Purine nucleotides.

Purine nucleotides. The existence of nonadrenergic, noncholinergic nerve components in the autonomic nervous system is now well established. They are strongly represented in the gastrointestinal tract of all vertebrates and have been identified in a variety of other organs, including lung, trachea, bladder, esophagus, eye, seminal vesicles, and possibly parts of the vascular and central nervous systems. Their ultrastructural identification and transmission properties are known and their physiological role is beginning to be understood, at least in the gastrointestinal tract. Evidence that ATP is the transmitter released from nonadrenergic, noncholinergic (purinergic) nerves includes: (a) synthesis and storage of ATP in nerves; (b) release of ATP from the nerves when they are stimulated; (c) exogenously applied ATP mimicking the action of nerve-released transmitter, both producing a specific increase in K+ conductance; (d) the presence of Mg-activated ATPase, 5'nucleotidase, and adenosine deaminase, enzymes, which inactivate ATP; (e) drugs (including 2-substituted imidazolines, 2,2'-pyridylisatogen and dipyridamole), that produce similar blocking or potentiating effects on the response to exogenously applied atp and nerve stimulation.


Glucocorticoid regulation of the serotonergic system of the brain. Glucorticoids at concentrations above 10(-7) M stimulate the uptake of tryptophan by brain synaptosomes. Furthermore, in both developmental and drug-induced (reserpine and ethanol) increases of brain tryptophan hydroxylase activity, glucocorticoids are required in order for the increases to occur. If de novo enzyme synthesis is assumed to take place in such increases, we could adopt the working hypothesis that two loci of glucocorticoid action may exist in serotonergic neurons with respect to their specific function: one in the cell body presumably in a sequence of macromolecular events, and the other directly on nerve terminals. Thus, in its immediate action, the hormone may rapidly regulate 5-HT synthesis through an increased uptake of tryptophan by nerve terminals. In the slow action, the hormone may mainly play a permissive role in the induction of tryptophan hydroxylase by factors yet to be identified.


A neurobiological role for a protein activator of cyclic nucleotide phosphodiesterase. This work supports the idea that PDEA is bound and stored in brain particulate fraction. The release of PDEA into cytosol where the activator-sensitive PDE is located, is the first event in the process of the regulation of cAMP metabolism and inactivation. PDEA is released by cAMP-dependent phosphorylation of the activator-binding sites. This process is Ca2+ independent and does not occur in the presence of cGMP and cGMP-dependent phosphorylation. The free, soluble PDEA activates the high Km PDE in the presence of micromolar concentrations of Ca2+. This protein decreases severalfold the Km for cAMP of the high Km activator-sensitive PDE. PDEA regulates cAMP metabolism when the concentration of cAMP is elevated by a transsynaptic activation of adenylate cyclase. The rate of synthesis and the release of PDEA might be a part of the process of receptor sub- and supersensitivity, which has been reported during denervation or as a result of chronic treatment with drugs.


Cellular depolarization and cyclic nucleotide content in central nervous system. There is ample evidence indicating that cAMP and cGMP content in mamalian brain is markedly influenced by cellular depolarization. The relationships between the two cyclic nucleotides and the complex process of cellular depolarization, which in turn leads to extensive functional and metabolic consequences are incompletely understood, however. Much data, which are reviewed in the present chapter, suggest that cellular depolarization leads to elevation of cAMP and cGMP levels by two different and unrelated mechanisms. With regard to cAMP, it seems reasonable to conclude that cellular depolarization in CNS causes a release of adenosine, and it is the subsequent action of this substance which accounts for a portion of the elevation of cAMP levels. At present, one cannot ascertain whether or not other factors contribute to the increase of cAMP content resulting from cellular depolarization. It is probable that it is not cellular depolarization, per se, but the influx of Ca2+ into intracellular spaces caused by cellular depolarization which increases the content of cGMP. The specific effects of Ca2+, Mg2+, and other divalent cations on the cGMP response suggest a close relationship between the cyclic nucleotide and some process associated with release of one or more yet undefined neurotransmitters. Wether the involvement is in presynaptic or postsynaptic mechanisms also remains to be determined.


Long-term regulatory mechanisms for tyrosine hydroxylase in sympathetic ganglia and carotid body. The delayed activation of TH elicited by hypoxia in carotid body indirectly suggests that the release of dopamine is part of the responses elicited by hypoxia. The TH activation appears to be independent from nicotinic receptor stimulation and can be abolished by dopaminergic receptor stimulation. Dexamethasone also increases the activity of TH. Since both hypoxia and dexamethasone fail to change the kinetic constants, the long-term increase of TH could be viewed as an expression of new synthesis of enzyme molecules. This assumption is supported by the evidence presented on the prompt kinetic change in PDE, which according to the model proposed by Uzunov et al. (42) expresses the participation of a prompt and sustained response of the second messenger in postsynaptic cells. It remains to be ascertained whether the change in the catalytic activity of PDE which metabolizes the second messenger can be suppressed by dopamine-receptor blockers. Immediate changes of PDE coupled with the delayed increase in TH activity may contribute to a better understanding of the neuronal mechanisms controlling the chemoreceptor function. It is hoped that the continuation of these studies will help to define the function of type I cells in the carotid body.


[Sexual development of boys, treated surgically for dystopia of testicles (author's transl)]. A clinical follow-up examination was made in a group of 208 children and adolescents who had undergone surgical treatment for congenital cryptorchid testis (unilateral or bilateral) between 2 and 10 years before. The puberal growth and maturing process of the primary and secondary sex characteristics of this morbidity group was compared to that of normal juveniles. The examination was made according to clinical criteria: length and circumference of the penis, volume of testes, general development of the genitals, pubic hair. Compared to normal juveniles, the genital development of the children and adolescents of the follow-up examinations series was retarded.


Study of the priming effect of interferon in L cells. II. Metabolic requirements for the development of primed state. Actinomycin D at a concentration inhibitory for nucleolar RNA synthesis did not influence the development of priming. Higher concentrations of the drug and long-term alpha-amanitin or cycloheximide pretreatments, inhibitory for heterogenous nuclear RNA synthesis, prevented the establishment of the primed state. The inhibition of mitochondrial DNA and protein synthesis did not inhibit the priming effect of interferon. Attempts to prime cells with pretreatments other than interferon were not successful.


Influence of polypeptide molecular and side chain lengths and of side chain steric location on the kinetics of basic polypeptide-induced sensitization of primate cells to transfection. Kinetics of sensitization of chimpanzee cell sheets to transfection by poliovirus RNA was determined for 5 basic polypeptides. With basic olypeptide hydrobromide at 100 microng/ml, initial sensitization rate was faster for poly-L-ornithine of average molecular weight (AMW) 15500 than of AMW 105000, and much faster for poly-L-lysine of AMW 1700 than of AMW 140000. Desensitization phases were observed with the 2 shorter polypeptides. Sensitization was much faster and sensitivity maxima were considerably higher for the polyornithines than for the polylysines. Poly-D-lysine of AMW 160000 sensitized cells slightly faster than poly-L-lysine of AMW 140000, but gave about the same sensitivity maximum. Analysis of the slow cell sensitization by poly-L-lysine of AMW 140000 revealed 2 steps: a fast step 1 (attachment) and a slow step 2 (processing).


Susceptibility of various cell lines to virulent an attenuated strains of pseudorabies virus at supraoptimal temperature. Replication of virulent and attenuated strains of pseudorabies virus (PRV) at supraoptimal temperature was studied in rabbit lung (ZP), pig kidney (PS), and BHK-21 cells adapted and non-adapted to 40 degrees C and L, HeLa and human amniotic (Am) cells non-adapted to 40 degrees C. The temperature of 40 degreeC did not influence the type cytopathic effect (CPE) in either adapted or non-adapted cells. According to the susceptibility at 40 degrees C the cells could be divided into three groups: cells permissive for all PRV strains (adapted and non-adapted ZP and non-adapted BHK-21 and PS); cells non-permissive for all PRV strains (L, HeLa and Am); and cells permissive for virulent and non-permissive for attenuated PRV strains (adapted BHK-21 and PS). The virus titres in permissive cells at 40 degrees C did not differ from those obtained at 37 degrees C. The attenuated strain CK-PRV X was found to be a tsmutant of PRV with host range character.


