The blockade of serotonin uptake into synaptosomes:relationship to an interaction with monoamine oxidase inhibitors. To test the hypothesis that the hyperpyrexia produced by meperidine and detromethorphan in rabbits pretreated with a monoamine oxidase inhibitor is related to inhibition of neuronal uptake of serotonin (5-hydroxytryptamine (5-HT)), fluoxetine (Lilly 110140) was studied. This potent and specific 5-HT neuronal uptake blocker was administered to phenelzine-pretreated rabbits and found to produce a lethal hyperpyrexia in doses equal to or greater than 2.5 mg/kg. The order of potency in blocking 5-[14C]HT uptake into synaptosomes prepared from rabbits was: fluoxetine greater than meperidine = dextromethorphan = levorphanol greater than anileridine greater than alphaprodine greater than morphine. Since fluoxetine, meperidine, and dextromethorphan produce hyperpyrexia in phenelzine-pretreated rabbits, whereas anileridine, alphaprodine, and morphine do not, there appears to be some correlation between the hyperpyrexic response and inhibition of 5-HT uptake. The exception is levorphanol, which is not hyperpyrexic despite being equipotent with meperidine and dextromethorphan in inhibiting 5-HT uptake. The ineffectiveness of levorphanol in producing hyperpyrexia may be due to its marked depressant properties, since the addition of another depressant drug (pentobarbital) antagonized the hyperpyrexic effect of meperidine.


Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum. Three, two major and one minor, distinct RNA polymerases have been isolated and partially purified from heterotrophically grown Rhodospirillum rubrum, a facultative photosynethetic bacterium. Associated with each of these three enzymes is a distinct polyadenylic acid polyemrase. All of these enzyme activities are dependent on DNA templates and are resistant to rifampicin and streptovaricin. The structural subunit composition, the response to various chemical compounds and DNA templates, and the properties of the products of these enzymes are studied in detail and compared with those of similar enzyme activities from other bacterial systems. Several unique features have been observed in the R. rubrum enzyme systems, such as an uneven incorporation of purine and pyrimidine nucleotides by the RNA polymerases, and the presence of a lag period in the polyadenylic acid polymerase activities.


Preenrichment procedure for detection of Salmonella in gelatin. Trypticase soy broth was superior to nutrient and lactose broths as a preenrichment medium for the detection of Salmonella in artificially and naturally contaiminated gelatin. The detection rate for Salmonella were further enhanced when homogenization of the gelatin-broth mixture was accomplished by the use of gelatinase rather than by heating of 45 degrees C. Detection rats were also increased by adjusting the pH of the gelatin-broth mixture of 7.0, optimum pH for gelatinase (EC 3.4.23.2) activity.


The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul. Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.


Clostridium perfringens--specific lysin. A mitomycin C induced lysate of Clostridium perfringens strain KZ219 was lytic to 50 strains of C. perfringens of types A-E, and three strains of C. plagarum. The lysin was active against only 2 out of 87 strains of 51 other clostridial species. The optimum pH of the lytic agent was 5.5. The activity was largely inactivated by proteolytic enzymes, and nearly completely inactivated by heating at 60 degrees C for 5 min.


Effects of the herbicides fluometuron and prometryn of Rhizoctonia solani in soil cultures. Responses of Rhizoctonia solani to herbicides in soil cultures were assessed by measuring soil enzyme activity and other growth-related factors. Both beta-galactosidase (EC 3.2.1.23) and phosphatase (EC 3.1.3.1.3.1.3.2) activities were highly correlated with amounts of mycelium in soil. Both enzyme activities were reduced significantly by either fluometuron or prometryn at 40 microgram/g of soil; the pathogen was more distinctly suppressed by fluometron and showed a stronger tendency to overcome the effects of prometryn with time. Inhibition was also reflected in reduced ultilization of glucose and less CO2-C evolved. Except for an increase in beta-galactosidase activity in the presence of 1 microgram fluometuron, low levels of either herbicide had little effect on the pathogen.


Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus. Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was  tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for  protecting against inactivation of cold- and heat-injured cells.


Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae. The antimetabolite sulfanilamide inhibits sporulation in Saccharomyces cerevisiae strain AP1. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the drug, although accumulation of sulfanilamide by the cells increases with time. Vegetative growth of AP1 is practically unaffected by sulfanilamide; pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can occur in cells exposed to the drug for greater than 24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the sporulation process when returnedto vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is approximately 90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected.


Enzymatic hydrolysis of agar: purification and characterization of beta-neoagarotetraose hydrolase from Pseudomonas atlantica. Agarose is degraded by a beta-agarase from Pseudomonas atlantica to neoagarooligosaccharides of degree of polymerization (DP), 4, 6, 8, and 10. A beta-neoagarotetraose hydrolase cleaves the central beta-linkage in neoagarotetraose and the beta-linkage near the nonreducing end in neoagarohexaose and -octaose to yield neoagarobiose. The beta-neoagarotetraose hydrolase was localized on or outside the cytoplasmic membrane, in the cell wall region. The enzyme was activated by NaCl, KCl, CaCl2, MnCl2, and MgSO4, has a Km of 3.4 X 10(-3) M for neoagarotetraose, was free from beta-agarase and alpha-neoagarobiose hydrolase activity, and showed no transglycosidic activity.


