Serum levels of pancreatic polypeptide in Zollinger-Ellison syndrome, and hyperparthyroidism from families with multiple endocrine adenomatosis type I. Mean fasting levels of pancreatic polypeptide (PP) in 24 patients with Zollinger-Ellison syndrome (ZES) and in 12 patients with hyperparathyroidism originating from families with multiple endocrine adenomatosis type I (MEAI-HPT) were significantly higher than in 72 normal controls. The overlap between the 3 groups, however, was large. In patients with ZES, increased PP levels were not related to the presence of MEAI or metastases; nor was there a correlation between serum PP and gastrin concentrations. The post-prandial PP release in 10 ZES patients and in 10 patients with MEAI-HPT was lower than in 9 normal controls. The physiological significance of the present findings is unclear.


Coupled secretion of gastric lipase and pepsin in man following pentagastrin stimulation. Secretion patterns of gastric lipase and pepsin were studied in duodenal ulcer patients before and after vagotomy and in healthy subjects following continuous administration of pentagastrin in graded doses. The lipase output increased in response to pentagastrin stimulation in similar fashion as that of pepsin. The output of the two enzymes were correlated with each other and with hydrogen ion output. Evaluation of dose-response relationships showed that in duodenal ulcer patients the secretion of gastric lipase and pepsin is more sensitive to pentagastrin stimulation than that of hydrogen ions.


Pulmonary function in inflammatory bowel disease. Pulmonary function has been assessed in patients with ulcerative colitis and Crohn's disease and compared with a healthy population. No statistically significant differences were found in the measurements observed within the three groups.


Physical chemical and technical limitations to intragastric titration. Intragastric titration, introduced in 1973, has become more popular than in the past and is being applied to the study of gastric secretion under a variety of conditions using NaHCO3 and NaOH as titrant. Physical chemical and technical considerations point to limitations on the conditions under which quantitative results can be obtained. At low end point pH, the limitation relates to volume expansion by secretions and titrant. Errors increase exponentially so that at pH 1.11 a 66% error is incurred and at pH 0.93 a 100% error would result. Neutralization with NaHCO3 poses limitations when CO2 is not effectively removed from the stomach, and even at pH 5.5 errors as large as 20% can be incurred. Errors increase markedly at high end point pH. Use of end point pHs above the pKa' of carbonic acid could lead to large amounts of factitious acid secretion if the intralumental contents are in equilibrium with CO2 entering from the gastric wall. There seems no reason that intragastric titration should not be a useful and quantitative method within the end point pH range of 4.0--4.5. In vivo validation of each experimental condition is needed.


Aspirin-stimulated intestinal electrolyte transport in rabbit ileum in vitro. Because aspirin and the heavy metal salts of related anions have effects on intestinal ion transport and on diarrheal states, we have studied the effect of aspirin (ASA) on the in vitro rabbit ileum in an attempt to understand its mechanism of action. Ten millimolar of aspirin increased the conductance of rabbit ileum by 10--50% but did not change the permselectivity of the shunt path as determined by measurement of diffusion potentials. In Cl-free and HCO3-free solutions, aspirin reduced both Na absorption and the short-circuit current (Isc), which suggests an effect on electrogenic Na transport. Such an effect was not unexpected, since aspirin interferes with ATP production. However, in Ringer solution, aspirin in concentrations as little as 1 mM in the serosal solution reduced the Isc as before but also stimulated Na and Cl absorption and reduced JRnet (? HCO3 secretion) to zero. Aspirin had no effect on Na transport in the absence of Cl and no effect on Cl transport in the absence of Na, which suggests that aspirin stimulated a coupled transport process. Although these effects of ASA resemble those of alpha-adrenergic agents, ASA's effect was not blocked by alpha-adrenergic blockers such as phentolamine or phenoxybenzamine. The exact mechanism of ASA-stimulated NaCl absorption remains to be determined.


[Telemetric monitoring of delivery when inducing labour by prostaglandin E2 tablets (author's transl)]. The article reports on 75 patients in whom labour had been introduced by means of oral administration of prostaglandin E2 tablets (35 patients were controlled at the bedside and 40 patients were monitored telemetrically.) Telemetric monitoring is preferable in oral application of ecbolics, since this entails a saving in ecbolics (4.7 as against 5.4 tablets), regular labour is induced more rapidly (after 57 as against 72 minutes) and total delivery time is shortened (4 hours 24 minutes as against 5 hours 15 minutes). There were no serious side effects neither in the mother nor in the neonate. 6 of the 75 patients complained of slight gastrointestinal side effects. The Apgar scores and pH levels of the children were in the normal range. The post-partum period was identical in both groups, namely, 11 minutes.


Postprandial gastric function in pancreatic insufficiency. Abnormalities in postprandial gastric function could contribute to the maldigestion of pancreatic insufficiency. To measure simultaneously postprandial gastric secretion and emptying and correlate these measurements with intraluminal duodenal changes, we performed intestinal intubation and duodenal perfusion during feeding of a solid-liquid test meal in 10 healthy controls and 10 patients with documented pancreatic insufficiency before and after replacement therapy. In pancreatic insufficiency, intraduodenal pH was significantly decreased late in the postprandial period while simultaneously measured duodenal acid loads were normal, confirming that reduced bicarbonate output rather than increased acid delivery was responsible for higher duodenal acidity in these patients. Significant (P less than 0.05) reductions in postprandial acid, pepsin, and total secretory outputs were noted in untreated patients only during the first postprandial hour. Absolute gastric emptying rates were lower in patients (P less than 0.05) than in healthy subjects, but fractional rates of emptying were similar. Fasting and postprandial hypergastrinaemia were consistently observed in the patients with pancreatic disease. There are postprandial disturbances of secretory function but no primary gastric motor defect in patients with exocrine pancreatic insufficiency.


Effect of human placental lactogen upon the human corpus luteum of late pregnancy. To acertain whether human placental lactogen (HPL) functions as a luteotropin during pregnancy in humans, studies were performed to determine if receptors for HPL are present in cells of the human corpus luteum of late pregnancy. Preparations of 125I-HPL which demonstrated specific binding to late pregnant rabbit mammary gland cell homogenates showed specific binding of less than 2.5% to homogenates of human corpora lutea of late pregnancy. These studies indicate that HPL is not luteotropic at this stage of pregnancy in humans. The action of HPL upon the corpus luteum appears to vary considerably according to species.


NADPH generating enzymes in Leydig cells from diabetic rats. Impaired testosterone biosynthesis in Leydig cells from streptozotocin treated rats is correlated with the reduced activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase. The results shown demonstrate that in the diabetic state the activity of these enzymes is reduced by almost 50 to 59% from normal levels. Insulin treatment restored their activities to normal levels. The diminished supply of NADPH in diabetic interstitial tissue is not the unique factor in the control of steroidogenesis, since the availability of large amounts of exogenous NADPH in the incubations of Leydig cell did not reduce the differences in testosterone synthesis observed when compared with normal cells.


The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L. The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.


Immunosupression of the primary and secondary immune response by an IgM plasmacytoma (TEPC-183). Previously we had established that TEPC-183 IgM(K) suppressed the primary immune response (IR) to both the T-dependent antigens 2,4-dinitrophenyl-haemocyanin (DNP-HCY) and T-independent pneumococcal polysaccharides. In the current investigation, the effect of TEPC-183 on an ongoing immune response to SS-III and DNP-HCY was examined. It was found that when TEPC-183 was injected 6 days after the initial antigen injection, at the height of the primary IR, the response was significantly suppressed to SS-III and to the DNP ligand. In addition, suppression of the secondary IR occurred when mice were injected with tumour as late as 35 days after the first antigen injection. Tumour removal lifted the immunosuppression to DNP and the tumour-removed group had a similar number of both direct and indirect anti-DNP-PFC, although HA levels were still reduced. When mice were pretreated with serum from normal mice or serum or ascites from TEPC-183 bearing mice, one day prior to and on the day of antigen injection, the immune response to DNP was reduced by TEPC-183 serum but not by normal mouse serum (NMS), while the anti-SSS-III response was reduced by both NMS and TEPC-183 serum. Thus, NMS selectively suppressed the T-independent response, but only TEPC-183 serum suppressed both types of responses. The suppressive effect of serum on the IR of normal mice indicates a role for soluble regulatory suppressive factors present in the serum of normal and tumour-bearing mice. The data are consistent with the idea that the tumour exerts its effect on the inductive as well as the proliferative phase of the immune response.