Effect of streptovirudin on parainfluenza 3 virus multiplication. Streptovirudin (SV) in a concentration of 100 microng/ml reduced the multiplication of parainfluenza 3 virus by about 3.5 log units. When uninfected WISH cells were treated for 2 and 12 hours with 100 mug/ml of SV, 3H-thymidineincorporation was decreased, but no marked differences were observed in the incorporation of 3H-uridine. The level of intracellular cyclic adenosine monophosphate (c-AMP) was found to decrease in SV-treated cells.


Sunday Canyon virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas. A new arbovirus was isolated from Texas, U.S.A., populations of the Cliff Swallow parasits Argas (Argas) cooleyi Kohls and Hoogstraal, 1960. The virus, named Sunday Canyon, is serological urelated to any of 185 arbovirus strains or 20 other viral agents with which it was compared. Morphologically it resembles Bunyamwera viruses and, in common with them, is sensitive to lipid solvents and acid pH, and apparently possesses RNA. Although considerably resistant to a temperature of 41.5 degrees C, it rapidly loses infectivity when incubated at 56 degrees C. It is lethal for newborn white mice and infective for the Vero and Antheraea eucalypti cell lines. Sundays Canyon virus is the second tick-associated, Bunyamwera virus-like agent known from North America and the third virus to be reported from A. cooleyi in Texas.


Appearance and some unusual features of Epstein-Barr virus antibody production in infectious mononucleosis and other health disorders. The indirect immunofluorescence (IF) test and the complement-fixation reaction (CFR) were used in examination of over 1500 sera obtained from patients with infectious mononucleosis (IM) and other health disorders. The evidence obtained supports a direct aetiological relationship between Epstein-Barr virus (EBV) and IM and points on a relationship of EBV to some other lymphadenopathies and health disorders. The incidence of the IgG type antibody against virus capsid antigen (EB-VCA) and soluble antigen (CF-SA) obtained from EBV genome-positive cells among different age groups of patients is described along with results of long-term examinations of serum samples from IM patients. The appearance and dynamics of production of both types of EBV antibody and their persistence in the organism varied. Long-lasting oscillations, in particular of the EB-VCA antibody levels were found in sera of patients with prolonged health disorders following IM. The diagnosis value of the IF test and the CFR is discussed.


Characteristics of poliovirus type 3 strains after passage through monkey central nervous system. Cercopithecus aethiops monkeys were inoculated intracerebrally with Sabin's type 3 polivoirus strain, and with strains isolated from subjects vaccinated with Leon 12a1b and from their contacts. Sample of brain, spinal cord, kidneys, lymph nodes, blood and spinal fluid were taken. From these materials, (except of kidneys) 23 poliovirus strains of type 3 were isolated. Genetic markers rct 40.3, rct 41, d and IST were determined for these strains. The changes observed in genetic markers of strains passaged through the cental nervous system (CNS) of monkeys are described.


6-MFA, an antiviral agent from Aspergillus ochraceus ATCC 28706: influence of body weight and mineral oil administration of the antiviral activity in mice. Sera obtained from mice treated with 6-MFA, an antiviral agent from Aspergillus ochraceus ATCC 28706 showing high interferon activity, could be transferred to healthy animals to make them resistant to Semliki Forest Virus (SFV) infection. Body weight of mice directly influenced, within limits, the proportion of treated mice surviving challenge virus infection as well as interferon production. Mineral oil (liquid paraffin, B. P.) administered prior to 6-MFA increased both the level of interferon in serum and the protection rate of the treated animals.


Disintegration of red clover mottle virus virions under different conditions of storage in vitro. Red clover mottle virus was kept in 0.1 M phosphate buffer, pH 7.0, at 4, --20 and --60 degrees C for 1 and 4 weeks and 3 months. The degree of virion disintegration was the highest at --20 degrees C and the lowest at 4 degrees C. Two cycles of freezing and thawing destroyed the virions. In frozen samples (at both --20 and --60 degrees C), the nucleoprotein sedimentation component M was disintegrated more than component B.


Effect of a proteolytic enzyme inhbitor on influenza virus replication. N-tosyl-L-phenylalanyl chloromethane patially inhibited the multiplicatiion of fowl plague virus (FPV) in infected cells. The capacity of the inhibitor to suppress the muliplication of FPV depended on the host cell system.


Structure of inclusion bodies of cabbage black ring virus. Inclusions in cells of Nicotiana glutinosa infected with a horse-radish strain of cabbage black ring virus (CBRV) were investigated on ultrathin sections. Large amorphous inclusions contained cellular constitutents showing usually no pathological alterations. Lamellar inclusions (bundles, circular inclusions, pinwheels), closely associated with endoplasmic reticulum, occurred frequently in areas of cytoplasm showing no vesiculation. Sac-like structures composed of concentric lamellae were observed in the cytoplasm or intruding into the vacuole. Spherosomes containing each a single crystal were found very often in the cytoplasm. Masses of virus particles in a parallel arrangement were usually located at the tonoplast.


An iridovirus from larvae of Culiseta annulata and Culex territans. Field-collected 4th instar larvae of Culiseta annulata and Culex territans were found to be infected with mosquito iridescent virus (MIV). Ultrathin sections revealed the presence in infected cells of different types of immature virus particles of an average diameter of 180 and 190 nm respectively.


Psychological aspects of cardiac arrhythmia. A review of data from a wide spectrum of research studies suggests that psychological-emotional factors can significantly influence and alter the incidence of cardiac arrhythmia. While the existing data are, in many cases, difficult to interpret because of theoretical and methodological problems, sufficient evidence does exist to warrant a concerted investigation into the total involvement of psychological factors in cardiac arrhythmia.


Internal mammary artery graft for ischemic heart disease. Effect of revascularization on clinical status and survival. Four hundred consecutive patients, 80 percent of whom had multiple vessel obstruction, received a single internal mammary artery graft (121 patients) alone or combined with vein grafts (279 patients) in 1971 and 1972. Four patients died during or within 30 days of the operation. Sixteen patients had intraoperative infarction; three died. Arteriography was performed postoperatively (mean 12 months) in 254 patients, and 248 of 261 internal mammary artery grafts (95 percent) and 195 of 237 vein grafts (82 percent) were patent. Follow-up was complete (mean interval 38 months); all 80 patients with single vessel disease are alive, and the 3 year survival rate for patients with double and triple vessel disease was 98.7 and 94.4 percent, respectively. Comparison of longevity of 741 patients who had received vein grafts in 1967 to 1970 with that of 400 patients with internal mammary artery grafts (1971 to 1972) indicates greater survival for the recent series (P less than 0.004). Factors responsible for improved survival include (1) reduced operative mortality, (2) fewer intraoperative infarctions, (3) more complete revascularization, and (4) higher patency rate of the internal mammary artery graft.


Enkephalin, drug addiction and acupuncture. From the results of clinical and basic research, there is clear evidence that acupuncture analgesia is closely associated with the nervous system, especially the central nervous system. Stimulation of certain acupuncture loci which have been used for analgesia during operations also can calm the withdrawal symptoms of morphine and heroin addicts. Acupuncture analgesia can be antagonized by the specific narcotic antagonist, naloxone. These findings suggest the factor or factors produced by acupuncture stimulation would also have agonist activity on opiate receptors. Moreover, the morphine receptors are most concentrated in those parts of the brain concerned with perception of pain and the pathway of acupuncture stimulation. Since the opiate receptors are associated with the synaptic fraction of brain cell membrane preparations, the natural ligand of these receptors may be a neurotransmitter. Enkephalin has stronger binding affinity to opiate receptors than morphine, which suggests that it is the natural ligand for these receptors. In other words, enkephalin might be the natural "pain killer" produced in the brain to suppress pain. If we summate all the information available now, it is possible to suggest that enkephalin may be the product of the nervous system released by acupuncture stimulation to create an analgesic effect as well as suppress opiate withdrawal symptoms.


The Silver-Russel syndrome: a case with sexual ambiguity, and a review of literature. A 38-month-old patient with Silver-Russel syndrome (SRS) and ambiguous genitalia had a 46,XY karyotype on leukocyte and fibroblast cultures. This is the third SRS child with ambiguous genitalia described in the literature. In a review of the findings in 148 reported cases of the syndrome, abnormalities occurring in over 50% of the cases are short stature, craniofacial dysproportion, low birth weight, term gestation, body asymmetry, incurved fifth digits, normal intelligence, short fifth digits, and down-curved corners of the mouth (shark mouth).


