaction rates greater than 10(4) sec-1. These values are consistent with a slow formation of a stable nucleus at the GC base pairs followed by a rapid propagation reaction at the AT base pairs. The line width of the (GC) central base pairs in the presence of phosphate is a direct measure of the lifetime of the total helix and yields an activation energy of 45 kcal for helix to coil conversion measured over a narrow temperature range. The exchange from the coil form with water is catalyzed by 0.1 M phosphate with a rate constant kHPO2-/4 = 0.2 times 10(6) 1. mol-1 sec-1.


Energy dependent hydrogen ion accumulation in submitochondrial particles. The fluorescence quenching of 9-aminoacridine (9AA) in suspension of beef heart EDTA submitochondrial particles was studied and was used to calculate the pH gradient between these particles and the medium. This pH gradient, which is energy dependent, is also dependent strongly on the presence of anion species in the medium. It is 2.2 pH units in acetate medium and can be as high as 3.6 units in the presence of other highly lyophilic anions. The anions tested were found to be effective in the following order: SCN- greater than I- greater than NO3- greater than Br- greater than Cl-. The validity of the deltapH calculations was confirmed by comparison with deltapH values calculated from NH4+ uptake. In contrast, calculations based on quinacrine (QA) fluorescence quenching under the same assumption used for 9AA did not agree with NH4+ measurements and show quantitative and in some cases even qualitative differences. Both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NH4+ decreased deltapH significantly. When the rate of electron transport is slow, i.e., with succinate as substrate or with NADH and low concentration of rotenone, very low concentration of nigericin (less than 20 ng/ml) decreased deltapH. Under these conditions, valinomycin antagonized the nigericin effect and restored deltapH to its original value. Upon increasing nigericin concentration (greater than 100 ng/ml) the valinomycin effect is gradually replaced by a slower response of further reduction of deltapH.


Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate. Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.


The binding of azide to human methemoglobin A0. Error analysis for the interpolative and noninterpolative methods. The binding azide to human methemoglobin A0 has been studied at 6 degrees, pH 7, and I = 0.2 by three spectroscopic methods: (1) the conventional interpolative method, (2) an interpolative dialysis technique, and (3) a noninterpolative method. The interpolative methods assume that the fractional spectral change equals the fraction of heme sites bound by ligand, while the noninterpolative method measures the extent of binding directly, i.e., without the interpolative assumption. Both experiment and error analysis show that method 1 has low precision, and consequently, gives an inherently unreliable binding isotherm. Method 2 achieves high experimental and intrinsic precision. However, method 3, which also has high precision, clearly proves that the interpolative assumption of method 2 is incorrect. That is, the true fractional extent of binding becomes equal to the fractional spectral change only after about 97% of heme sites have been bound with ligands.


Hydrogen-deuterium exchange in nucleosides and nucleotides. A mechanism for exchange of the exocyclic amino hydrogens of adenosine. The pH dependence of the apparent first-order rate constant for the exchange of the exocyclic amino hydrogens of adenosine with deuterium from the solvent was measured by stopped-flow ultraviolet spectroscopy. This dependence shows acid catalysis, base catalysis, and spontaneous exchange at neutral pH values. A study of the effect of several buffers on the rates of exchange reveals both general acid and general base catalytic behavior for the exchange process. We propose a general mechanism for the exchange which requires N-1 protonated adenosine as an intermediate for the acid-catalyzed exchange and amidine anion for the base-catalyzed exchange. In both cases the rate-limiting step is the base-catalyzed abstraction of a proton from the exocyclic amino moiety. Evaluation of the rate constants predicts the equilibrium for the exocyclic amino/imino tautomers to be 6.3 times 10(3):1.


Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium-sparing diuretic on aldosterone-stimulated sodium transport, lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder. A correlation study of the effects of two agents, 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]propionic acid (TPIA) and amiloride, on aldosterone-induced alterations in Na+ transport, lipid synthesis, and phospholipid fatty acid composition has been carried out in the toad urinary bladder. TPIA, an inhibitor of acetyl-CoA carboxylase, inhibits aldosterone-stimulated Na+ transport as well as hormone-induced lipid synthesis and the increase in weight percentage of phospholipid long-chain polyunsaturated fatty acids. Amiloride, a diuretic which blocks sodium entry into the transporting epithelium, does not alter aldosterone's effects on lipid and fatty acid metabolism but prevents the hormone-induced increase in Na+ transport. These results support the conclusion that aldosterone increases Na+ transport in the toad urinary bladder by altering membrane fatty acid metabolism and that the lipid biosynthetic events following aldosterone treatment are a primary response to the hormone and not secondary to increased Na+ transport.


Equilibrium measurements of cognate and noncognate interactions between aminoacyl transfer RNA synthetases and transfer RNA. The interaction of Escherichia coli isoleucyl-tRNA synthetase with its cognate and five noncognate tRNAs, and of yeast valyl-tRNA synthetase with its cognate and four noncognate tRNAs, has been measured directly by fluorescence quenching. The cognate associations are strongest (association constant of 10(8) M-1 or more at pH 5.5, 17 degrees). A wide variation is found in the strengths of the noncognate interactions; these have association constants smaller than that of these cognate association by a factor of less than 10 to over 10(4), depending on the enzyme-t-RNA pair. A more detailed study of the cognate isoleucyl-tRNA synthetase-tRNAIle association suggests that the strength of the interaction is markedly sensitive to a pH-dependent transition in the enzyme centered at pH 6 on the other hand, Mg2+-induced structural changes in tRNAIle at 17 degrees in low salt do not greatly affect the availability of the nucleic acid's receptor sites for enzyme...


A 13C nuclear magnetic resonance and circular dichroism study of the collagen-gelatin transformation in enzyme solubilized collagen. Natural abundance Fourier transform 13C nuclear magnetic resonance (13C NMR) were obtained for enzyme solubilized collagen at 1 degrees intervals through the transition region. The transition of collagen molecules from the rigid triple helical state to single-stranded, random-coil state is accompanied by a change from broadened carbon resonances unobservable under high-resolution conditions to narrow line spectra. Thus distinction can be made between helical and random-coil states of individual residues. The transition is monophasic, as determined by examination of 14 different carbon resonances, and the entire structure is found to melt cooperatively over a temperature interval of 5 +/- 1 degrees. All the residues seem to be involved in the unfolding process concurrently. The transition was also studied by examining the changes in the circular dichroism spectrum brought about by heating. The experiments corroborated the observation that the transition proceeded cooperatively over a temperature interval of 4 degrees. Enzyme soluble collagen is seen to melt less cooperatively than native collagen. The enthalpy change was determined by assuming an equilibrium between three random coil gelatin chains and tropocollogen molecules. From the enthalpy, the average length of the tripeptide sequences (70-85) involved in the transition can be estimated. The shortening of the cooperative unit could arise as a result of some alteration of the native conformation through proctase treatment.