Properties of 1-phosphofructokinase from Pseudomonas putida. The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis-Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 X 10(-4) M and 4.08 X 10(-4) M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3',5'-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.


Concentration of enteroviruses from large volumes of turbid estuary water. A method is described for the efficient concentration of viruses from large volumes of highly turbid estuary water. Virus in acidified seawater in the presence of aluminum chloride is adsorbed to a 10-in. (about 25.4 cm) fibreglass depth cartridge and 2- and 0.65-micron epoxy-fibreglass filters in series. This filter series is capable of efficiently adsorbing enteroviruses from 50 U.S. gallons (about 190) of estuary water of varying salinity and turbidity. Adsorbed viruses were eluted from the filters with glycine buffer (pH 11.5) and the eluate reconcentrated by using a precipitate formed by the addition of ferric chloride. Viruses were eluted from this precipitate with fetal calf serum. Using this procedure, four different enteroviruses in 50 gallons (about 190) of estuary water were concentrated 9 000- to 12 000-fold with an overall efficiency of 41%.


Isolation and characterization of a neuroblastoma cell line from peripheral blood in a patient with disseminated disease. Circulation in the blood stream of neuroblastoma cells was confirmed by establishment of a cell line from the peripheral blood of a child with disseminated disease. The morphologic, enzymatic, and chromosomal pattern of this cell line was similar to a cell line established from the primary tumor on a previous occasion. The peripheral blood smear did not demonstrate tumor cells but increased numbers of atypical monocytes; lymphoblasts were evident, which may have been unrecognized neuroblasts.


Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart. In an attempt to determine whether a subcellular compartmentation of creatine kinase exists and if so, whether there is a different distribution of the 3 isoenzymes of CK in the cell, studies were carried out with the guinea pig heart which had been subfractionated by either isopycnic density gradient centrifugation or differential pelleting. Isoenzyme analysis of CK in the isolated subcellular fractions by electrophoresis on agarose gels, revealed that the MM isoenzyme occurred in the cytosol, myofibrils, and the microsomes while the MB isoenzyme (which is cardio-specific) was only found in the cytosol. Trace amounts of the BB isoenzyme were detected in the cytosol. Considerable CK activity was associated with the mitochondria, this did not represent the MM, the MB, or the BB isoenzymes but was a distinct and additional mitochondrial-specific form of CK. PH optima and kinetic studies were carried out to characterise and distinguish the mitochondrial isoenzyme from other CK isoenzyme activity. The evidence for a differential compartmentation of MM, MB, BB, and mitochondrial CK is discussed in relation to their possible cellular roles.


Some new approaches to potential test systems for drugs against prostatic cancer. We have previously reported on test systems, based on 5alpha-reductase (5alpha-RA) and arginase activities and steroid deposition in animal prostates, potentially useful in screening drugs possibly effective in cancer of the prostate. Recently, we have concentrated on the development of other in vivo and in vitro systems which may prove of further value in testing such drugs. These systems include the following: (a) The effects of drugs on 5alpha-RA activity in human and animal non-malignant and human cancerous prostatic tissues in organ culture. The parameters necessary for the maintenance of optimal 5alpha-RA in such explants have been determined, and it has been shown that certain agents (estramustine phosphate, progesterone, estradiol-17beta) can inhibit 5alpha-RA under in vitro conditions, pointing to the potential use of such an approach in screening various cytostatic agents. In addition, 65Zn deposition, the histology, and the androgen metabolism in such tissues in organ culture are being determined as additional parameters. (b) The deposition of 65Zn in the rat dorsolateral gland, particularly as affected by prolactin and testosterone, and the effects of chemotherapeutic agents on such deposition. Methotrexate and CCNU have been shown to be potent inhibitors of 65Zn deposition in the dorsolateral gland. The parameters related to zinc metabolism in the prostate are being further investigated. (c) The demonstration of receptors for estrogens (estradiol-17beta, diethylstilbestrol) in the prostate of the baboon. The various parameters related to the specificity of such receptors have been established. The development and standardization of these approaches, some facets of which are reported in the present publication, may prove them to be more sensitive, specific, and optimal than other systems and should afford an opportunity to test various combinations of drugs potentially useful in cancer of the prostate with relative speed, efficacy, and ease of manipulation.


Aurinetricarboxylic acid: a potent inhibitor of glucose-6-phosphate dehydrogenase. Inhibition by aurinetricarboxylic acid (ATA) of glucose-6-phosphate (G6P) dehydrogenase was "competitive" with respect to G6P and "mixed type" with respect to NADP+. Inhibited enzyme bound two molecules of ATA. Kinetic constants, Km, Ki at varying pH suggested possible binding of the inhibitor by the sulfhydryl of the enzyme; of the several enzymes tested only milk xanthine oxidase and G6P dehydrogenase from bovine adrenal was inhibited by ATA.