Detection of heterozygotes for tyrosinase-negative oculocutaneous albinism by hairbulb tyrosinase assay. Five heterozygotes for ty-neg OCA had absent or markedly reduced hairbulb tyrosinase activity, when compared to normally pigmented control subjects. Clinically, all five were fully pigmented and could not be distinguished from the normal control subjects. It is proposed that the quantitative hairbulb tyrosinase assay can be used to detect heterozygotes for ty-neg OCA. A mechanism to explain the low levels of activity in the hairbulbs from heterozygotes is discussed.


Very low arylsulfatase A and cerebroside sulfatase activities in leukocytes of healthy members of metachromatic leukodystrophy family. Very low levels of arylsulfatase A (ASA) have been found in the leukocytes of healthy members of a metachromatic leukodystrophy (MLD) family. The cerebroside sulfate sulfatase (CSS) activities in the same individuals are about 10% of the control level. Arguments favoring a dominant mutation different from that of classical MLD are presented. This report reinforces the relationship between the two enzymatic activities.


Factors determining human chorion laeve permeability in vitro. An increased mean diffusion permeability across human chorion laeve in vitro was measured for meperidine (D = 5.26 x 10(-6) cm.2 sec.-1) and diazepam (D = 4.51 X 10(-6) cm.2 sec.-1). These values corresponded to large chloroform-buffer partition coefficients (49 and 29) measured for these two compounds. Diffusion permeability values of 3.98 and 2.18 x 10-6 cm.2 sec.-1 measured for urea and glucose corresponded to their relative insolubility in lipid, as indicated by chloroform-buffer partition coefficients of 0.05 and 0.0004, respectively. An increase in placental permeability in vitro to the weak organic acid 5,5-dimethyl, 2,4-oxazalidinedione at lower pH's corresponded to an increase in the fat-soluble nonionized fraction of this compound. These data support the concept that this tissue is most permeable to compounds of relatively small molecular size and/or with a high level of lipid solubility. The large diffusion permeability values measured for meperidine and diazepam suggest that these compounds will diffuse rapidly between mother and fetus at a maximal rate limited only by uterine blood flow.


Effects of calcium on renin and aldosterone in the rat. CaCl2 suppresses the plasma renin activity (PRA) response to Na+ deprivation in the rat. The purpose of the present study is:1) to determine if the effect of Ca2+ on PRA is modified by the anion delivered with Ca2+, and 2) to evaluate the effect of Ca2+ loading on aldosterone production. PRA and in vitro aldosterone production by adrenal quarters were measured after a 7-day balance study. On a low Na+ diet, PRA of animals drinking 1% CaCl2 (13.1 ng/ml per h +/- 1.3 SE), but not of animals drinking 1% calcium gluconate, was suppressed (P less than 0.05) compared to that of water-drinking controls (20.9 ng/ml per h +/- 2.1 SE). Aldosterone production of calcium gluconate and CaCl2-loaded animals was greater than that of controls (P less than 0.01). K+ balance of CaCl2 and calcium gluconate-drinking animals was more positive than that of controls (P less than 0.05). In conclusion, inhibition of PRA by CaCl2 but not by calcium gluconate indicates that the effect of Ca2+ on PRA is modified by the accompanying anion. Both CaCl2 and calcium gluconate stimulate aldosterone production, independent of changes in PRA, possibly due to an effect of Ca2+ on K+ balance.


Immunopathological aspects of hepatitis type B. Tissue samples from 180 unselected necropsy cases of various forms of hepatitis were examined by histopathology and immunofluorescence. The hepatitis forms studied included acute fulminant hepatitis (28 patients), subacute hepatitis (48 patients), acute fatal hepatitis (24 patients), chronic aggressive hepatitis (26 patients), liver cirrhosis (49 patients), and "minimal" hepatitis (5 patients). Hepatitis B surface antigen and hepatitis B core antigen were detected in 101 patients (56.1 per cent). In these, lesion-bound immune complexes of hepatitis B surface antigen were found in the liver and extrahepatic locations in 77 patients (76.2 per cent). The latter included activated germinal centers of lymph nodes and spleen, focal hyaline lesions of splenic and renal arterioles, necrotic and/or proliferative lesions of small and medium-sized arteries, and kidney glomeruli with mild proliferative and degenerative lesions. There was an inverse relation of the approximate amounts of hepatitis B surface antigen in the liver and the liver damage, the latter being directly proportional to the amount of HBS Ag immune complexes in the liver and indirectly proportional to their amount in extrahepatic locations and to the severity of lesions at these sites.


Metabolic features of isolated rat lung cells. I. Factors controlling glucose utilization. Isolated rat lung cell suspensions were prepared by collagenase digestion of the lung stroma. These cells were functionally competent as judged, among other criteria, by their constant rates of oxygen uptake and glucose utilization. An important metabolic feature of these cells is that they display very high glycolytic rates. At least 60% of the glucose utilized was converted to lactate, regardless of the glucose concentration in the medium. The state of reduction of the nicotinamide system, as indicated by the lactate-to-pyruvate ratio, was normal, thus indicating that the high glycolytic fluxes are not related to poor oxygenation of the preparation. Utilization of glucose displayed Michaelis-Menten saturation type kinetics with a Vmax of 331 nmol/10(6) cells per h and an apparent Km of 2.4 mM. These values were not affected by the presence of ouabain (0.1 mM), mannoheptulose (5 mM), or insulin (1 mU/ml), whereas phloridzin produced a drastic inhibition of glucose utilzation showing an apparent Ki of 0.4 mM. The substitution of sodium by K+ or Li+ as the predominant cations in the incubation medium does not alter rates of glucose utilization. Optimal pH for glucose utilization was within the physiological range with a more pronounced inhibitory effect at alkaline pH's. The intracellular concentration glucose was found to be low. This finding, in conjunction with a Q10 (27-37 degrees C) for glucose utilization above 2.0 and the differential effects of D- and L-glucose on production, seems to indicate that a stereospecific glucose transport system exists in lung cells. Several findings point to glucose transport into the lung cells as a probable rate-limiting step for its metabolism:1) the activity of the glycolytic enzymes largely exceeded the observed rate of glucose utilization;2) the decrease in enzyme activity during starvation was not accompanied by a decreased glycolytic flux, suggesting that factors other than enzyme activity, perhaps the supply of fuel, are rate limiting in the overall process of glucose breakdown;3) fructose was able to increase lactate production in the presence of saturating concentrations of glucose. These additive effects of glucose and fructose seem to support the point of view that it is not the glycolytic machinery but the supply of fuel which is rate limiting for glucose utilization by isolated rat lung cells.


Glucose and lactate kinetics in burn shock. We studied the glucose and lactate kinetics during burn shock by means of the primed constant infusion of [6-3H]glucose and Na-L-(+)-[U-14C]lactate. We found an early postburn hyperglycemia that was due to an increased rate of appearance (Ra) of glucose. Later, the ability of the tissues to extract glucose became impaired, and the plasma glucose concentration increased even more in spite of the return of Ra of glucose to the control level. The Ra of lactate rose rapidly postburn, whereas the rate of disappearance (Rd) of lactate stayed at the control level for 30 min. Consequently, plasma lactate rose approximately 350%. After 30 min, Rd of lactate increased to the same level as Ra; and Ra, Rd, plasma lactate concentration, and the percent of glucose derived from lactate remained elevated for the duration of the experiment. These results indicate an important role of lactate in burn shock metabolism and substrate kinetics.


Treatment of Bacteroides endocarditis with carbenicillin. A heroin addict developed acute bacterial endocarditis with Streptococcus viridans and Bacteroides melaninogenicus. Although blood cultures became negative during penicillin G and clindamycin therapy there was little clinical response. Prompt clinical improvement was achieved with intravenous carbenicillin in a dose of 40 g daily. In vitro testing supported the superiority of carbenicillin therapy in this patient.


pH gradients in the developing teeth of young mice from autoradiography of [14C]DMO. The distribution of the weak acid [14C]DMO was studied in mice, 10-19 days old, by an autoradiographic technique that does not translocate or remove the compound. Calculations of pH values in areas of the developing teeth of these animals were made by comparing the photometric density of the distribution of [14C]DMO, as an indicator of pH, with that of 14C]NAAP, as an indicator of water content, which was reported in the companion paper. The cellular layers, i.e., odontoblasts, ameloblasts, etc., had pH values of 7.1 +/- 0.1; the pH values in enamel, predentine, and dentine ranged from 7.3 to 8.5. It is suggested that an alkaline matrix promotes nucleation and growth of hydroxyapatite.