Augmentation of immune responses after methotrexate administration. A single intraperitoneal injection of 0.5 mg methotrexate (MTX) has been found to increase the immune reactivity of spleen cells from (C57B1/6 X DBA/2)F1 mice. Five days after injection, spleen cells from MTX-treated mice exhibited greater PHA responsiveness and GvH reactivity, and mice given SRBC at this time developed greater than normal direct PFC responses. This pattern of effects of MTX was particularly evident in mice that had been given high doses of BCG intravenously 14 days before testing, a treatment that generally depressed the measured activities. MTX enhancement of GvH was also observed in mice that had been depleted of short-lived T lymphocytes by adult thymectomy. We suggest that MTX-sensitive cells possible exert, particularly in BCG-treated mice, a suppressive action on the responding cells.


Some observations on human semen analysis. Semen analysis of 66 unmarried medical students in the age group of 17-21 years was carried out. A higher liquefaction time pH, motility, lower sperm count and abnormal forms were observed compared to reported values. Liquefaction time, pH and sperm count was found significantly different in non-vegetarians and vegetarians, perhaps due to difference in their dietary proteins.


Eosinophil chemotactic factor. Release from human polymorphonuclear neutrophils by arachidonic acid. An eosinophil chemotactic factor (ECF) can be released from human polymorphonuclear neutrophils by arachidonic acid (AA), its methyl ester, but not by other derivatives such as AA ethyl ester and arachidonyl acetate. The ECF is highly specific for eosinophils and does not attract polymorphonuclear neutrophils. A dose-dependent non-cytotoxic ECF release induced by AA can be obtained from human polymorphonuclear neutrophils from a lymphocyte-monocyte-basophil suspension, from rat basophil leukemia cells, but not from human lymphocytes. Kinetic studies demonstrate that ECF release occurs rapidly with an early rise and steep fall-off at later times of secretion. The amount of ECF release is dependent on pH, temperature and medium which is used for stimulation. Gel filtration analysis as well as subcellular fractionation studies suggest that the AA-induced ECF is a biological activity either distinct from AA and its split products or representing a known or unknown conversion product of AA with potent effects on eosinophils at minimal concentrations. AA may therefore represent an important mechanism of cell activation.


Neutrophil function and host resistance. The part played by the phagocytic cells against invading pathogens has been known since the work of Metchnikoff nearly a century ago. This review deals primarily with the role of the neutrophilic polymorphonuclear leukocyte in host defense against microbial infections. The overall function of these cells in protection from infection is dependent on a number of steps. First, an adequate number of functionally mature neutrophils have to be produced and released into the circulation by the bone marrow. Cells must circulate normally and be capable of adhering to capillary and venule walls overlying inflammatory sites. The next step involves the exit of phagocytes from the blood stream through the capillary wall and emigration into the tissues to establish contact with the invading pathogens. This process is accomplished by the locomotive characteristics of these cells and chemotaxis. Most organisms must then be phagocytized to be killed. Two discrete phases are involved in phagocytosis; the "recognition" and attachment phase followed by the ingestion phase. After phagocytosis a series of coordinated morphologic and biochemical events are set into motion which leads to eventual death and lysis of the ingested microbes. A variety of antimicrobial mechanisms are involved in this final step and indicate that these cells have an appreciable reserve capacity if one mechanism is impaired. Recent evidence which clarifies mechanisms involved in all these stages is discussed.


Evidence for circadian rhythmicity in guanyl cyclase of the rat intestinal epithelial cell. The activity of the enzyme, guanyl cyclase, associated with the rat intestinal brush border membrane, has an endogenous circadian rhythm which is observed in the absence of oral intermittent feeding and of a dark period. This rhythm is cued by the feeding schedule but is essentially unaffected by a light-dark cycle.


The effects of X-irradiation on lens reducing systems. Studies have been made of the effects of X-ray on various lens reducing systems, including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS) and of certain enzymes, including GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PG). It was found that during several weeks following X-irradiation but prior to cataract formation, there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the X-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the X-rayed lens was found to decrease significantly relative to controls 1 week prior to cataract formation, and the ratio of NADPH to NADP+ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the X-rayed lens. A corresponding decrease occurred in the activity of the HMS in X-rayed lenses as measured by culture in the presence of 1-14C-labeled glucose, G-6-PD was partially inactivated in the X-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to X-irradiation. The data indicate that X-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high-molecular-weight protein aggregates may be initiated.


Interaction of hemoglobin with membrane lipids: a source of pathological phenomena. The binding of hemoglobin to phosphatidylserine liposomes was studied by Hb quenching of the fluorescence intensity of 12-(9-anthroyl) stearic acid embedded in the lipid membrane. The interaction is basically electrostatic. Hb A2 interacts more strongly than Hb A. The binding always includes an irreversible fraction. Interaction of Hb with normal red blood cell membranes was studied with the same technique. The main difference between the two systems is that the binding of Hb to the inner surface of the red cell membrane is based on the interaction of Hb with the membrane proteins and is reversible. The inner side of the red blood cell membrane is composed of phosphatidylserine lipids but these are normally masked by membrane proteins. In cases where Hb A2 is higher or when membrane lipids are abnormally exposed, Hb might interact irreversibly with the lipid layer and distort the membrane.


[The bacterial flora of preputial space]. The bacterial flora of the preputial space of 210 healthy males (43 children aged between 2 and 11 years, 137 males between 12 and 60 years and 30 men over 60 years) was determined by smears and cultueres from glans penis, sulcus coronarius and the adjacent prepuce. The results were grouped according to various criteria, e.g. glans covered or uncovered and age of males. Differences in the distribution of germs could be found in relationship to age. In the case of an uncovered glans penis the presence of microbial flora corresponds to the grampositive saprophytic bacteria in areas rich in sebaceous glands. In the case of a covered glans the density of microorganisms increases. Prevalent are gramnegative anaerobes, especially Bacterioides melaninogenicus, also enterococci, enterobacteria and coagulase-positive staphylococci.


Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity. Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigating in vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.


Oxidative enzymes and myosin-ATPase in the trunk musculature of the river lamprey (Lampetra fluviatilis). The distribution and activities of several oxidative enzymes and of myosin-ATPase have been studied in the trunk musculature of the river lamprey. The strongest activity of oxidative enzymes and of myosin-ATPase was shown by the parietal fibre bundles of the myotomal subunits, whilst the central fibres reacted to a lesser extent. Pre-incubation of sections in media containing formaldehyde and glycine or with buffers of different pH, followed by incubation for myosin-ATPase, also differentiated the muscle fibres into a specific distribution pattern within the myotomes, resembling that of the oxidative enzymes to some extent. The parietal fibres were found to be of the slow type (type I) and the cental fibres to be fast (type II A).


Simple, accurate equations for human blood O2 dissociation computations. Hill's equation can be slightly modified to fit the standard human blood O2 dissociation curve to within plus or minus 0.0055 fractional saturation (S) from O less than S less than 1. Other modifications of Hill's equation may be used to compute Po2 (Torr) from S (Eq. 2), and the temperature coefficient of Po2 (Eq. 3). Variations of the Bohr coefficient with Po2 are given by Eq. 4. S = (((Po2(3) + 150 Po2)(-1) x 23,400) + 1)(-1) (1) In Po2 = 0.385 In (S-1 - 1)(-1) + 3.32 - (72 S)(-1) - 0.17(S6) (2) DELTA In Po2/delta T = 0.058 ((0.243 X Po2/100)(3.88) + 1)(-1) + 0.013 (3) delta In Po2/delta pH = (Po2/26.6)(0.184) - 2.2 (4) Procedures are described to determine Po2 and S of blood iteratively after extraction or addition of a defined amount of O2 and to compute P50 of blood from a single sample after measuring Po2, pH, and S.