[The effect of toxin II isolated from sea anemones (Anemonia sulcata) on the isolated guinea pig ileum]. When the guinea-pig isolated ileum is treated with the Toxin II from Anemonia sulcata, it releases not only acetylcholine but also serotonin. These transmitters are both involved in the spasmogenic action of the toxin.


[Demonstration of N-acetylgalactosaminyltransferase activity in the murine lymphoma YC8]. The N-acetyl-galactosaminyltransferase activity has been studied in the biological fluids of YC8-lymphoma bearing Balb/c mice. It is enhanced during tumor development from 1 to 30 times in peritoneal fluids and from 1 to 10 times in sera. This activity is nil in urines. Optimal requirements for activity have been determined. Results suggest the existence, during tumor process not only of an enhancement of enzymatic activity, but also of a new molecule synthesis, molecules which are endogenous acceptors for the enzyme.


Continuous-flow determination of dialyzable calcium in serum. We describe a procedure for measuring dialyzable calcium in 60 sera per hour with use of AutoAnalyzer (Technicon) modules. The serum is buffered, then dialyzed in a regular 6-inch (15-cm) dialysis block. A constant proportion of the dialyzable calcium is fluorometrically measured. Analysis of samples containing protein, prepared by an equilibrium dialysis technique, shows that protein-bound calcium is not detected. The method requires less than 0.4 ml of serum, and is accurate, precise, and durable. No unusual care or special techniques are required during sample collection or analysis.


Continuous-flow determination of serum inorganic phosphate with a single reagent--the vanadomolybdate method re-evaluated. The merits of the vanadomolybdate method in the determination of inorganic phosphate are highly underestimated with regard to the simplicity of the method and the stability of both the reagents and the color that is formed. The absorption curve of the phosphovanadomolybdate complex, with its maximum at 335 nm, extends into the visible range of the spectrum. This permits measurements with inexpensive tungsten-lamp colorimeters. On-stream dialysis is best done in a nitric acid medium, 0.15 mol/liter. Paralleled by the change in the H2PO4-/H3PO4 ratio, appreciable protein binding and poor dialysis efficiency are seen at lower acid concentrations (pH greater than 1.0). Optimum reaction-mixture concentrations of vanadium and molybdenum appeared to be respectively 0.2 and 5 mmol/liter up to 3 mmol of phosphate per liter, in a final acid medium of 0.2 mol/liter, concentrations considerably lower than those used in some studies published earlier. Color development with the stable combined reagent is complete after only 20 s at room temperature and the color is stable for at least 2 h. Figures on precision and accuracy demonstrate the reliability of the method.


Simultaneous measurement of phenobarbital, phenytoin, primidone, ethosuximide, and carbamazepine in serum by high-pressure liquid chromatography. We present a method for simultaneously determining five anticonvulsants [phenobarbital, phenytoin (diphenylhydantoin), primidone, ethosuximide, and carbamazepine] in as little as 25 microliters of serum. The proteins are precipitated with an acetonitrile solution containing hexobarbital as an internal standard. The anticonvulsants are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer (19/81 by vol) at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm, and quantities estimated from their peak heights. Each analysis requires about 14 min. at an optimum column temperature of 50 degrees C. The lower unit of detection for all of these drugs is less than 10 ng. Sensitivities, for serum samples, of 1.0 mg/liter for all the drugs analyzed are attained routinely. Analytical recoveries for the five drugs varied from 97 - 107%, with good day-to-day precision (CV between 3.9 and 5.9%). Of more than 30 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.


Children who do not talk at school.

Children who do not talk at school. This paper contains two case reports illustrating the use of behaviour modification in the treatment of elective mutism. The programme was carried out by two social workers in the natural environment of the home and school.


Recent developments in the clinical assessment of the metabolism of aromatics by high-performance, reversed-phase chromatography with amperometric detection. High-performance liquid chromatography with electrochemical detection has developed into a tool with excellent capability to monitor picomole amounts of individual metabolites in both tissue and body-fluid specimens. Significant technical advances in the area of microparticle chemically-bonded stationary phases have led to dramatic improvements in both sensitivity and resolution. Reversed-phase systems can be modified to include charged exchange sites by addition of detergents to the mobile phase. Adjustment of the surface charge in this manner permits capacity factors for ionic sample components to be increased or decreased. This concept is quite compatible with electrochemical detection and has provided the foundation for several clinical assays now in routine use. Examples of additional applications are presented for the determination of catecholamines in tissue, 3-methoxy-4-hydroxyphenylglycol in urine, and dopamine-beta-hydroxylase activity in human serum.


Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-beta-naphthylamine derivatives. Enzyme activity can be measured in single cells or in cell suspensions by either static or flow microfluorometry when a fluorogenic substrate is used. We have used amino acid derivatives of arylamines as fluorogenic substrates for tagging cellular proteinases. The liberated aromatic amine, which can diffuse from the cell, is trapped as a fluorescent insoluble Schiff-base product with 5-nitrosalicylaldehyde, with the peak of fluorescence emission shifted from lambdaem 425 nm to lambdaem 530 and 595 nm. Although the reaction is faster at pH 4 than at higher pH's, the equilibrium during the assay of certain peptidase activities is such that the liberated aromatic amine is trapped in the cell at pH values as high as 7.5. 5-Nitrosalicylaldehyde causes almost no inhibition of substrate hydrolysis at 1 mmol/liter, a concentration exceeding that required for complete trapping of reaction product. The kinetics of enzymatic reactions with four synthetic substrates are demonstrated in intact Balb 3T3 cells and sonicated preparations in the presence of 5-nitrosalicylaldehyde. Hydrolytic rates for the substrate, CBZ-ala-arg-arg-4-methoxy-beta-naphthylamine, are given for single 3T3 cells by microfluorophotometry and for suspensions of 3T3 cells by flow cytometry. The clinical value of the method is demonstrated for differentiating mixed populations of leukocytes.


Inorganic phosphorus in human bile.

Inorganic phosphorus in human bile. The inorganic phosphorus concentration has been measured in fresh bile from patients undergoing cholecystectomy, usually because of cholelithiasis. The amount in common duct bile for patients with cholesterol on the stone surface was significantly higher than that for patients with some calcium carbonate in this area. For all patients with functioning gallbladders, inorganic phosphorus in gallbladder bile was always higher than that in the corresponding common duct bile. In bile from functioning gallbladders, inorganic phosphorus was linearly related to pH for cholesterol stone-formers, but values for calcium carbonate stone-formers were below the regression line and not related to pH. The results show that bile from calcium carbonate stone-formers is more dilute with respect to inorganic phosphorus.


A practical chromogenic procedure for the diagnosis of Krabbe's disease. Krabbe's disease is caused by a deficiency of galactocerebrosidase in organs and tissues. Determinations of galactocerebrosidase activity had required the use of galactocerebroside labeled with radiocarbon or radiohydrogen. These materials are expensive and their use is restricted to laboratories with radioactive counting facilities. An analogue of galactocerebroside, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, was synthesized. The hydrolysis of this analogue by extracts of tissues and cells from patients with Krabbe's disease is greatly reduced from normal levels. Cultured skin fibroblasts preparations derived from heterozygous carriers of Krabbe's disease have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Krabbe's disease and for the detection of heterozygous carriers of the Krabbe trait.


Urinary alpha-glucosidase analysis for the detection of the adult form of Pompe's disease. Two alpha-glucosidases from human heart, liver, muscle, kidney and urine have been separated by means of Sephadex G-100 gel filtration. The first peak (Peak I) was neutral alpha-glucosidase and the second peak (Peak II) was lysosomal acid alpha-glucosidase. Peak II was absent in a patient with the adult form of Pompe's disease. KCl stimulated the activity of the Peak II enzyme but it strongly inhibited the activity of the Peak I enzyme measured at pH 4.0. Decreases in the urinary alpha-glucosidase activity measured at pH 4.0 with added KCl and the ratio of the activity at pH 4.0 with added KCl/the activity at pH 6.5 without KCl may aid in the detection of homozygotes or heterozygotes with the adult form of Pompe's disease.


Serum gamma-glutamyl transferase activity in chronic renal failure during regular hemodialysis and after successful renal transplantation. Serum gamma-glutamyl transferase activity (gamma-GT) was measured in 108 uraemic patients, 110 patients on regular haemodialysis and 71 sucessfully transplanted patients. The incidence of abnormal results was 11, 14 and 28%, respectively. The results in the non-dialyzed and dialyzed patients indicate that in uraemia serum gamma-GT activity remains normal and as such, significant hepatic microsomal enzyme induction does not appear to occur. Raised serum gamma-GT in uraemic patients and after renal transplantation should suggest concurrent, added, pathology such as hepatobiliary disease or the administration of hepatic microsomal enzyme inducing drugs.


Interaction of calcium ions with serum albumin in chronic renal failure. In order to study the effect of the uremic environment on serum calcium-albumin interaction, ten adult subjects with chronic renal failure (creatinine clearances below 20 ml/min) and five adult normal subjects, had their serum ionic calcium (Orion SS-20) determined at three different serum pH values. At similar serum protein levels, the correlations between serum ionic calcium pH were: (a) normals: number of pairs = 15, r = -0.90, p less than 0.01, Ca2+ = 4.54-0.484 pH. (b) uremics: number of pairs=15, r=-0.97, p less than 0.01, Ca2+=4.02-0.429 pH. These regressions were similar as far as the slopes (Fslope=0.62, p greater than 0.05) but had different intercepts (Fintercepts=64.34, p less than 0.01). These results confirm that uremics, when compared to normal subjects, present an ionic hypocalcemia at any level of serum pH. They also show that serum calcium-albumin interaction is normal in uremia. In view of the above results, it can be suggested that, among several possible causes, alterations in the binding-affinity of calcium ions to serum albumin can be excluded as an additional factor of secondary hyperparathyroidism in uremia.