Computer simulation of ischemic rat heart purine metabolism. II. Model behavior. The behavior of a model for the partial depletion of adenine nucleotides in the perfused rat heart has been compared for ischemic and high coronary flow anoxic conditions. The accumulation of noradrenaline in the interstitial fluid greatly activates adenylate cyclase ultimately resulting in the degradation of 11.02 micronmol/g dry wt of ATP to adenosine, inosine, and hypoxanthine in 30 min. The high coronary flow rate during anoxic perfusion promotes washout of the noradrenaline from the interstitial fluid so that the hormone accumulates to only one fifth of its highest level in ischemia. This results in only slight activation of adenylate cyclase and in insignificant degradation of ATP in 2 min. The behavior of the model has been examined for two aerobic conditions--a transition from light to heavy work (2 min) and a transition from substrate-free to glucose perfusion (12 min), In both cases adenylate cyclase was not activated above its basal activity, and insignificant depletion of adenine nucleotides is predicted by the model.


Psychoneuroendocrinology of affective disorder: an overview. Neuroendocrine function has been reported by several workers to be abnormal in affective disorder. It has been shown that neurotransmitters (noradrenaline, dopamine, and serotinin) are involved in the regulation of neuroendocrine function. Several biological hypotheses of affective disorder have implicated a defect in neurotransmitter function, but these hypotheses have been found lacking in part over the years. The study of neuroendocrine abnormalities found in various types of affective disorder may clarify some aspects of this complex issue by reflecting neurotransmitter activity in this disorder. Such studies should help further explain affective illness.


Spinal cord injury: a role for the psychiatrist. The initial psychiatric evaluation of individuals with spinal cord injuries should focus on the patients' coping strategies; this information is an essential part of the treatment and rehabilitation plan. Psychiatrists can also work effectively with staff members in groups designed to ease the strain of working with severely disabled patients. Such patients may have psychiatric illnesses that are not secondary to the injury; careful selection of antidepressant or antipsychotic medication is essential. The author points out that the psychiatrist has much to offer in the treatment of individuals with spinal cord and other severely disabling injuries.


Characteristics of release of duodenal gastrin. Duodenal gastrin release in the dog was studied after mucosal antrectomy with intact duodenal innervation, following which basal gastrin levels fell. Acetylcholine at pH 7 but not at pH 1.5 and insulin hypoglycemia but not 2-deoxyglucose release duodenal gastrin. Glycine (pH7), physiologic saline (pH 7), and balloon distention failed to release duodenal gastrin. These findings suggest differences in release characteristics between canine antral and duodenal gastrin, and point out species differences between man and dog in respect to release of duodenal gastrin.


Intestinal brush border enzymes after short-term mesenteric ischemia. The total activities of sucrase, trehalase, amino-peptidase, and gamma-glutamyltransferase in the isolated brush border of the entire small bowel are reduced to 35, 55, 33, and 21 per cent, respectively, of control values (p less than 0.001) 2 hours after a 45 minute occlusion of the superior mesenteric artery. Since brush border proteins are also reduced by ischemia to 42 per cent of control, enzymatic activity when expressed as U/mg protein is significantly reduced only in the case of gamma-glutamyltransferase, to 48 per cent of control.


Rapid intubation with fazadinium. A comparison of fazadinium with suxamethonium and alcuronium. The speed of onset and intubation conditions have been compared for suxamethonium 1 mg/kg, alcuronium 0-32 mg/kg and fazadinium 1 mg/kg and 1-25 mg/kg. Fazadinium 1 mg/kg was not significantly different in times to intubation from suxamethonium although the latter gave a highly significant greater number of patients with excellent conditions. The higher dose of 1-25 mg/kg fazadinium did not give significantly better intubating conditions than the lower dose. Although the times to intubation for the two dose levels of fazadinium did not differ statistically, the higher dose was significantly slower in onset than suxamethonium. Alcuronium was slower than either suxamethonium or fazadinium 1 mg/kg in producing satisfactory conditions. The nondepolarizing drug, fazadinium, may be a useful neuromuscular blocking agent in emergency cases where rapid intubation is required and when it is wished to avoid possible or probable adverse effects from the depolarising drug suxamethonium.


Depression of phase-transition temperature in a model cell membrane by local anesthetics. Disordering, fluidizing and dilating effects of anesthetics upon cell membranes are well recognized. The fluidization can be precisely measured with phospholipid membranes. When phospholipids are dispersed in water, they form globules of bilayer structure. These model membranes undergo transition between crystalline (ordered and less fluid) and liquid crystalline (less ordered and fluid) phases according to the temperature, the degree of packing of each molecule, and the chemical environment. The phase transition occurs in a cooperative fashion and the turbidity of the dispersion changes abruptly, clear in liquid crystalline phase and turbid in crystalline phase. The present study was undertaken to quantitate the fluidizing effects of local anesthetics on dipalmitoyl lecithin (DPL) bilayer dispersion by measuring the turbidity change. Tetracaine, bupivacaine, lidocaine, and procaine were studied. They all depressed the phase-transition temperature. The binding of the drugs to the model membrane followed unsaturable kinetics, and the pH titration curve showed that only uncharged molecules were active. The freezing point depression was analyzed according to the Van't Hoff model. From this model, the partition coefficients of the uncharged molecules between DPL and water were estimated: lidocaine 76, procaine 159, bupivacaine 812, and tetracaine 1,405. The concentration of local anesthetics in the DPL phase needed to decrease the phase-transition temperature 1 degree C showed a constant value of 0.132 M. The concentration of local anesthetics in the DPL phase is a function of pH, partition coefficient, and volume ratio between the DPL and aqueous phases. The normalized values of the fluidizing action of these drugs at physiologic conditions correlated well with their nerve-blocking potencies. The present results indicate that the uncharged molecules fluidize the lecithin membrane by unsaturable nonspecific binding. The possible effect of the charged molecules upon the fluidity of natural membranes remains to be established.


Mutagenicity of halogenated ether anesthetics. An in vitro microbial assay system employing two histidine-dependent strains of Salmonella typhimurium, TA1535 and TA100, was used to test the mutagenicities of enflurane, methoxyflurane, isoflurane and fluroxene. Enflurane, isoflurane and fluroxene in concentrations ranging from 0.01 to 30 per cent and methoxyflurane in concentrations ranging from 0.01 to 7 per cent were incubated with bacteria in the presence or absence of homogenates of liver prepared from rats pretreated with the enzyme inducer, Aroclor 1254. Enflurane, methoxyflurane, isoflurane, and urines from patients anesthetized with these agents were not mutagenic. Fluroxene, however, was highly mutagenic, and therefore poses a possible hazard for operating room personnel and patients.


Mechanisms of chronotropic effects of volatile inhalation anesthetics. The chronotropic effects of all currently available volatile anesthetics were investigated in isolated rat atrial preparations. Anesthetic ethers, diethyl ether, methoxyflurane, and enflurane elicited a dose-dependent positive chronotropic effect. Fluroxene produced a slight depression at low concentrations. The halogenated hydrocarbon anesthetics, halothane, chloroform, and trichloroethylene, did not show a uniform pattern. Halothane's effect was small and biphasic. Chloroform caused a dose-dependent decrease in heart rate, and trichloroethylene caused a marked positive chronotropic effect. The dose-response curves in all anesthetics remained unaltered in the presence of either 3 x 10(-7) M dl-propranolol or 1 x 10(-6) M atropine. It is concluded that volatile anesthetics elicit significant direct chronotropic actions on rat atrial preparations. The mechanism of their actions does not involve stimulation of beta-adrenergic or cholinergic receptors.


[Study of a new anesthetic agent: Etomidate (R 26490). Unusual electroencephalic aspects]. Etomidate or R 26490 is a new hypnotic agent produced by JANSSEN and al. in 1971. The first human experimentation was performed in 1972. The authors used Etomidate for 300 anaesthetics given for neuroradiological investigations. Two protocols were used for study: 25 anaesthetics were given with Etomidate as sole anaesthetic to outline its specific properties; 275 angiographies were performed with a combination of Etomidate and fentany1. In protocol no 1, the mean consumption was for 1,85 mg/kg/h, in protocol no2, the mean consumption was of 0,9 mg/kg/h. Pharmacological study of this drug defines it as a short acting hypnotic agent of low toxicity. Clinical observation shows cardiovascular and respiratory stability. However, frequent myoclonias were noted, when Etomidate was given as a sole anaesthetic. EEG recording and evoked response encephalography helped to outline some effects of Etomidate on central nervous system.