Abdominal cryptorchidectomy in the horse, using inguinal extension of the gubernaculum testis. Removal of abdominal testes was accomplished in 32 horses ranging in age from 6 to 72 months. Twenty were unilateral and 12 were bilateral abdominal cryptorchids. An incision was made over the superficial inguinal ring, and the extension of the gubernaculum testis was identified and grasped with forceps. Traction was applied to this structure until the vaginal process was everted beyond the superficial inguinal ring. The vaginal process was incised and the protruding structure, usually the epididymis, was grasped. The testis was drawn out by gentle traction on the epididymis, and castration was performed in the routine manner.


Simultaneous localization of LHRH and catecholamines in rat hypothalamus. The PAP unlabelled antibody enzyme method of Sternberger was used for the histochemical demonstration of LHRH and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) in the hypothalamus of the adult male and pregnant female rat. The sections for light and electron microscopy were serially treated with normal goat serum, LHRH antiserum and/or TH antiserum, goat anti-rabbit IgG, PAP complex and 3,3'-diaminobenzidine (DAB) or 4-Cl-1-naphtol. LHRH-positive cell bodies were discernible in the medial preoptic area. The LHRH-positive terminals were densely localized in the organum vasculosum of the lamina terminalis and in the perivascular region of the median eminence (PVME). Dopamine (DA)-positive cell groups (TH-positive perikarya) were discernible in the arcuate nucleus, and its terminals were densely localized in the PVME. The simultaneous identification of LHRH and DA in the distinctive neuronal system of the median eminence was possible with the PAP double staining technique, in which LHRH is revealed as a brown precipitate with DAB, and TH is revealed as a blue reaction product with naphtol. The LHRH neuronal system did not contain TH and vice versa. The ultrastructural study revealed that LHRH was localized in large vesicles with a diameter of 100 nm within the axon terminals, while TH was localized in the endoplasmic reticulum, the neurotubules and small vesicles with a diameter of 50 nm within the DA neuron. The axo-axonic contact of LHRH and DA terminals was demonstrated in close proximity to portal vessels, suggesting the synaptic influence of DA on the release of LHRH into these vessels.


Mammalian and microbial cell-free conversion of anthracycline antibiotics and analogs. Cell-free preparations of Streptomyces nogalater and rat liver catalyze reduced pyridine nucleotide dependent conversion of nogalamycin to 7-deoxynogalarol and nogalose (Scheme 1). The mammalian process requires TPNH and has a specific activity of 85 nmoles of 7-deoxynogalarol formed per hour per mg of protein while the bacterial process prefers DPNH and has a specific activity of 5. The oxygen-sensitive conversions have pH optima of 7.5 (rat) and 9 (S. nogalater). Other anthracycline substrates converted to their 7-deoxyaglycones by both systems include nogamycin, 7(R)-O-methylnogarol, 7(R)-O-methylnogalarol, doxorubicin (Adriamycin), steffimycin, and steffimycin B.


Enzymes of agmatine degradation and the control of their synthesis in Klebsiella aerogenes. The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.


Morphology-associated expression nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Mucorracemosus. The in vivo regulation of glutamate dehydrogenase (GDH) was studied in Mucor racemosus as a function of nutritional conditions and morphological state. Both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)-dependent GDH activities were found. The effect of carbon and nitrogen source on the specific activity of the NAD-dependent GDH suggests that its role is primarily catabolic. The NAD-dependent activity was generally an order of magnitude greater in mycelial cells than in yeast-phase cells grown on the same medium. During yeast-to-hyphal morphogenesis the increase in NAD-dependent activity preceded the appearance of hyphal cells both under aerobic and anaerobic conditions. Exogenous dibutyryl-cyclic AMP prevented the increase in NAD-dependent GDH concomitantly with the suppression of morphological differentiation. The NADP-dependent activity did not change appreciably during morphogenesis.


Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation. Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.


Relation between the adenylylation state of glutamine synthetase and the expression of other genes involved in nitrogen metabolism. We have partially characterized the biochemical parameters of glutamine synthetase from Klebsiella pneumoniae and have shown that the differential affinity of adenylylated and unadenylylated glutamine synthetase for adenosine diphosphate provides a convenient means of determining the adenylylation state. Using this assay procedure, we examined the relationship between the adenylylation state and the expression of other genes involved in nitrogen assimilation. We observed no correlation between the adenylylation state and the expression of histidase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, and urease in aerobic cultures.


NgoII, a restriction endonuclease from Neisseria gonorrhoeae. EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.


Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa. The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.


Derepression of nitrogenase activity in glutamine auxotrophs of Rhodopseudomonas capsulata. In contrast to wild-type cells, glutamine auxotrophs of the photosynthetic bacterium Rhodopseudomonas capsulata synthesize nitrogenase, produce H2 (catalyzed by nitrogenase), and continue to reduce dinitrogen to ammonia in the presence of exogenous NH4+. The glutamine synthetase activity of such mutants is less than 2% of that observed in the wild type. It appears that glutamine synthetase plays a significant role in regulation of nitrogenase synthesis in R. capsulata.


Combined use of strain construction and affinity chromatography in the rapid, high-yield purification of 6-phosphogluconate dehydrogenase from Escherichia coli. A rapid, high-yield method for purification of 6-phosphogluconate dehydrogenase from Escherichia coli K-12 is described. Sonic extracts prepared from heat-induced cultures of strain RW184, doubly lysogenic for the specialized transducing bacteriophage lambdacI857St68h80dgndhis and bearing a deletion of the gene for glucose 6-phosphate dehydrogenase, contained levels of 6-phosphogluconate dehydrogenase 15- to 20-fold higher than cultures of wild-type cells. Affinity chromatography on blue dextran-Sepharose with batchwise elution with 1 mM nicotinamide adenine dinucleotide phosphate affected a further 10-fold purification. Enzyme prepared in this manner was homogeneous according to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and immunoelectrophoresis using antiserum directed against it. Fructose 1,6-diphosphate is an inhibitor of enzyme activity.


Respiration-linked proton transport, changes in external pH, and membrane energization in cells of Escherichia coli. The kinetics of respiration-dependent proton efflux and membrane energization have been studied in intact cells of logarithmic-phase Escherichia coli. Parallel measurements of the rate and extent of proton efflux into the external medium (half-time, about 10 s; ratio of H(+) to O, about 0.5) and the oxidation of E. coli cytochrome b (half-time, </=1 s; about 6% oxidized) after a pulse of 5.5 ng-atoms of O indicate that the rate of proton efflux is at least 10 times slower than expected from the time required for the cells to reduce the oxygen added in the pulse. The kinetics of formation and dissipation of the transmembrane electric potential (deltapsi) after an O(2) pulse were estimated from changes in the fluorescence properties of the cell envelope-bound probe N-phenyl-1-naphthylamine. Under anaerobic conditions, a small pulse of oxygen induced a rapid (half-time, </=1 s) partial decrease in the fluorescence intensity of the probe, followed by a slower relaxation of the fluorescence change to the original intensity. The extent of the initial rapid decrease was linearly dependent upon the amount of oxygen added in the pulse (0 to 11 ng-atoms of O per pulse), whereas the rate of the subsequent relaxation was accelerated by the uncoupler p-trifluoromethoxycarbonylcyanidephenylhydrazone and the K(+) ionophore colicin E1. This suggests that the initial fluorescence decrease after an O(2) pulse reflects the energization of the membrane, whereas the relaxation of the fluorescence decrease reflects the subsequent deenergization of the membrane arising from counterion redistributions. The fact that the efflux of H(+) into the external medium after an O(2) pulse was inefficient and much slower (half-time, about 10 s) than the reduction of the added O(2) (half-time, </=1 s) and the energization of the membrane (half-time </=1 s) suggests that some of the protons translocated across the cytoplasmic membrane during a brief pulse of respiratory activity are accumulated in a region of the cell which is not in rapid equilibrium with the external medium.


Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. Nitrogen control in Salmonella typhimurium is not limited to glutamine synthetase but affects, in addition, transport systems for histidine, glutamine, lysine-arginine-ornithine, and glutamate-aspartate. Synthesis of both glutamine synthetase and transport proteins is elevated by limitation of nitrogen in the growth medium or as a result of nitrogen (N)-regulatory mutations. Increases in the amounts of these proteins were demonstrated by direct measurements of their activities, by immunological techniques, and by visual inspection of cell fractions after gel electrophoresis. The N-regulatory mutations are closely linked on the chromosome to the structural gene for glutamine synthetase, glnA: we discuss the possibility that they lie in a regulatory gene, glnR, which is distinct from glnA. Increases in amino acid transport in N-regulatory mutant strains were indicated by increased activity in direct transport assays, improved growth on substrates of the transport systems, and increased sensitivity to inhibitory analogs that are trnasported by these systems. Mutations to loss of function of individual transport components (hisJ, hisP, glnH, argT) were introduced into N-regulatory mutant strains to determine the roles of these components in the phenotype and transport behavior of the strains. The structural gene for the periplasmic glutamine-binding protein, glnH, was identified, as was a gene argT that probably encodes the structure of the lysine-arginine-ornithine-binding protein. Genes encoding the structures of the histidine- and glutamine-binding proteins are not linked to glnA or to each other by P22-mediated transduction; thus, nitrogen control is exerted on several unlinked genes.


Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro. Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 43 degrees C but was only slightly affected by variation of the pH between 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin E1 or colicin E3.


Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation. The efficiency of transformation of point mutations depends upon base pair mismatches during the recombination process. For low-efficiency markers, the genetic information carried on the donor deoxyribonucleic acid is preferentially lost. To understand this elimination process, we investigated the effect of the size of donor deoxyribonucleic acid on the relative efficiency of low-efficiency point mutations. The deoxyribonucleic acid was shortened either by mechanical shearing or by restriction enzyme treatments. The results indicate that transformation by low-efficiency markers was not affected by shortening the distance between them and the end of the molecule any more than was transformation by the other markers. Moreover, no lethal event could be detected for either cell or chromosomal marker survival. These data do not exclude the double-strand-break hypothesis that was proposed to explain the loss of genetic information for low-efficiency markers, but they offer no support for it.


Enzymology of butyrate formation by Butyrivibrio fibrisolvens. Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine and ovine rumen. The enzymology of butyrate formation from pyruvate was investigated in cell-free extracts of B. fibrisolvens D1. Pyruvate owas oxidized to acetylcoenzyme A (CoA) in the presence of CoA.SH and benzyl viologen or flavin nucleotides. The bacterium uses thiolase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, and crotonyl-CoA reductase to form butyryl-CoA from acetyl-CoA. Reduction of acetoacetyl-CoA to beta-hydroxybutyryl-CoA was faster with NADH than with NADPH. Crotonyl-CoA was reduced to butyryl-CoA by NADH, but not by NADPH, only in the presence of flavin nucleotides. Reduction of flavin nucleotides by NADH was much slower than the flavin-dependent reduction of crotonyl-CoA. This indicates that flavoproteins rather than free flavin participated in the reduction of crotonyl-CoA. Butyryl-CoA was converted to butyrate by phosphate butyryl transferase and butyrate kinase.


Uptake of circular deoxyribonucleic acid and mechanism of deoxyribonucleic acid transport in genetic transformation of Streptococcus pneumoniae. Deoxyribonucleic acid (DNA) from the covalently closed circular DNA molecules of Pseudomonas phage PM2 was found to enter normally transformable cells of Streptococcus pneumoniae as readily as linear bacterial DNA. In a mutant of S. pneumoniae that lacks a membrane nuclease and is defective in DNA entry, as many molecules of PM2 DNA as of linear DNA were bound on the outside of cells at equivalent DNA concentrations. Bound DNA suffered single-strand breaks, but circular DNA with preexisting breaks was bound no better than closed circles. In the presence of divalent cations, DNA bound to cells of a leaky nuclease mutant showed double-strand breaks. At least the majority of PM2 DNA that entered normal cells was single stranded. These results are consistent with a mechanism for DNA entry in which DNA is first nicked on binding, then a double-strand break is formed by cleavage of the complementary strand, and continued processive action of the membrane nuclease facilitates entry of the originally nicked strand. Although the bulk of circular donor DNA appeared to enter in this way, the results do not exclude entry of a small amount of donor DNA in an intact form.


Biochemistry of L-proline-triggered germination of Bacillus megaterium spores. The mechanism by which L-proline triggers germination in Bacillus megaterium QM B1551 spores was investigated. First, brief exposure of spores to L-proline, followed by dilution, was sufficient to trigger germination. Once germination was triggered, the spores continued initiation of germination and did not require high concentrations of L-proline. Triggering of germination was pH and temperature dependent. Second, enzymes for L-proline catabolism were absent in spores, and several non-metabolizable analogs of L-proline were effective trigger compounds. Third, triggering of germination occurred in the presence of inhibitors of proton motive force production, oxygen uptake, and metabolism. Fourth, uptake of L-proline occurred after the triggering of germination. These results argue that neither uptake nor metabolism of L-proline was necessary to trigger germination. Instead, L-proline probably causes a biophysical alteration in the spores that triggers the biochemical changes in germination.


Sensitivity of Escherichia coli to cephaloridine at different growth rates. Steady-state populations of Escherichia coli B/r were treated with cephaloridine at minimal inhibitory concentrations. The antibiotic sensitivity of the cells and the localization of spheroplast emergence along the cell surface were examined as a function of cell length and growth rate. In fast-growing populations (greater than 1 division per h) the sites of cephaloridine interaction occurred preferentially at the cell pole in the smaller cells and at the cell center in dividing cells. At decreasing growth rates the cells became more resistant to cephaloridine, and a gradual shift from the cell pole toward the cell center was observed for the sphere position. A similar growth rate-dependent change in localization was found for sucrose-induced plasmolysis vacuoles.


Deletion mapping of the polA-metB region of the Escherichia coli chromosome. A lambdacI857 prophage inserted into one of the genes of the rha locus was used to select deletions unambiguously ordering the markers polA-glnA-rha-pfkA-tpi-metBJF. Transduction with phage P1 indicates at least 70% linkage between glnA and polA. The order of the pfk and tpi markers is reversed from that previously published. Despite the relatively large distance separating the glnA and rha loci, deletions removing this entire region have no obvious phenotype. The isolation of Tn10 transposons integrated at different sites between rha and glnA greatly facilitated this work.


Activation of rat liver guanylate cyclase by proteolysis. Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.


Identification and partial purification of an ATP-stimulated alkaline protease in rat liver. Extracts from rat liver contain a sulfhydryl-dependent endoprotease which degrades [methyl-14C]globin or 125I-hemoglobin to acid-soluble peptides. This enzyme was isolated from the 100,000 x g supernatant of the homogenate. It showed a pH optimum between 7.5 and 9.5 and very little activity below pH 7.0. The enzyme has an apparent molecular weight of 550,000 as determined on Sepharose 6B column chromatography and sucrose density gradient centrifugation. ATP, at physiological concentrations, as well as pyrophosphate, stimulated the protease activity in these partially purified preparations up to 3-fold. Nonionic detergents such as Triton X-100 increased proteolytic activity and the stimulation by ATP. Other nucleotide triphosphates and ADP also increased proteolysis but less effectively than ATP. Sodium phosphate, creatine phosphate, and EDTA had no stimulatory effect.


A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney. Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.


Immunological studies on 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes. An apparently homogeneous preparation of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme from Escherichia coli was used as the antigen for antibody production in New Zealand white rabbits. The antibodies were monospecific as judged by immunodiffusion and immunoelectrophoresis. Antigen . antibody complexes maintained full enzyme activity and were inhibited by phenylalanine, indicating that neither the active site nor the feedback-inhibitor binding site is mechanistically connected to amino acid sequences which are antigenic determinants. While phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase could be quantitatively removed from solution by immunoprecipitation with soluble or immobilized antibodies, neither the tyrosine-sensitive nor the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, the other two isoenzymes catalyzing the first step in the biosynthesis of aromatic compounds, formed any detectable complexes with the antibodies. This indicated less structural similarity than would be expected for isoenzymes. Also, the antibodies did not cross-react with 5-dehydroquinate synthase, the enzyme catalyzing the second step of the common aromatic biosynthetic pathway.