A micro-radiochemical assay for alpha-1,4-glucosidase and its use in the assessment of type II glycogenosis (Pompe's disease). An assay for alpha-1,4-glucosidase (acid maltase) activity which is deficient in Pompe's disease is described. The assay can be used to measure the enzyme in cultured skin fibroblasts, cultured amniotic cells and peripheral blood leucocytes. [U-14 C]Maltose is used as the substrate in a total assay volume of 8 microliter. The product, [U-14C]glucose, is separated from the substrate by cellulose thin-layer chromatography. The procedure permits replicate assays from 400 microliter whole blood and from amniotic cells in primary culture. Discrimination of the heterozygous Pompe state appears to be facilitated.


A radioimmunoassay for the R-type binders of cobalamin. A radioimmunoassay of R-type binders of cobalmin was devised and tested. The assay incorporated an antibody against purified human salivary R binder as the binding reagent. The labeled ligand was cyano[57Co]cobalamin bound to the R binder of pooled human saliva. The standard source of unlabeled ligand was also from human saliva of known R binder content. The assay was responsive to R binders of several sources, to either pure R or that of crude sources and equally to R binder saturated or unsaturated with cobalamin. It was not responsive to transcobalamin II. The assay was reproducible and reliable.


An improved method for the determination of acetylcholinesterase activity in rectal biopsy tissue from patients with Hirschsprung's disease. A method is described for the assay of acetylcholinesterase in tissue samples using the selective cholinesterase inhibitor, lysivane (10-(alpha-diethylaminopropyl) phenothiazine hydrochloride). Significantly increased enzyme activity is found in rectal biopsy specimens from patients with Hirschsprung's disease (p less than 0.001) indicating the diagnostic usefulness of the assay.


Respiratory acidosis and alkalosis.

Respiratory acidosis and alkalosis. The physiology of respiratory control of acid-base balance is reviewed. The pathophysiological mechanisms during hypercapnia and hypocapnia are discussed in the light of the causes and clinical manifestations of these disturbances. In addition to the role of the kidney in the compensatory processes of these disturbances, renal functional changes during acute and chronic pulmonary acid-base derangement is discussed. Some of the difficulties encountered in the patient with chronic renal disease in whom respiratory abnormalities may be present are also discussed.


Metabolic acidosis and alkalosis.

Metabolic acidosis and alkalosis. The factors controlling renal bicarbonate reabsorption and acid excretion under normal conditions and in the presence of metabolic acidosis and alkalosis are reviewed. The methods used to assess distal acidification and its limitations are also discussed. Measurement of urinary pCO2 in maximally alkaline urine (pH greater than 7.8) is a very useful qualitative method to assess distal acidification. The finding of a low urinary pCO2 in maximally alkaline urine indicates a distal acidification defect. We propose that both the secretory and gradient defect types of distal renal tubular acidosis are associated with a low urinary pCO2 when the urine is maximally alkaline. Sodium sulfate and neutral phosphate infusion may allow distinction between a secretory and gradient defect. Sodium sulfate lowers urine pH in the gradient defect but fails to produce the same response in the secretory defect. Neutral phosphate infusion when urine pH (6.8-7.4) is close to the pK of phosphate (6.8) results in an increase in urinary pCO2 in the gradient defect but not in the secretory defect. The mechanisms of generation, maintenance and treatment of metabolic alkalosis are also discussed.


Methadone maintenance: effect of urinary pH on renal clearance in chronic high and low doses. The subjects were 12 male patients stabilized on methadone for many months or years. A comparison was made of the plasma levels and renal clearance of methadone between patients on "high" doses (80 to 110 mg/day) and those on "low" doses (15 to 40 mg/day). A general trend to higher renal clearance was seen in the "high" -dose group, but on more detailed examination there was a direct correlation only when the patients were categorized by urinary pH. At low pHs, there was nearly a 3-fold increase in renal clearance which was associated with a decreased major metabolite to methadone ratio. No evidence for a difference in rate of metabolism between the two groups was found nor were there differences in hepatic function. It was concluded that urinary pH was a major factor in renal clearance of methadone.


The crystalline salts of calcium bilirubinate in human gallstones. 1. The acid, neutral and "intermediate" salts of calcium bilirubinate have been synthesized. 2. All are crystalline, and the intermediate salt which has 1 1/2 molecules of bilirubin per calcium atom probably has a crystal structure containing molecules of both acid and neutral salts. 3. The acid or intermediate salts have been found in 14 out of 70 gallstones analysed recently by X-ray diffraction.


Autoimmunity, oncornaviruses, and lymphomagenesis. The interactions between aberrant immune responses and C-type oncornaviruses in mice are complex. These viruses may be activated during certain immune responses, e.g., histocompatibility reactions, in the face of chronic immunosuppression. Oncornaviruses, themselves, may induce autoaggressive cell-mediated responses in certain lymphoid subpopulations, and these autoimmune reactions may be important in subsequent lymphomagenesis. Parallel events may occur in other animal models, e.g., the NZB mouse, and in certain human disorders, e.g., Sjögren's syndrome, systemic lupus erythematosus, and myasthenia gravis.