Arrhythmic doses of epinephrine and dopamine during halothane, enflurane, methoxyflurane, and fluroxene anesthesia in goats. The cardiac arrhythmicity of epinephrine and dopamine was compared in awake goats and during approximate equivalent levels of halothane, enflurane, methoxyflurane, and fluroxene anesthesia. The arrhythmic threshold dose for epinephrine and dopamine was significantly (p less than 0.05) reduced during halothane anesthesia when compared to values determined in awake animals. Enflurane anesthesia had no significant affect on the arrhythmic threshold dose for either catecholamine. However, methoxyflurane and fluroxene anesthesia significantly (p less than 0.05) elevated the arrhythmic threshold dose for dopamine. Epinephrine produced greater elevations in mean arterial pressure than dopamine with all anesthetics except enflurane, and dopamine produced significantly (p less than 0.05) higher heart rates in the awake animals and those anesthetized with halothane and enflurane. The authors conclude that, in terms of arrhythmic potential, there is no advantage in the use of dopamine rather than epinephrine for the reversal of halothane-induced myocardial depression during halothane or enflurane anesthesia.


[Effects of Etomidate on ventilation and blood gases]. 17 surgical patients, agent 49 to 87, are studied. Their anaesthetic induction was performed by an intravenous injection of Etomidate on a 0,23 mg/kg body weight basiis. Respiratory frequency and minute ventilation were put on continuous recording, PO2, PCO2 and arterial blood pH were measured 1,3 and 5 minutes after injection of the drug. Anaesthetic induction, occuring 10 seconds after injection of the drug, was followed by an increase in respiratory frequency (32 p 100) and a rather modest increase of minute ventilation (9,2 p. 100). Among blood gases measurements, only PaO2 showed a decrease of 6,7 p. 100, when PO2 and pH remained practically unchanged. The small increase in minute ventilation, compared with a relatively important increase in respiratory frequency, suggests some decrease in efficient alveolar ventilation without however affecting blood gases in an important fashion.


[Hemodynamic effects of Etomidate].

[Hemodynamic effects of Etomidate]. Hemodynamic variations due to Etomidate administration were studies in 10 patients submitted to artificial ventilation. Etomidate was given at a 0.37 mg/kg B.W. dosage. Cardiac rate, mean arterial B.P., C.V.P., mean arteial pulmonary pressure, mean pulmonary capillary pressure and cardiac output were investigated. From values found, systolic output, systemic vascular resistance, and pulmonary arteriolar resistance were calculated. Results show that Etomidate has little effect on hemodynamics. There is only a 5 p. 100 increase in cardiac rate, a 16p. 100 decrease in cardiac output, 1 18 p. 100 decrease in systolic output and a 12p. 100 decrease in mean arterial B.P. Variations of mean capillary pressure, of systemic vascular resistance and of pulmonary arteriolar resistance are not significant.


[Use of the diazepam-pentazocine (pentazepam) combination in anesthesiology]. The authors report on 50 surgical or endoscopic cases in which local or regional anaesthesia was completed with  a combination of diazepam and pentazocine (Pentazapam). This "diazanalgesia" was performed either as a routine or as a complement to regional anaesthesia (mostly an epidural) in which restlessness or insufficient analgesia were noted. Both drugs are injected separately. Diazepam being used to combat anxiety and pentazocine to allay pain. Sedation was considered to be sufficient in 43 cases out of 50 and analgesia in 47 cases out of 50. In all but one case, recovery was calm. In 24 cases, the patients regained consciensness quickly. There was only little effect on respiratory frequency, blood gases, blood pressure or cardiac rate. Thus, pentazepam combination can be considered as a useful method of "wake diazanalgesia" as far as it is used to complement a local or regional anaesthesia. This technique seems particularly useful in the aged or bad risk patient because of its moderate cardiovascular or respiratory aciton.


[Toxico-pharmacologic study of a curarimimetic, AH 8165, in relation to storage time]. The toxicological and pharmacological study of a non depolarizing curariform drug AH 8165 was performed under various conditions of storage, duration and temperature (duration: 1 month 2 years; temperature: 4 degrees C to 70 degrees C). It shows that this drug ought to be kept a low temperature for storage (in the range of + 4 degrees C). In the sam way, this study demonstrates a better stability of this drug when stored lyophilized form instead of a solute. The interest of standardized animal experimentation in studying the activity of a therapeutic substance depending on the duration of storage is shown.


[Effects of Alfatesine (ct 1341) on cerebral blood flow and metabolism in man. Global and regional changes]. The effects of Alfatesine on local cerebral blood flow  and on cerebral oxygen consumption were studied in 5 normal subjects and in 6 patients suffering from diffuse vascular repercussion of cerebral distress. There is a 75 p. 100 fall in cerebral  blood flow in the gray substance and a parallel fall in oxygen consumption. At rest, the caracteristic anaesthetic action modifies the caracteristical distribution of blood flows in the cortex. A relative hyperemia appears in the area of the central sulcus. Those focal changes could not be found in the group of pathogical patients.


[Correction of the alphamimetic effects of metaraminol by nicergoline]. In this work, the authors are studying the cardio-vascular effects of nicergoline. To do so, the drug is made to compete with metaraminol whose dominant alphamimetic effects are known. This experimentation was performed in 7 patients in coma stage III or IV and whose vascular receptors were active. Those 7 patients were submitted continuous metaraminol perfusion and a steady state vasoconstriction was thus obtained. A single intravenous injection of 10 mg nicergoline resulted in vasodilation with a fall in vascular resistance and a moderate fall in mean blood pressure. Cardiac output remained unchanged. This work confirme thus the alphasympatholytic effects of nicergoline.


[Continuous measurement of arterial pressure by means of an arterial catheter. Its value in secondary transport]. It is of no much use to try to assess the cardiovascular condition of patients with impending or confirmed shock by taking the blood pressure with the classical arm cuff, especially if arterfacts such as noise, vibrations, shaking etc... are interfering. Instant continuous measurement of blood pressure by means of arterial cannulation avoids those drawbacks and is a sure and efficient method of assessment of an often unstable, precarious and sometimes aggravated cardio-vascular condition by the very transportation. The S.A.M.U. of Montpellier used this technique in 15 cases. The authors are reporting their experience and also are evoking some technical problems that may occur.


[Use of constant perithoracic negative pressure in the transport of newborn infants with severe forms of idiopathic respiratory distress]. The authors used for transportation of neonates in severe idiopathic respiratory distress the continuous perithoracic negative pressure method suggested by OUTBRIGE and STERN in neonate intensive care. The authors are describing the material used and are presenting their first encouraging results. This technique improves conditions and therapeutic efficiency in thus treated infants. The easy handling and accessibility of apparatus constitue an efficient and not too aggressive mean of treatment of those particularly frail and difficult to treat infants during transportation.


[Do enzyme determinations have any prognostic value in neurotraumatology?]. In 34 cases of skull traumas and in 14 cases of acute cerebral accidents, CK, GOT, GPT, LDH and its isoenzymes were assessed in the patient's serum and CSF. Results show that the increase of the enzymatic level in the CSF seems due to cerebral tissue damage for the main part, when in the serum the increase is influenced by combined traumas. The CSF dosages allow to define a threshold of severeness, unlike serum dosages without any pronostic value.


[Value of cerebral metabolic exploration in post-traumatic coma states in the acute phase]. The authors report the values of mean hemispheric blood-flow and cerebral arterial consumption they found in 34 neurosurgical comatous cases in acute state. In basal conditions, mean values of mean hemispheric bloodflow and oxygen consumption are lowered. There seems to be a relation between the values found and the comatous stage on one hand, the prognosis on the other hand. The cerebral response to hypercapnia (16 assays) allows to separate 2 groups, one with a noticeable improvement of cerebral bloodflow, the other with only a minimal response. There was no significant variation of cerebral oxygen consumption in both group. Cerebral response to CO2 seems to be clearly related to the stage of coma (low in the most severe cases) but pronostic incidence remained uncertain. A hypertensive test by means of Aramine (18 assays) allows to separate 3 groups : 1 group (8 cases) where the mean hemispheric bloodflow remained stable during hypertension as did the cerebral oxygen consumption -(autoregulation remained unchanged), 1 group (4 cases) where mean hemispheric bloodflow and cerebral oxygen consumption were lowered (excessive autoregulation), 1 group (6 cases) where mean hemispheric bloodflow increases clearly while under Aramine perfusion (loss of autoregulation). Those dynamic tests, either hypercapnic or hypertensive, allow, in comparing oxygen consumption variations with cerebral bloodflow variations, the distinction between : patients where metabolic autoregulation seems maintained (good prognosis) - (10 cases), patients where metabolic regulation is lost with either "luxury perfusion" (14 cases) - poor prognosis, or "insufficient perfusion" (10 cases). The authors are discussing the treatment concerning those last mentioned patients.