The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer. A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.


Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding. The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.


The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria. Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.


Identification of the covalently bound flavin prosthetic group of cholesterol oxidase. Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.


Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases. Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. A method is described which enables one to assay simultaneously the NAD- and NADP-linked reactions of dehydrogenases which can utilize both coenzymes. The method is based on the fact that the thionicotinamide analogs of NADH and NADPH absorb light maximally at 400 nm, a wavelength sufficiently far removed from the absorbance maximum of NADH and NADPH to permit measurements of the simultaneous reduction of NAD+ (or NADP+) and the thionicotinamide analog of NADP+ (or NAD+). Application of the method to glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reveals differential effects of glucose 6-phosphate concentration on the NAD- and NADP-linked reactions catalyzed by this enzyme which can not be detected by conventional assay procedures and which may have regulatory significance.


Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase. The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.


Adsorption of human serum albumin to Amberlite XAD-7 resin. In the present study the conditions leading to tight binding of human serum albumin to the Amberlite XAD-7 resin without the use of chemical coupling agents have been defined. Optimal binding (10.97 mm/kg dry XAD-7) was achieved at pH 5.0 and adsorption conformed to a Langmuir isotherm. Theoretical analysis of the data suggest adsorption of a monolayer of albumin which is supported by the absence of visual surface coating on scanning electron micrographs. Binding of human serum albumin was reduced when two chemical coupling agents, glutaraldehyde and carbodiimide were included in the human serum albumin solution, the elution of adsorbed HSA from the resins under severe flow conditions was reduced, and the amount eluted was minimal in all instances.


Simple deep hypothermia for open-heart surgery. Two hundred fifty patients more than 2 years of age having correction of congenital heart diseases by simple deep hypothermia alone were investigated in respect to metabolic abnormalities, post-operative complications, intellectual development and postoperative EEGs. LOS in lethal complications was attributed to the difficulty of resuscitation, indicating the application of this method is ideal for patients less than 6 years in age or less than 20 kg in weight. No impairment of intellectual development was observed when compared IQ before the operation and at the time of long term follow-up in serial study, but electroencephalographic assessment indicated that postoperative abnormalities might occur more frequently than previously suspected. Conclusively, it would appear that hypothermic intracardiac surgery is a safe method, provided circulatory arrest time is not allowed to exceed a limited period and the procedure is reasonably performed having a good understanding in the pathophysiology of hypothermia.


Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events. A particulate fraction isolated from human neutrophils by homogenization, then centrifugation at 27,000 g, was demonstrated to generate chemiluminescence. This luminescence required the addition of reduced pyridine nucleotide and was very low in fractions from resting normal cells. Stimulation of neutrophils with opsonized zymosan, phorbol myristate acetate, or ionophore A23187 resulted in marked enhancement of the chemiluminescence measured in subsequently isolated particulate fractions. Stimulation did not boost the luminescence produced by fractions from cells of patients with chronic granulomatous disease. The chemiluminescence of particulate fractions from stimulated neutrophils was linear with increasing protein concentration, had a pH optimum of 7.0, and was higher with NADPH as substrate than with NADH. These results confirm previous studies suggesting that the enzyme system responsible for the respiratory burst in neutrophils is present in this fraction. The particulate fraction was used to examine the nature and origin of neutrophil luminescence by investigating the effect on this phenomenon of certain chemical and enzymatic scavengers of oxygen metabolites. Results suggest that the energy responsible for the luminescence of particulate fractions and, presumably, the intact cell, is derived from more than one oxygen species and that luminescence is a product of the interaction of these species and excitable substrates within the cell.


Plasma digoxin and magnesium concentration in open heart surgery. Plasma concentrations of various parameters were determined before, during and for 24 hours after cardiopulmonary bypass surgery in 12 patients. Digoxin levels decreased uniformly during perfusion in all cases. Plasma magnesium levels decreased significantly during pump and remained low for 24 hours. Potassium and calcium levels were kept in the high normal range as a result of exogenous administration of these ions. Small amounts of magnesium added to the circuit in 7 patients did not change the pattern of hypomagnesemia. More vigorous magnesium replacement during bypass surgery may be required if hypomagnesemia is to be avoided.


pH and bicarbonate effects on mitochondrial anion accumulation. Proposed mechanism for changes in renal metabolite levels in acute acid-base disturbances. Mitochondria from rabbit and dog renal cortex were incubated with 1 mM (14)C-weak acid anions in media containing low (10 mM) or high (40 mM) concentrations of bicarbonate and the steady-state accumulation of labeled anion in the matrix was measured. In the absence of an energy source, no concentration of (14)C-anion in the mitochondrial matrix space was present, but the anion concentration was significantly higher at low- than at high-bicarbonate concentration. Addition of an energy source, usually ascorbate plus tetramethyl-p-phenylenediamine, led to increases in matrix space anion levels and to accentuation of the difference in anion uptake between low- and high-bicarbonate media, so that two to four times as much anion was present at low- than at high-bicarbonate concentrations. The anions affected included substrates for which inner membrane carriers are present in mitochondria, such as citrate, alpha-ketoglutarate, malate, and glutamate, as well as substances which diffuse passively across the inner membrane such as acetate and formate. When a nonbicarbonate medium buffered with Hepes was used, pH change did not alter anion uptake although anion concentrations exceeding those in the medium still developed when an energy source was present. The difference in mitochondrial anion accumulation between low- and high-bicarbonate levels diminished with decreasing temperature or with increasing anion concentration in the medium. Estimation of intramitochondrial pH with [(14)C]5,5-dimethyl-oxazolidine-2,4-dione showed that the pH gradient across the inner mitochondrial membrane was significantly greater with 10 than with 40 mM bicarbonate in the medium.A hypothesis is described that relates this effect of pH and bicarbonate on mitochondrial anion accumulation to the very rapid changes in substrate levels in renal cortex, which develop when acute metabolic acidosis or alkalosis is produced in the intact animal. It is suggested that an abrupt fall in systemic pH and bicarbonate is associated with a shift in substrate in renal cortex out of the cytoplasm and into mitochondria, where some of the added substrate is metabolized. Reduction in the size of the cytoplasmic pool of substrate occurs with relatively little accompanying change in the size of the mitochondrial pool, thus causing a net reduction in the total tissue pool. This mechanism accounts for the reduction in tissue levels of many mitochondrial substrates observed acutely in metabolic acidosis. In metabolic alkalosis, reversal of these effects leads to expansion of the cytoplasmic pool, thereby resulting in the rise in tissue levels of substrates which occurs in this condition.


An interfering factor in the automated analysis of calcium. The widespread use of stainless steel for sample probes, connectors, and sample stream splitters in autoanalyser systems can lead to significant differences in the amount of calcium available for analysis. At alkaline pH calcium can be adsorbed onto the metallic components of the sampling system, and under more acidic conditions calcium can be released. Thus an alkaline sample will have an apparently reduced calcium content, while a more acidic sample may have an apparently increased concentration of calcium. By using non-metallic components in the sampling system this source of error an be eliminated.


Effect of pH on sporicidal and microbicidal activity of buffered mixtures of alcohol and sodium hypochlorite. The effect of pH on the activity of buffered sodium hypochlorite solution, and a buffered methanol/sodium hypochlorite mixture, against Bacillus subtilis spores was investigated. The best results, considering both sporicidal activity and stability, were achieved in the pH range 7.6--8.1. The sporicidal activity and stability of five alcohol/hypochlorite mixtures, each containing a different alcohol and buffered to pH 7.6 and of hypochlorite alone buffered to pH 7.6, were compared. The mixtures were marginally more sporicidal than hypochlorite alone when fresh but were much less stable. An unbuffered methanol/hypochlorite mixture, a methanol/hypochlorite mixture buffered to pH 7.6, and hypochlorite alond buffered to pH 7.6 were all found to be effective against six vegetative organisms and spores of B. subtilis and Clostridium sporogenes. By buffering alcohol/hypochlorite mixtures or hypochlorite solution alone in the pH range 7.6--8.1, high sporicidal activity can be achieved with low concentrations of alcohol and hypochlorite. Such formulations show promise for the disinfection of heat-sensitive medical equipment.