Predictability of arterial pH and PCO2 from right atrial blood samples in experimental animal. Arterial and right atrial blood samples were drawn simultaneously under acutely changing acid-base and ventilatory conditions in a series of experiments carried out in nine pigs. The pH, PCO2, bicarbonate concentration and base excess from arterial and right atrial blood are compared. The predictability of the arterial values from the right atrial values is discussed. It is concluded that right atrial blood samples are approximately as informative as arterial blood samples for determining the acid-base status under both normal and acutely changed acid-base and ventilatory conditions.


Fiberoptic bronchoscopy and culture bacteria from the lower respiratory. Forty-five specimens were obtained by sequential translaryngeal aspiration and fiberoptic bronchoscopy from 31 clinically unifected patients with lung cancer in order to evaluate the reliability of routine fiberoptic bronchoscopy for culture of the lower respiratory tract. Bacteria were recovered brom 98 percent (44) of the specimens obtained via fiberoptic bronchoscopy and from 58 percent (26) of the specimens obtained by the preceding translaryngeal aspiration. The microorganisms grown from cultures of specimens obtained by fiberoptic bronchoscopy consisted of mixtures of both nonpathogenic and potenitally pathogenic bacteria. Potentially pathogenic bacteria were present in 87 percent (39) of the specimens from fiberoptic bronchoscopy and 31 percent (14) of specimens from translayngeal aspiration. The results of cultures from the two procedures agreed completely in only a single instance. Culture of washings or secretions obtained by routine fiberoptic bronchoscopy is not recommended because it provides inaccurate and clinically confusing information about the presence or types of bacteria in the lower respiratory tract prior to instrumentation.


Patterns of antipsychotic drug use in four Southeastern state hospitals. The drug prescriptions of patients in the four mental hospitals of a state were obtained as of a specific date and subjected to various analyses. The results disclose a high frequency of antiparkinson drug orders (41.5% of the patients), their relationship to polypharmacy (30% of the antipsychotic drug prescriptions were for two or more to be administered simultaneously), the direct relationship between the antipsychotic dose level and antiparkinson drug and PRN orders and the incidence of multiple dose schedules (75%). Also other prescription practices which have received little or no attention are described and discussed.


The ventilatory response in uncomplicated diabetic ketoacidosis. The ventilatory response to uncomplicated sub-acute metabolic acidosis was studied by measuring arterial blood CO2 tension (PaCO2) and pH in 27 episodes of diabetic ketoacidosis without coexisting lactic acidosis. From those data, regression equations and 95% confidence bands were calculated for the relations between PaCO2 and pH. These relations were very similar to those previously described by others in patients with chronic uremic acidosis.


Isolation and identification of the major metabolite of albuterol in human urine. The major urinary metabolite of albuterol in man was isolated and purified by ion-exchange chromatography and countercurrent distribution. On the basis of proton magnetic resonance, infrared, and mass-spectral analysis, enzymic hydrolysis, and assay of inorganic sulfate after acid hydrolysis, the metabolite has been identified as the 4'-O-sulfate ester of albuterol.


Pharmacokinetics of intravenously injected chloramphenicol in baby pigs. A sensitive fluorometric assay procedure for chloramphenicol and its metabolites was used to measure distribution in tissues and elimination of the drug after injection of 22 mg/kg iv in baby pigs. Disappearance of chloramphenicol from plasma was biphasic; an initial component was rapid (half-life about 9 min) and a second component had a half-life of 55 min. The total chloramphenicol levels in tissues and body fluids had half-life periods of 1-1.5 hr and dropped below limits of detectability within 9 hr after injection. About 80% of the injected dose was recovered from urine collected over the first 6 hr, and none thereafter. Fecal elimination, if occurring, was negligible.


Biotransformation of cannabidiol in mice. Identification of new acid metabolites. The in vivo metabolism of cannabidiol (CBD) was investigated in mice. Following the ip administration of CBD to mice, livers were removed and metabolites were extracted with ethyl acetate prior to partial purification on Sephadex LH-20 columns. Fractions from the columns were converted into trimethylsilyl, d9-trimethylsilyl, and methylester-trimethylsilyl derivatives for analysis by gas-liquid chromatography-mass spectrometry. In addition, metabolites containing carboxylic acid and ketone functional groups were reduced to alcohols with lithium aluminum deuteride before trimethylsilation. A total of 22 metabolites were characterized, 14 of which had not been reported previously. The metabolites could be categorized as follows: monohydroxylated (N=2), dihydroxylated (N=3), CBD-7-oic acid, side chain hydroxy-GBD-7-oic acids (N=3), side-chain acids (N=3), 7-hydroxy-side-chain acids (N=4), 6-oxo-side-chain acids (N=3) and glucuronide conjugates (N=3). The most significant biotransformations were glucuronide conjugation and, to a lesser extent, formation of CBD-7-oic acid.