Characteristics of two new reticuloendotheliosis virus isolates of turkeys. Reticuloendotheliosis (RE) virus strains MN81 and MN67 isolated from epiornithics of RE in turkeys were partially characterized. Strains MN81 and MN67 replicated in chicken embryo fibroblast,duck embryo fibroblast and turkey embryo-fibroblast cultures and produced syncytial cytopathic effects in duck embryo fibroblast and turkey embryo fibroblast cultures. The virions of MN81 and MN67 measured approximately 100 nm in diameter, resembled RE virus strain T, and could be distinguished from avian leukosis viruses morphologically. The buoyant density of strain MN81 was found to be 1.15 g/cm3 in sucrose gradients. Strains MN81 and MN67 were inactivated by heat, acid pH, ether, and chloroform treatments. These strains were serologically unrelated to avian leukosis virus but were related to RE virus strains T, CS, DIA, and SN.


Body position and mode of ventilation influences arterial pH, oxygen, and carbon dioxide tensions in halothane-anesthetized horses. Effects of body position and type of ventilation were determined on arterial blood gases (PaO2, PaCO2) and pH during and immediately following clinical halothane anesthesia in 36 young, physically conditioned horses. Horses in dorsal recumbency had a lower PaO2 than did similarly breathing horses in a lateral position. Predictably controlled positive-pressure ventilation inproved arterial oxygenation and permitted maintenance of a normal PaCO2. Most horses, regardless of type of ventilation and operative body positioning, were hypoxemic in the immediate postanesthetic period.


Effects of electroanesthesia and a phenothiazine tranquilizer on thermoregulation in the sheep. The effects of giving propiopromazine alone and of electroanesthesia-propiopromazine treatment on thermoregulation (body temperature regulation) were studied in 3 sheep at ambient temperatures of 5, 25, and 35 C. Measures of thermoregulation during a 120-minute treatment and 120-treatment recovery period included rectal temperature, respiratory frequency, respiratory evaporative heat loss, metabolic heat production, multiple skin temperatures, and shivering. During cold exposure (5 C), both the propiopromazine administration and the electroanesthesia-propiopromazine treatment resulted in hypothermia which was attributed to increased peripheral and respiratory heat losses, a transient inhibition of shivering thermogenesis, and a reduction in metabolic heat production. At 35 C ambient temperature, both resulted in hyperthermia caused principally by a reduction in respiratory evaporative heat loss. The effects of electroanesthesia-propiopromazine treatment on thermoregulation appeared to be additive at both the cold (5 C) and the hot (35 C) environments, in that simultaneous administration resulted in a more profound thermoregulatory impairment. Nevertheless, shifts in body temperature during electroanesthesia are partly attributable to phenothiazine premedication.


Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis virus in bovine serum. A microimmunodiffusion test (MIDT) specific for detection of antibodies to infectious bovine rhinotracheitis (IBR) in bovine serum has been developed. The antigen used in the MIDT was prepared from IBR virus-infected Madin-Darby bovine kidney cells grown in tissue culture. The antigen was stable, and relatively high yields were obtained readily. Results of the MIDT were obtained within 48 hours and agreed with those of the serum-neutralization test.


The syndrome of inappropriate secretion of antidiuretic hormone associated with anaerobic thoracic empyema. The syndrome of inappropriate secretion of antidiuretic hormone has been associated with many pulmonary diseases, including tuberculosis and bacterial and viral pneumonia: however, it has not been reported with anaerobic infections or empyema in the absence of pneumonia. We report a patient with empyema due to Bacteroides melaninogenicus, Bacteroides oralis, and Peptostreptococcus who developed the syndrome. Eight hours before the start of therapy, his serum sodium concentration was 127 mEq per liter; serum osmolality, 255 mOsm per kg; urine osmolality, 522 mOsm per kg; urinary sodium concentration, 39 mEq per liter. The creatinine clearance and the adrenocorticotropic hormone stimulation test were normal, and there was no evidence of dehydration. No other causes of the syndrome of inappropriate secretion of antidiuretic hormone were apparent. With drainage and antimicrobial drug therapy, the empyema cleared, and the syndrome resolved in 8 days. The patient has been well, without evidence of inappropriate secretion of antidiuretic hormone, for 9 months. Anaerobic infections and/or empyema without pneumonia can be associated with the syndrome of inappropriate secretion of antidiuretic hormone.


[Study on L-malic acid catabolism by "Lactobacillus casei" cells immobilized into polyacrylamide gel lattice (author's transl)]. Study on L-malic acid catabolism by Lactobacillus casei cells immobilized into polyacrylamide gel lattice has shown that the pH profil of malic acid decarboxylase activity does not differ significantly from that of free cells. The apparent enzymatic constant value as determined by the Warburg manometric method is 5 X 10(-3) M for free cells and 1.25 X 10(-2) M for immobilized cells. Malic acid decarboxylase activity can be preserved in immobilized cells over a 12 month period when the reaction vessel is fed continuously with the growth medium. It is noteworthy that the reaction vessel is similar to a constant activity reactor whose apparent enzymatic constant value, of 6,25 X 10(-3) M, does not differ significantly from that of free cells. Labelling of the cells with 14C-glycin has shown that the immobilized cells released 14 per cent of the radioactivity over a 65 hour period, indicating that the cells are possibly in a non proliferating state. After 9 months of working, a large fraction of viable bacterial cells can be isolated again. The use of such a reactor for producing metabolites is proposed.


[Cross reactions of higher type-specific capsular polysaccharides of Klebsiella in antipneumococcal sera]. Qualitative and quantitative precipitin reactions are given of the polysaccharides K60, K61, K65, K67-69, K70, K71, K73, and K83, also of depyruvylated K1. As expected, the last reacts more strongly in antipneumococcal (anti-Pn) VIII and X than does intact K1. K65 and K67 precipitate much of the same large fraction of anti-PnXXIII as does K47, showing that K65 and K67 also possess lateral non-reducing end-groups of L-rhamnose. The massive reaction of K83 in anti-PnII confirms the chemically demonstrated non-reducing end-groups of D-glucuronic acid in the repeating unit, Such groups, or 1,2-linked D-glcA, will probably also be found in K71.


[Natural antiallergenic factors in bile]. Two groups of substances having an antihistaminic activity were found on purifying bovine bile. It has already been found that bile acids inhibit contraction of guinea pig ileum; another principle has been isolated which is one thousand times more active than the bile acids; this (these) substance(s)--SAN (" substance antiallergique naturelle ")--seems to have more inhibitory effect on smooth muscle contraction induced by SRSA than histamine, acetylcholine, bradikinin and serotonin. SAN is also able to inhibit the Schultz-Dale reaction. These findings indicate that SAN could be a possible antiallergic agent.


[Role of adherent cells in GVH induced immunosuppression]. Two different mechanisms seem to be involved. In early GVH there is apparently an amplification of a normal inhibitory function of a cells. In more advanced GVH immunosuppression seem to be caused by a GVH induced suppressive activity of A cells. Both activities seem to be mediated by soluble factors.


[Spleen reactivity after BCG treatement: test and strain dependency of the response]. According to the strain used, spleen cells from mice injected two weeks before with 3 mg BCG, when they are engaged in a graft-versus-host reaction show a decrease in reactivity (C57Bl/6or AKR), an increase (DBA/2, BALB/C or SJL/J) or no modification (CBA, C3H). The in vitro responsiveness to PHA and conA of the treated spleen cells was lower than controls for all the strains studied. However, nylon column purified T cells from BCG treated mice showed a dissociated response, being less stimulated than normal cells by PHA and more so by ConA.


[Macrokinetic equations of pH effect on the growth of Actinomyces aureofaciens and biosynthesis of tetracycline]. The effect of pH on the culture respiration rate at different concentrations of glucose in the medium was studied. It was found that the hydrogen ions showed their effect irrespective of the subsrate concentration in the medium. In this connection a type of macrokinetic equations of the effect of pH on the growth and antibiotic biosynthesis was chosen. The constants of the model were determined.


[Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin]. The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK''1 6.1 and pK''2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.


[Chromatographic separation of rifamycins on a thin sorbent thin layer]. A method of simultaneous separation of rifampicins O, S and rifamicin derivates S, SV in a thin layer of silica gel KSK No. 2 impregnated with acetate buffer, pH 4.6 in a solvent system chlorophorm-methanol (100:5) was developed. The minimum detectable amount of the compounds studied was 1--2gamma, when the coloured zones were estimated visually.


[Penicillin amidase from E. coli. A direct spectrophotometric method of determining the enzyme's activity]. A method for determination of the enzymatic activity of penicillinamidas (PA) based on spectrophotometric estimation of the stained product amount produced in hydrolysis of 4-phenylacetamido-2-nitrobenzoic acid (PANBA) catalyzed by the enzyme is proposed. Some physico-chemical properties of the substrate and the stained product were studied. The kinetic parameters of the PANABA enzymatic hydrolysis were determined. Catalytic activity of some enzyme products of PA of different purity levels was studied comparatively in reactions of PANBA and benzylpenicillin hydrolysis.


[Simplified liquid nutrient media for controlling antibiotic activity, the spectrum of their antibacterial action and the sensitivity of microorganisms]. Beaf-peptone broth and some of its modifications, one of which is a simple and in expensive one to a leser extent binding to antibiotics, such as penicillin, oxytetracycline and streptomycin and providing sufficient growth of the test microbes were used to determine the antibiotic activity with the methods of serial dilutions. The simple modification was recommended for practical use. The MIC of the antibiotics in the above simple medium was less than that in the control. The results of the antibiotic activity determination on both media coincided.


[Experimental study of a gentamicin aerosol]. The study of gentamicin aerosol showed its relative innocuousness: it did not inhibit the growth and development of young animals, did not induce pathological changes in the upper respiratory tract, kidneys, liver, heart and spleen on its prolonged use. Pathohistological examination revealed slight irritating effect of the gentamicin aerosol in the lungs after its use in a dose of 8 or 25 mg/kg for 6 weeks. A procedure for investigating the effect of the aerosol on the activity of the trachea ciliated epithelium of warm blooded animals was developed. The gentamicin aerosols prepared from solutions of different concentrations (1 to 50 mg/ml) induced ingibition of the ciliated epithelium function at average from 15 to 35 per cent which was associated with the solution acidity (pH 4.54 to 4.82). Such a decrease in the function of the ciliated epithelium due to the antibiotic aerosol use was a factor prolonging the antibiotic retention time in the respiratory organs. It was found that aqueous solutions of drugs used for inhalation, such as ephedrin, euphelin, dimedrol, N-acetyl-L-cystein and others had no effect on the activity of gentamicin and may be used with it in a form of aerosols.


Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1. The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by  the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.


Growth characteristics of a new methylomonad. A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.


Association of hydrogen metabolism with methanogenesis in Lake Mendota sediments. Lake Mendota sediments were studied to determine the role of H2 in sediment methanogenesis. H2 was generally not detectable in sediment. The addition of H2 to sediment significantly increased methanogenensis. The amount of methane produced was proportional to the concentration of hydrogen added. H2 addition stimulated the reduction of CO2 to methane, but did not significantly stimulate the conversion of methanol or the methyl position of acetate to methane. Various organic compounds also stimulated sediment methanogenesis. Formate, ethanol, and glucose were shown to serve as electron donors for CO2 reduction to methane. The addition of formate to sediment resulted in H2 evolution. H2 was not deith the phenomenon of interspecies hydrogen transfer. The results indicate that hydrogen is an important intermediate and a rate-limiting factor in sediment methanogenesis.


Media for identification of Gibberella zeae and production of F-2-(Zearalenone). Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.


Wilm's tumour, hypospadias, and cryptorchidism in twins. Twin boys, both of whom had hypospadias and bilateral cryptorchidism, each developed a left-sided Wilms's tumour. The first twin was found to have an advanced multifocal tumour at the age of 15 months and died with local recurrence and pulmonary metastases. The diagnosis was made in the second twin one month later and at nephrectomy the tumour was found to be encapsulated without metastases; he is disease-free 12-years later. Although the histological appearances were similar in each twin, the tumours did not develop at the same rate and did not show the 'mirror-image' pattern suggested for embryonal tumours in identical twins.


Effects of beta-adrenoceptor blocking agents, pindolol, alprenolol and practolol on blood pressure and heart rate in conscious renal hypertensive dogs. The effects of pindolol, 10 mg/kg, alprenolol, 50 mg/kg, and practolol, 50 mg/kg, given by mouth, on blood pressure and heart rate were investigated over a 24-hr period in 5 conscious renal hypertensive dogs, using a cross-over design. Pindolol and alprenolol caused significant falls in blood pressure which averaged 22 mm Hg (at 3-hr after p.o. administration) and 20 mm Hg (at 3-hr). However, practolol failed to produce any significant changes in blood pressure. Heart rate increased by 67 beats/min (at 1-hr) and 39 beats/min (at 3-hr) after pindolol and alprenolol, respectively, but did not show any significant increase when practolol was given orally. The pindolol-induced tachycardia and hypotension were not suppressed significantly by propranolol (1 mg/kg i.v.) which blocked completely the tachycardia and hypotension induced by isoprenaline (3 mg/kg p.o.). The hypotension and tachycardia observed after oral administration of D-32 (50 mg/kg) or after intravenous infusion of p-OH D-32 (1 mg/kg per min for 5 min) were also not modified significantly by propranolol (1 mg/kg i.v.). Based on these results and other published data, mechanisms pertaining to the hypotension exerted by beta-adrenoceptor blocking agents were discussed.


Selective activation of cardiovascular adenylate cyclase in vivo. Cyclic AMP levels were measured in cardiac and vascular tissues removed from pentobarbitone-anesthetized rats pretreated with theophylline 45 mg/kg i.p. Noradrenaline, 20 microng/kg i.v., increased cardiac but not vascular cyclic AMP levels; this selective effect of noradrenaline was not altered by pretreatment with phenoxybenzamine, 1 mg/kg i.v. Tazolol, 120 microng/kg i.v., selectively increased cardiac cyclic AMP levels whereas fenoterol, 15 microng/kg i.v., selectively increased vascular cyclic AMP levels. The results suggest that adenylate cyclase activation in cardiac and vascular tissued in vivo is mediated by beta adrenoceptors which consist of different subtypes.


Comparative subcutaneous absorption of local anesthetics: lidocaine, procaine and tetracaine. The pH partition hypothesis was applied to the absorption of ionizable local anesthetic amines through the subcutaneous (s.c.) route. Solutions of lidocaine hydrochloride (LHCl), procaine hydrochloride (PHCl) and tetracaine hydrochloride (THCl) at various pH were implanted on s.c. animal tissue through a glass absorption cell. The s.c. absorption of these drugs was evaluated from the clearances' slopes evolved by determination of the drugs' concentrations in the cell at definite intervals. The higher the pH level, these conjugated bases become more unionized and lipid partitioned resulting in increased rates of s.c. absorption. The extent of unionization is governed by the pka of each of these drugs; at the high feasible pH--procaine which is least unionized--its s.c. absorption rate is nearly a third of that of lidocaine or tetracaine. The pH effect on s.c. absorption of local anesthetics was substantiated by estimation of lethal time50 (LT50) in mice.


Acidostability of speroplasts prepared from Thiobacillus thiooxidans. Thiobacillus thiooxidans was acidostable even in the absence of its respiratory substrate, elementary sulfur. This suggests that the acidostability of the bacterium was enery-independent. The organism was subjected to osmotic shock with 0.75 M sucrose at 0 degrees C and then treated with snail intestinal juice in the presence of 0.3 M sucrose. The decrease in the optical density of the sample thus prepared on dilution with deionized water and electron microscopic observation of the sample showed that spheroplasts were formed from the bacterium by this procedure. Spheroplasts were able to respire sulfur and their respiratory activity was acidostable. Spheroplasts, when treated with Nagase, proteolytic enzyme, lost their acidostability, and some protein components disappeared from the membrane fraction. This suggests that the acidostability of the bacterium may be related to protein conponents of the membrane.