Desensitization of beta receptor mediated cyclic AMP response of cultured fibroblasts by partial agonists. Two novel beta-adrenergic myocardial stimulants of general structure: Formula: (see text), where R is a phenyl or benzyl group were investigated for their ability to stimulate and desensitize the cyclic AMP response of C6 glioma cells. Compound ICI 89, 963 (R:phenyl) which elicited less than 1% of the maximum increase in cAMP produced by isoproterenol, was strikingly effective as a desensitizer of the isoproterenol response. This desensitization was markedly reduced by propranolol. Compound ICI 119,033 (R: benzyl) which was a more effective stimulant of cAMP synthesis than ICI 89,963, was also a more effective desensitizer of the isoproterenol response of C6 cells. The kinetics of the desensitization by ICI 89,963 were comparable with those for isoproterenol reaching a maximum in 2 to 3 hours. The data indicate that beta-adrenergic agonists are more potent as desensitizers of the cyclic AMP response than as stimulants of that response.


Effect of discussion seminars on the attitudes of dental students to clinical periodontal problems. The effect of discussion seminars on the attitudes toward clinical problems within four affective domains in periodontics was studied in undergraduate dental students. Specially prepared case presentations which highlighted clinical problems were used in the seminars as well as for the evaluation. The tests were based upon the assumption that the viewpoints of the periodontal teachers are the correct ones and can be used to identify the affective objectives. The results of the present study indicate that the students' attitudes changed and became similar to the viewpoints of the faculty as an effect of the discussion seminars. This observation speaks in favor of the use of such seminars for effective teaching as a supplement to didactic teaching and clinical instruction.


Controlled fall in natremia and risk of seizures in hypertonic dehydration. The aim of the study was to derive some practical measurements which might help in defining a "safe" infusion rate in order to avoid seizures during treatment of hypernatremic dehydration. Forty seven infants with hypernatremic dehydration were rehydrated on a 160 ml/kg/24 h basis: 9 developed seizures during treatment (group I), 22 matched for age did not convulse (group II). Nine subsequent cases were prescribed a 120 ml/kg/24 h regimen: none convulsed (group III). The three groups were comparable in many respects, including initial plasma Na and pH. Fluids were comparable regarding (Na), their rates of administration were respectively 216, 181 and 123 ml/kg/24 h. The rate of infusion affected slopes of decreases in natremia. It was suggested that the decrease in plasma Na should not exceed 0,5 mEq/1/h.


Effect of the sweet-tasting protein monellin on growth of a cariogenic bacterium: a microbiological study. Monellin is a protein with an intensely sweet taste and could be considered as a potential alternative sweetener to sucrose. The effect of monellin on the in vitro growth of the cariogenic microorganism Streptococcus mutans was investigated.


Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure. We have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.


Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies. Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.


Immobilization of neuraminidase and its potential application in the modification of cell surfaces. Vibrio cholerae neuraminidase was immobilized on the inside of 1.0 mm inner diameter nylon tubing with retention of enzyme activity, when assayed at 37 degrees C and pH 5.5 with mucin as substrate. The stabilities of the immobilized and soluble enzymes were similar for up to 3 hr at 37 degrees C. Preliminary data indicated that immobilized neuraminidase will release sialic acid from the surface of leukemic AKR mouse thymus and spleen lymphocytes; however, the level of immobilized enzyme activity needs to be increased for practical applications. With this improvement immobilized neuraminidase could become a novel preparation for carrying out cell surface modifications with minimal enzyme contamination of the cell.


Intracellular pH-determination by fluorescence measurements. A method was developed to determine the intracellular pH (pHi) of individual cells by use of fluorescence measurements. The method is based on the observation that the fluorescence excitation spectrum of fluorescein is pH-dependent. Fluorescence excitation spectra from individual rat bone marrow cells treated with fluorescein diacetate (FDA) were compared with those of fluorescein solutions of known pH values. Cells which were suspended in media of pH between 4.0 and 8.1 with high to normal buffering capacities had pHi values equal to those of the media. Cells suspended in media with low buffering capacities maintained a pH,i of 6.7 +/- 0.2. Preliminary results indicated that the pHi of individual cells may also be determined by using flow cytometry.


Etiology and antimicrobial therapy of acute maxillary sinusitis. Eighty-one adults with symptoms of acute sinusitis were studied by direct needle puncture and aspiration of the maxillary sinus (105 sinuses). Fifty-nine bacterial strains were isolated in titers of greater than or equal to 10(4) colony-forming units/ml; Streptococcus pneumoniae and Haemophilus influenzae accounted for 64% of the isolates. Other bacteria recovered included anaerobes (12%), Neisseria species (8.5%). Streptococcus pyogenes (3%), alpha-hemolytic Streptococcus (3%), non-group A beta-hemolytic Streptococcus (3%), Staphylococcus aureus (2%), Pseudomonas aeruginosa (2%), and Escherichia coli (2%). Viruses were isolated from 11 sinuses; these isolates included rhinovirus (six), influenza A (H3N2) virus (three), and two types of parainfluenza virus (one each). The efficacy of therapy with orally administered ampicillin, amoxicillin, or trimethoprim-sulfamethoxazole was evaluated by a repeat sinus puncture and culture. Clinical and bacteriologic responses to all three regimens were good.


Factors causing the clumping reaction of streptococcal strains with human plasma. Fresh isolates of 204 strains of Streptococcus haemolyticus, 75 strains of viridans group Streptococcus, and 45 strains of Streptococcus pneumoniae were studied for their clumping reactions with human plasma. The plasma and serum factors that clumped the streptococcal strains were compared with those that clumped a Staphylococcus aureus strain. One hundred eleven strains of S. haemolyticus, 10 strains of viridans group Streptococcus, and none of the strains of S. pneumoniae tested were clumped by human plasma. However, clumping activity was remarkably unstable after subculturing on plates containing Todd-Hewitt blood agar. The strain with the highest level of activity was clumped by fibrinogen and normal human serum. Results of tests of the clumping reactions of staphylococci and streptococci with human serum indicated that the serum factors responsible for those reactions may be identical.


Rapid diagnosis of meningitis with use of selected clinical data and gas-liquid chromatographic determination of lactate concentration in cerebrospinal fluid. The usefulness of determination of lactate concentration in cerebrospinal fluid (CSF) for differentiation between various types of meningitis was evaluated. Lactate concentration in the CSF was assayed by gas-liquid chromatography for 115 patients, 17 of whom had serous meningitis and 38 had bacterial meningitis. The mean lactate concentration in the CSF of patients with bacterial meningitis was significantly higher than in the CSF of patients with serous meningitis and in a control group. The mean concentration in patients with serous meningitis was significantly higher than in controls. The highest lactate level in serous meningitis overlapped with the lowest level in bacterial meningitis. Elevated lactate concentrations in CSF were found also in patients with noninfectious disorders of the central nervous system. Misleading results may therefore be obtained if the lactate concentration in CSF alone is used to distinguish between serous and bacterial meningitis. The study suggests, however, that measurements of lactate levels in CSF, when combined with clinical and conventional laboratory observations, can increase the reliability of rapid diagnosis of bacterial meningitis.


Type I interferon induced in mice by infection with Trypanosoma equiperdum. Type II interferon is known to be produced in mice infected with Trypanosoma cruzi. Therefore, interferon production was investigated in mice infected with Trypanosoma equiperdum. Interferon was found in the sera of mice infected with T. equiperdum from day 1 after infection until death on day 5. The maximal level of interferon appeared before parasitemia was detected, a finding showing that the endogenous interferon probably had no significant influence on the course of infection. Unexpectedly, the serum interferon was characterized as type I by its stability at pH 2 and by its neutralization by antiserum to virus-induced mouse interferon. The production of two different types of interferon in mice infected with T. cruzi and T. equiperdum may have been due to the dissimilar life cycles of the two trypanosomes in the white mouse.