Absorption, excretion and disposition of cyclindole in laboratory animals and human volunteers. Radioactivity from orally administered single doses of cyclindole-14C was excreted primarily in the urine of the rat (99%/48 hr), monkey (80%/120 hr), and dog (70%/144 hr). Approximately 38-58% of a daily dose of cyclindole was recovered from 24-hr human urine, as determined by gas chromatography. Substantial amounts of unchanged drug were voided by dogs. Cyclindole was metabolized primarily by N-demethylation and/or hydroxylation in the 7-position. Hydroxylation at the 6-carbon atom was of minor importance in humans only; none of the animal species excreted the 6-hydroxy metabolite. Dogs were capable of N-demethylation, but no metabolites oxidized at the 6- or 7-carbon atoms were detected in dog urine.


The effects of delta9-tetrahydrocannabinol and cannabidiol on the metabolism of gonadal steroids in the rat. The effects of delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on hydroxylation of estradiol and testosterone in rat liver microsomes were investigated. Acute and chronic treatment of male and female rats with either cannabinoid, 2 or 10 mg/kg, significantly elevated steroid hydroxylase activity. Acute administration of THC or CBD, 10 mg/kg, evoked no detectable changes in cytochrome P-450 levels, but a significant decrease in those of cytochrome b5. Chronic administration of THC or CBD, 2 mg/kg, decreased levels of cytochrome P-450, whereas cytochrome b5 levels appeared normal. Acute doses of THC or CBD at 10 mg/kg significantly depressed testosterone formation in testis microsomes. Chronic THC, 2 mg/kg, but not CBD, evoked the most dramatic decrease in testicular enzyme activity.


Glucuronidation and deglucuronidation reactions in hepatic and extrahepatic tissues during perinatal development. The relative activities of uridine diphosphoglucuronyltransferase (UDPGT) and beta-glucuronidase (betaG) were measured during perinatal development of hepatic and extrahepatic tissues to determine the balance between glucuronidation and deglucuronidation reactions at different developmental stages. Liver, lung, kidney, intestine, and placenta were studied in guinea pigs and rabbits. In general, betaG activities exceeded those of UDPGT in fetal tissues, whereas the converse was evident in adults. There were significant species and age differences in the onset of betaG and UDPGT activities and the occurrence of developmental peaks. A dramatic betaG developmental peak was observed in fetal guinea pig intestine and newborn rabbit intestine. Both microsomal and lysosomal betaG exhibited similar developmental patterns in all tissues tested. Hepatic nonsteroid UDPGT activities were higher at parturition than in adult animals, whereas no such developmental peak occurred for steroid UDPGT. Triton X-100 activated fetal UDPGT in vitro by approximately the same factor as it did for adult UDPGT.


Metabolism of 7,12-dimethylbenz[a]anthracene in mouse skin homogenates analyzed with high-pressure liquid chromatography. The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) in epidermal homogenates from 3-methylcholanthrene-pretreated mice was analyzed with high-pressure liquid chromatography. Metabolism was undetectable in the absence of pretreatment. Specific activities in epidermal homogenates from pretreated mice were found to be approximately 100 to 1000 times lower than those observed in comparable incubations containing hepatic microsomes from MC-pretreated rats. The major metabolite formed was identified as 7-hydroxymethyl-12-methylbenz[a]anthracene. Each of the known hydroxymethyl metabolites also was present in detectable quantities. The K-region diol was not measurably present in incubations with mouse skin homogenates or rat liver microsomes from MC-pretreated animals. 7,8-Benzoflavone, 5,6-benzoflavone, and 17-beta-estradiol were found to be potent inhibitors of the metabolic transformation of DMBA by epidermal homogenates in vitro, whereas butylated hydroxytoluene and 1,1,1-trichloro-2,3-propene oxide had little effect on or enhanced metabolite formation from DMBA in vitro.


[Splenic infarcts in Goodpasture's syndrome: its significance for diagnosis, prognosis, and treatment (author's transl)]. Necrotising splenic arteritis with numerous extensive anaemic splenic infarcts was found in four cases of Goodpasture's syndrome with the necrotising variant of rapidly progressive glomerulonephritis. The disease was rapidly fatal. Early scanning of splenic infarcts for diagnostic, prognostic and therapeutic purposes of the fulminating form of Goodpasture's syndrome is recommended.


Microsomal N-hydroxylation of trans-4'-halo-4-acetamidostilbenes. Hamster liver microsomes catalyze the N-hydroxylation of trans-4-acetamidostilbene and its 4'-Cl and 4'-F analogs. Other metabolites which were detected and quantitated were the 4'-OH derivative of trans-4-acetamidostilbene and the glycolamides of the 4'-Cl and 4'-F compounds. The kinetics of metabolism of the acetamidostilbenes were also determined. The apparent KM values of N-hydroxylation of the four substrates were similar, but the maximal velocities showed marked variations. The Vmax for the 4'-Br analog was 16% of that of trans-4-acetamidostilbene, and the rate of N-hydroxylation of the 4'Cl and 4'-F analogs was only 3% of that of the parent compound.