Effect of external factors on phototaxis of Chlamydomonas reinhardtii. II. Charbon dioxide, oxygen and pH. 1. The effects of carbon dioxide, oxygen and pH on the inversion intensity of phototaxis of Chlamydomonas reinhardtii have been investigated. 2. With decreasing with CO2 tension the inversion intensity is decreased. 3. The gassing with CO2 can be substituted by hydrogencarbonate only to a small extent (20%). On the other hand, the effect of decreasing CO2 tension can be prevented also only in part by adjusting the pH to about 6.5-7.0. Thus the effect of CO2 on the inversion intensity of phototaxis is obviously a composite of a true CO2 effect and an effect of the concomitant pH change. 4. Oxygen has only a slight effect. In presence of oxygen (air) the phototactic reaction values are somewhat lower than in its absence. 5. Under certain conditions circadian rhythms seem to be initiated by changing oxygen as well as CO2 tensions. 6. Based on these results some contradictory results of older investigations are discussed.


Penicillinase (beta-lactamase) formation by blue-green algae. Beta-Lactamase (penicillinase) activity was found in a number of strains of blue-green algea. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of beta-lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO3 as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO2 in N2 or by growth under conditions of N2 fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalosporins. Some differences were observed in the "substrate proliles" of penicillinases from the two strains against different penicillins.


Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrifcans grown on glucose/nitrate. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate : NADP+ L-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD+-and NADP+- linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg2+ requirement. Kinetics for both NAD(P)+ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD+-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP+-linked activity. Neither of the two activities are inhibited by 100 muM NADH but both are inhibited by NADPH. The NAD+-linked activity is far more sensitive to inhibition by NADPH than the NADP+-linked activity.


Cellular proteases increased in paramyxovirus (Sendai virus) carrier cells possibly responsible for enhanced formation of cowpox virus-specific cell surface antigen. Cowpox virus (CPV) growth and its S-ag (cell surface antigen) formation  in HVJ (Sendai virus) carrier cells pre-treated with Actinomycin D or Puromycin were not affected as much as those in parent cells. These suggest the different cellular functions of carrier cells. The activity of carrier cell extracts causing a characteristic degradation of CPV reacted with them in vitro disappeared after the pre-incubation of extracts with hemoglobin or casein. Measurements of cellular protease activities including lysosomal enzymes demonstrated significant increases in the carrier cell extracts compared to those in parent cells; The CPV, thus reacted in vitro with the extracts or lysosomal fraction from carrier cells, acquired more rapidly and markedly the enhanced ability of S-ag formation parallel to virus infectivity alteration in the reaction. These results indicate that the enhancement of CPV S-ag formation in the HVJ carrier cells may be due to their increased cellular enzymes, possibly proteolytic ones, capable of promoting the first step of CPV uncoating or degradation in the cells.


Overlay dentures. Philosophy and practice. II. The practical stages in the construction of mandibular overlay dentures, with and without precision attachment anchors, are described and illustrated in detail. The importance of post-insertion maintenance, continuing effective oral hygiene and patient care, as a prerequisite for a successful overlay denture experience, is emphasized.


Fatty liver and kidney syndrome in chicks. II. Biochemical role of biotin. The role of biotin-dependent enzymes in the fatty liver and kidney syndrome of young chicks was studied. Under conditions of a marginal deficiency of dietary biotin, the level of biotin in the liver has differing effects on the activities of two biotin-dependent enzymes, pyruvate carboxylase and acetyl-CoA carboxylase. The activity of acetyl-CoA carboxylase is increased, but when the dietary deficiency of biotin produces biotin levels which are below 0-8 mug/g of liver, the activity of pyruvate carboxylase may be insufficient to completely metabolize pyruvate via gluconeogenesis. There is an increase in liver size and in the activities of enzymes involved in alternate pathways for the removal of pyruvate. Blood lactate accumulates and there is increased synthesis of fatty acids, and an accumulation of palmitoleic acid; these steps are accomplished by increased activities of at least the following enzymes: acetyl-CoA carboxylase, malate dehydrogenase (decarboxylating) (NADP+) and the desaturase enzyme. When the biotin level is below 0-35 mug/g of liver and the chick is subjected to a stress, physiological defence mechanisms of the chick may be inadequate to maintain homeostasis and they finally collapse, resulting in accumulation of triacylglycerol in the liver and blood; the chick is unable to maintain blood glucose levels and death occurs, often only a few hours after the imposition of the stress.


Changes in plasma enzyme concentrations following intramuscular injections and gastroscopy. The effects of intramuscular injections on plasma creatine kinase (CK), aspartate amino-transferase, lactate dehydrogenase, and hydroxybutyrate dehydrogenase concentrations were examined in 19 patients given intramuscular premedication for gastroscopy, and 18 patients given other intramuscular injections. Only CK concentrations showed significant increases which were as high as four times the upper limit of normal, and affected a maximum of 51% of patients at 12 hours after the first injection. Elevated CK concentrations persisted for up to 72 hours, and followed injections of diazepam, various antibiotics, and the combination of a narcotic analgesic with atropine. Gastroscopy did not appear to increase plasma enzyme concentrations in six patients who were given intravenous premedication. The significance of these findings to the diagnosis of myocardial infarction is discussed.


Effects of changes in pH of bath fluid on isolated guinea pig ileum preparation. The effects of changes in the pH of Tyrode's solution on the responses of isolated guinea pig ileum preparation to acetylcholine and histamine were studied. At higher levels of pH (increased alkalinity), the responses of the tissue to both acetylcholine and histamine were increased. At lower pH levels (increased acidity), the response of the tissue to acetylcholine was increased but that to histamine was reduced.


Glycosidases from the culture medium of Physarum polycephalum. Eight exo-glycosidase activities were detected in the axenic culture medium of the myxomycete, Physarum polycephalum. The secretion of each enzyme examined followed the growth curve and continued during the stationary phase after the cessation of growth. Two or more forms of each enzyme were detected after electrophoretic separation. The beta-N-acetyl-D-hexosaminidase activity was readily separated into its two electrophoretic forms, X and Y, which were purified 145- and 306-fold respectively. These beta-N-acetyl-D-hexosaminidases had several similar characteristics. Evidence is presented that the major electrophoretic form of alpha-D-galactosidase is heterogeneous. The possible functions of extracellular glycosidases in teir occurrence and properties.


A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi. The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.


A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates. 1. Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C. The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. 2. Acetaldehyde forms a u.v.-absorbing complex with glycine. This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work. 3. The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex. 4. The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx. 7.1) and the enzyme-NADH complex (pK approx. 8.4). The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex. 5. Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated. The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH. 6. The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates.


The use of gadolinium as a probe in the Fc region of a homogeneous anti-(type-III pneumococcal polysaccharide) antibody. The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.


Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit. Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.


Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b. 1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.


Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans. 1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.


Isolation and characterization of sheep pepsin. Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.


Properties of rat liver microsomal stearoyl-coenzyme A desaturase. 1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.


The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid. The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.


Purification and properties of an alpha-D-galactoside galactohydrolase from the seeds of Trifolium repens (white clover). Five alpha-D-galactosidases (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) have been identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidase I has been purified to homogeneity with an approx. 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the alpha-D-galactosidic activity. The pH optimum was found to be between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans has been examined, and it has been found that alpha-galactosidase I exhibits two enzymic activities, namely alpha-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick & Smith (1968), and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the mol.wt. has been estimated to be 43 000 and 41 000 respectively. These results indicate that alpha-galactosidase I is a monomeric protein and that both enzymic activities associated with the enzyme reside on the same polypeptide chain.


An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase. 1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.


Large fragments of human serum albumin.

Large fragments of human serum albumin. Large fragments of human serum albumin were produced by treatment of the native protein with pepsin at pH3.5. Published sequences of human albumin [Behrens, Spiekerman & Brown (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591; Meloun, Moravek & Kostka (1975) FEBSLett.58, 134-137]were used to locate the fragments in the primary structure. The fragments support both the sequence and proposed disulphide-linkage pattern (Behrens et al., 1975). As the pH of a solution of albumin is lowered from pH4 to pH3.5, the protein undergoes a reversible conformational change known as the N-F transition. The distribution of large fragments of human albumin digested with pepsin in the above pH region was critically dependent on pH. It appeared that this distribution was dependent on the conformation of the protein at low pH, rather than the activity of pepsin. The yields of the large fragments produced by peptic digestion at different values of pH suggested that the C-terminal region of albumin unfolds or separates from the rest of the molecule during the N-F transition. The similarity of peptic fragments of human and bovine albumin produced under identical conditions supports the proposed similar tertiary structure of these molecules.