Characterization and determination of the activity of biliary beta-glucuronidase in rats. beta-Glucuronidase activity determined in 100 diluted bile samples from 12 rats with bile duct fistula by using phenolphthalein glucuronide as substrate incubated at 56 degrees C and pH 6 was 636 +/- 650 (mean +/- S.D.) modified Sigma units/ml. The enzyme had an optimal pH of 6.0 and was inhibited slightly by cholate by markedly by chenodeoxycholate and deoxycholate. The biliary beta-glucuronidase had, thus, low activity under normal physiologic condition because of the high pH (8.1) and high bile salt content (20 mumoles/ml) of the bile. The enzyme kinetic studies revealed that the direct bilirubin was a competitive inhibitor to phonolphthalein glucuronide for the enzyme. The affinity of the former to the enzyme was 163 times that of the latter. The studies provided a method for measuring the true activity of biliary beta-glucuronidase (Vmax) devoid of interfering factors by measuring the enzyme velocity (v) in the diluted bile with at least five different concentrations of substrate (s). The plotting of (1/v) vs. (1/s) should yield the y intercept or (1/Vmax).


Interaction of amiloride and lithium on distal urinary acidification. The interaction of amiloride and LiCl administration on renal HCO3 handling was studied in hydropenic rats. Amiloride administration resulted in a significant increase in Na, Cl, and HCO3 excretion, whereas K excretion decreased significantly. LiCl administration resulted in a significant increase in Na, Cl, K, and HCO3 excretion. LiCl administration to animals receiving amiloride led to a significant increase in HCO3 excretion but failed to cause an increase in Na or K excretion. Addition of amiloride to animals receiving LiCl resulted in a significant increase in Na and HCO3 excretion. The net increase in fractional HCO3 excretion seen in this group was greater than that seen in all other groups. The finding that the net increase in FEHCO3 was greater in animals receiving amiloride after administration of LiCl than in animals receiving LiCl after amiloride administration indicates that amiloride blunted the effect of LiCl on HCO3 excretion. Administration of amiloride to both normal rats and to rats infused with Li during HCO3 administration resulted in a significant decrease in U-B Pco2 which could not be explained by the decrease in urine HCO3 concentration. These data demonstrate that amiloride inhibits distal acidification in vivo. LiCl administration also resulted in a decrease in U-B Pco2 which could be explained by the decrease in urine HCO3 concentration. LiCl administration also resulted in a decrease in TcH2O which could be prevented by prior administration of amiloride. These data indicate that amiloride blunts the effect of LiCl on urinary acidification, an effect similar to that observed on urinary concentration. These data suggest that the effect of Li on urinary acidification is in part dependent on Li entry into the cell.


[A simple gas chromatographic method for the determination of hypnotics in the serum (author's transl)]. In the extract of the serum, prepared as described by Külpmann (1979) (this J. 17, 89-96), other hypnotics, in addition to the barbiturates can be determined by gas chromatography; these are: carbromal, 2,2-diethylallylacetamide, ethinamate, glutethimide, methyprylone and pyrithyldione. Methaqualone can be detected qualitatively. The coefficient of variation for the precision in the series is dependent on the hypnotic investigated and ranges from 2.1 to 8.5%, the recovery from 76 to 92%; the detection limit is estimated to be 1.6 up to 4.6 mumol/l. The specificity was proved by comparison 1) of analyses of sera from poisoned patients or animals before and after the additional purification of the extract by thin-layer chromatography, 2) with the retention times of about 100 drugs under the gas chromatographic conditions that were used. The method allows the determination of 18 barbiturates and 7 non-barbiturates within one to two hours.


The effect of thioacetamide on urea cycle enzymes of rat liver. The urea cycle enzymes, carbamoyl-P-synthetase, ornithine transcarbamylase, arginase and other enzymes related to ammonia metabolism, such as glutamate dehydrogenase, glutamine synthetase and alanine and aspartate aminotransferases,have been studied in thioacetamide-induced liver disease in rats. Urea and ammonia were determined both in serum and in liver extracts. Glutamate and aspartate were determined in liver extracts. There was a marked decrease (in brackets: fraction of control) in carbamoyl-P-synthetase (0.23), ornithine transcarbamylase (0.36) and arginase (0.62). The accumulation of ammonia (3.22) and the decreased urea level (0.80) are well known indications of liver failure. Glutamate dehydrogenase and glutamine synthetase increased respectively to 1.50 and 1.33, and the changes in glutamate and aspartate levels were respectively 1.68 and 0.92; this indicates that the metabolic route: 2-oxoglutarate leads to glutamate leads to glutamine is increased, and thereby compensates for the low rate of urea formation. Aminotransferase activities were respectively 0.43 and 0.25. No significant differences were found in serum aminotransferases, or in the concentrations of ammonia and urea.


Dopaminergic neuronal responses to a non-amphetamine CNS stimulant. The present study compares the effects of d-amphetamine (d-AMP) and the potent non-amphetamine CNS stimulant, amfonelic acid (AFA), on the firing rate of single midbrain dopaminergic (DA) neurons and on neostriatal DA metabolism (dihydroxyphenylacetic acid--DOPAC). The results indicate that AFA, like d-AMP, reduces the firing rate of DA neurons, although unlike d-AMP, AFA does not cause a decrease in neostriatal DOPAC content and, in fact, enhances that produced by haloperidol (HALO). The AFA-induced decrease in firing rate, like d-AMP, is reversed by the DA receptor blocker HALO, but again unlike d-AMP, the decrease in firing rate is not prevented by catecholamine synthesis inhibition with alpha-methyl-para-tyrosine. Thus, both amphetamine and amfonelic acid have identical electrophysiological effects on DA neurons but act by different mechanisms.


Long-term haloperidol-treatment of mice: a change in beta-adrenergic receptor responsiveness. Mice administered haloperidol 3 mg/kg/day in their drinking water for 21 days were tested for their locomotor responsiveness to saline or acid vehicle, dl-, l- or d-propranolol, metoprolol, butoxamine or practolol. Haloperidol-treated animals administered saline or acid-vehicle were, in five of six experiments, more active than animals withdrawn from vehicle-treatment. Haloperidol- and vehicle-treated animals responded differently to the non-selective beta-adrenoreceptor antagonists (dl-propranolol and l-propranolol) and selective beta1-adrenoreceptor antagonists (practolol and metoprolol), but not to a selective beta2-adrenoreceptor antagonist (butoxamine). With dl-propranolol (4 mg/kg) the locomotor activity of haloperidol-treated animals was significantly (0.01 less than P less than 0.02) greater than that of the vehicle-treated animals. Similar effects in the same direction were seen with l-propranolol (1 mg/kg, 0.005 less than P less than 0.01), practolol (10 and 100 mg/kg, 0.025 less than P less than 0.05 and 0.01 less than P less than 0.025 respectively) and metoprolol 8 mg/kg, 0.005 less than P less than 0.01). The d-isomer of propranolol which is about 50 times less active as a beta-adrenoreceptor antagonist than the l-isomer, although having equal membrane stabilizing effects, did not differentially affect haloperidol- or vehicle-treated groups. The results suggest that there has been a change in beta 1-adrenoreceptor responsiveness in animals withdrawn from long-term haloperidol treatment.


Effects of magnesium and fluoride on ion exchange and acid resistance of enamel. Labial surfaces of bovine incisor enamel, after weak acid demineralization, were exposed for 24 h in solutions that contained trace levels of calcium as 45Ca, 0.4 mM NaF, and 1.0 mM MgCl2 at pH 7.0. The solutions approached saturation with apatites in the absence of NaF, and saturation with fluorapatites in the presence of NaF. NaF greatly increased the exchange of 45Ca. MgCl2 decreased 45Ca exchange, but had no effect on F- exchange in the surface minerals. MgCl2 decreased, while NaF increased the acid resistance of the exchanged surface minerals on later exposure to 10 mM acetic acid at pH 4.5. These results indicated that magnesium in oral fluids and tooth minerals may promote caries.


Mechanism of chloride corrosion of dental amalgam. Polarization of conventional dental amalgam in Ringer's solution produces a dissolution of the gamma 2 phase at--250 mV (SCE) and formation of Sn-Cl and Sn-O-Cl. This study was undertaken to determine the electrochemical nature of these reactions. Potentiostatic polarization scans were run at 4, 37 and 50 degrees C in several dilutions of aerated Ringer's solution. Manipulation of reactant concentrations and electrolyte temperature produced shifts in the potentiostatic profiles. At 50 degrees C, a gamma 2 dissolution peak shift to -300 mV was observed, and at 4 degrees C the peak shifted to -200 mV. Dilution of Ringer's solution with respect to [Cl-] over three orders of magnitude produced a linear shift in the noble direction of 500 mV. Cathodic polarization appears to be limited by the oxygen half cell reaction. Changes of [Cl] affect the shape of the peak, round out its approach to the current maximum. Shifts in peak potential due to temperature and concentration changes agreed with calculations based on critical pitting potential for gamma2 dissolution due to a chloride reaction. Polarization profiles in pooled saliva agreed with calculated potentials based on known saliva [Cl-].


Fluorometric determination of clobazam, a 1,5-benzodiazepine, in human plasma. A fluorometric procedure for clobazam, a 1,5-benzodiazepine, based on a fluorophore formed upon irradiation of the drug using short wavelength UV light (254 nm) for 35 min is presented. Fluorescence is linear over a 100-6400-ng/ml range using excitation and emission wavelengths of 350 and 400 nm, respectively. Application of the method to the determination of clobazam in spiked human plasma samples revealed that the drug can be determined at nanogram per milliliter levels with an accuracy of 1-5%. The procedure is specific for clobazam in samples containing its major plasma metabolite, N-desmethylclobazam, and also in samples containing 1,4-benzodiazepines and other selected drugs. A plasma level-time profile after oral administration of a single 40-mg dose of clobazam to a healthy adult male is also illustrated.


GLC analysis of theophylline, hydroxyethyltheophylline, and diphenylpyraline hydrochloride syrup. A GLC method for determining theophylline, hydroxyethyltheophylline, and diphenylpyraline hydrochloride is presented. The method permits the separation and quantitative determination of the therapeutically active ingredients with a single injection in an overall time of approximately 2.5 hr.


Determination of oxycodone in plasma and identification of a major metabolite. An electron-capture GLC method is described for oxycodone and its major metabolite, noroxycodone, in plasma and urine. The method involves extraction of the two substances into benzene-isopropranol at pH 10.4, followed by back-extraction into 0.1 N HCl. The acid phase is washed with hexane and made alkaline prior to reextraction into benzene-isopropanol. The solvent is removed by evaporation, and the heptafluorobutyryl derivatives of the test substances are formed. After removal of excess reagent, oxycodone and noroxycodone are quantitated by GLC. The characteristics of both substances, with respect to plasma levels in dogs and analgesic activity in mice, are reported. Isolation of noroxycodone from human urine and its identification by TLC, GLC, and mass spectrometry are described.


Renal handling of zomepirac sodium (McN-2783-21-98) in the rat. The plasma and renal clearance of zomepirac, a weak organic acid, was investigated in anesthetized CR Wistar rats after administration of single bolus i.v. injections or continuous i.v. infusions of the 14C-labeled compound. Adjustment of urine pH to the alkaline range caused more than an 8-fold lowering of the plasma elimination half-life (from 7.0 to 0.8 hr) and enhanced renal clearance by a factor of 53 compared to control. Acidification of the urine or probenecid administration increased the elimination half-life (to 10.9 and 17.5 hr, respectively), and decreased renal and plasma clearance of zomepirac. Since zomepirac is highly bound to plasma proteins (approximately 98%), only a small fraction of the drug is available for filtration at the glomerulus. Therefore, the renal elimination of zomepirac is accomplished mainly by active tubular secretion. Passive nonionic reabsorption is a major factor in determining the net clearance of the drug.


Acute and chronic methadone exposure in adult rats: studies on arterial blood gas concentrations and pH. The effects of different dosages of methadone on respiration were determined by evaluating arterial blood pCO2, pO2 and pH in naive and opioid-addicted animals. Male Sprague-Dawley rats were treated (i.p.), acutely or chronically, with either 2.5, 5.0 or 7.5 mg/kg of dl-methadone hydrochloride; appropriate saline controls were utilized. Blood was sampled from the tail artery before injection and 15, 30, 60, 120, 180 and 240 min postinjection. Animals exposed to methadone in acute experiments exhibited a respiratory depression that involved hypoxemia, hypercapnia and/or acidosis. In addition, the magnitude of this respiratory depressant action was dose-dependent and reached a maximal point 15 to 30 min after drug administration. Rats receiving chronic methadone exposure showed few alterations from control blood gas concentrations and pH. This study demonstrates that acute methadone administration is associated with respiratory depression, with the extent of reductions in pCO2, pO2 and pH related to drug dosage. In addition, chronic methadone treatment confers a substantial tolerance to the respiratory depressant action of methadone.


An analysis of the inhibitory post-synaptic current in the voltage-clamped crayfish muscle. 1. Inhibitory post-synaptic currents (i.p.c.s) were recorded from the feed-back current through a wire electrode inserted longitudinally into the opener muscle fibre of the claw in the crayfish (Cambarus clarkii). 2. I.p.s.c. rose to its peak in about 3-4 msec and decayed approximately exponentially. The decay time constant at -100 mV was 9.4 msec. 3. The decay time constant decreased as the membrane was hyperpolarized and increased during depolarization. The time constant (tau) depends on voltage (V) according to the relation tau = a exp (AV), with a = 18.6 msec and A = 0.0065 mV-1. Voltage dependence was opposite in direction to that seen at frog end-plates, but in the same direction as that of e.p.s.c. in crayfish muscle. 4. At lower temperatures, the rise and fall times of i.p.s.c.s were prolonged. Q10 for the decay time constant was 2.4 between 22.6 and 12.5 degrees C. 5. When pH was decreased from 7.2 to 5.5, the decay time constant increased by about 50%, with little change in the voltage dependence of the time course. 6. When chloride in the solution was changed to iodide, the decay time constant was increased by a factor of 3, while voltage dependence of the time course was not changed. In bromide solution the decay time constant increased by about 50%. 7. Peak amplitudes of i.p.s.c.s were approximately linear as the membrane was depolarized, but they levelled off as the membrane was hyperpolarized beyond reversal potential. The non-linear I-V relation did not result from inadequate voltage clamping, nor from a change in the inside concentration of chloride. After equilibration with iodide solution the I-V relation was approximately linear. 8. The decay time constant was increased after repetitive nerve stimulation. This prolongation became more pronounced at lower temperatures. 9. The kinetic process of the transmitter action is discussed. It is suggested that the rate limiting process for i.p.s.c. is binding and unbinding of the transmitter to the receptor.


Polyarteritis nodosa masquerading as temporal arteritis. The unusual occurrence of polyarteritis nodosa presenting as non-giant cell temporal arteritis and a clinical picture suggestive of acute cholecystitis is reported.


Pathophysiologic effects distal to a tourniquet in the dog. Intramuscular tissue pO2, pCO2, and pH were monitored distal to a pneumatic tourniquet in a dog hindleg preparation. A severe state of tissue hypoxia, hypercarbia, and acidosis was quantitated. The recovery time for tissue gases following release increased with increasing tourniquet time. Elevated c.p.k. and lactic acid values were noted at 2 hours of ischemia, reflecting the muscle changes at that time. Histologic sections revealed early signs of degeneration by 1 hour which progressively increased with increased tourniquet ischemia. On the basis of this study, we conclude that ischemia should not exceed 1 to 1 1/2 hours if significant pathophysiologic tissue changes are to be avoided. If longer time is required, intermittent release of the tourniquet for 10 minutes at the end of each  hour of inflation will avoid complications.


Germinal cell tumors of the testis after orchiopexy. A review of the literature revealed 220 cases of germinal cell tumor of the testis that has occurred after orchiopexy. Of these cases 97 were accurately documented in regard to scrotal placement of the undescended testis and the subsequent tumor. Only 6 cases were reported after orchiopexy in children less than 10 years old. Since the number of patients at risk is unknown the possible protection of early orchiopexy against subsequent malignant degeneration cannot be established. The small number of cases reported after orchiopexy in children less than 10 years old is encouraging. The accumulated reports of germinal cell tumors of the testes after orchiopexy in patients between 10 and 20 years old militate against such a procedure in patients with a unilateral underscended testis.