[Stimulation of H+ secretion and serum gastrin by intra-operative electrical vagal stimulation before and after proximal selective vagotomy (author's transl)]. H+ secretion and gastrin concentration were measured before and after electric vagal stimulation during proximal selective vagotomy in 29 patients with duodenal ulcers. Before vagotomy, H+ secretion and serum gastrin concentration significantly rose after stimulation, while after complete vagotomy H+ secretion remained below basal values, although serum gastrin concentration was significantly increased. These results indicate that without vagal innervation there is no gastrin secretion within physiological levels. The method is suitable for testing the completeness of the vagotomy, the results not agreeing with those obtained by pressure measurements (Burge's method).


Hepatic enzymes, their induction and usefulness in predicting maximal serum bilirubin levels in premature newborn infants. Sixty-seven babies were utilized to (a) document the serum bilirubin lowering effect and safety of a phenobarbitone and nikethamide combination in neonatal hyperbilirubinaemia of non-hemolytic origin; (b) determine whether birthweight and/or SGOT, SGPT or SGGT activity on day one of life correlated with the maximum serum bilirubin level achieved; and (c) investigate the pattern of hepatic enzyme levels in serum under normal conditions anf following drug induction. Results indicate a significantly lower serum bilirubin level in the treated group of babies. Birthweight and day one SGGT levels, and SGGT/birthweight ratio correlated well with the maximum serum bilirubin reached, the latter ratio being particularly useful in predicting the degree of hyperbilirubinaemia.


In vitro pharmacology of the opioid peptides, enkephalins and endorphins. In the guinea-pig ileum methionine-enkephalin, normorphine and morphine are equipotent in depressing electrically evoked contractions; leucine-enkephalin has about 25% of the activity. The mouse vas deferens is more sensitive to the enkephalins which are 30 to 60 times more potent than morphine. Fragments of beta-lipotropin61-91 (beta-endorphin) having sequences up to LPH76 are more potent in the mouse vas deferens than in the guinea-pig ileum but beta-endorphin is about equipotent in the two preparations. None of the peptides has antagonist activity. Methionine-enkephalin and normorphine are equipotent in inhibiting [3H]-naloxone binding by homogenate of guinea-pig brain in the absence of Na+ while leucine-enkephalin has only 25% of this activity. In the guinea-pig ileum, naloxone antagonises normorhine and the enkephalins equally well whereas in the mouse vas deferens about ten times more naloxone is required for the enkophalins that for normorphine. Methionine-enkephalin depresses output of acetylcholine in the guinea-pig ileum and of noradrenaline in the mouse vas deferens.


The role of adrenoceptors in norepinephrine-stimulated VO2 in muscle. The purpose of this study was to characterize the norepinephrine (NE)-evoked calorigenic response of an isolated perfused rat muscle preparation into alpha-and/or beta-adrenergic components. Epinephrine was as effective as NE in evoking the increase in oxygen consumption. The alpha-agonist, phenylephrine, was also as effective as NE in eliciting an increase in oxygen consumption by the muscle. Isoproterenol (beta-agonist) had no effect on the VO2 of the preparation. Phentolamine, an alpha-blocker, completely blocked the NE-stimulated VO2. Propranolol (beta-antagonist) could also completely block the NE induced VO2 but this drug was found to be less potent. It was concluded that the NE-induced calorigenic response of muscle cannot be classified either strictly as an alpha-or beta-response, but unlike brown adipose tissue, the alpha-response seems to predominate in skeletal muscle. Also, it appears as though different mechanisms of action may be involved in the NE-stimulated VO2 in skeletal muscle and brown fat.


[Effect of a schedule of administration of chlorpromazine, majeptil and trisedyl on the synthesis of proteins in different brain structures of rats]. Following a course-wise introduction of chlorpromazine, majeptil and trisedyl to rats the methionine-35S incorporation in the protein was diminished in most of the brain segments.


[Effect of nonachlazine on the processes of uptake and release of nordrenaline]. Nonachlazine (2 micrometer) blocked significantly the uptake 1 of endogenic noradrenaline in the myocardial tissues and in adrenergic neurons of vas deferens. The uptake II, as well as the intensity and dynamics of the noradrenaline release were not affected by nonachlazine. These processes resulted in an increase of free noradrenaline concentrations in the synaptic space. The latter seems to be a result of the mediator's passage into the nerve fibers (through axonal membranes) and into the granule, this being due to the nonachlazine-induced block. It is suggested that the metabolic and functional nonachlazine effects are related to the beta-adrenergic receptors stimulation, mediated through the agency of noradrenaline.


[Stabilization of biological membranes with various acetylene amines]. In experiments with mitochondrial membranes and membranes of the endoplasmatic reticulum of the rats' liver the membrane-stabilizing action of a number of unsaturated amines was evaluated. The acetylene compounds studied reduced the extent of the membranes damage following UV-irradiation, treatment with detergents and their incubation in a hypoosmotic medium. Such an action displayed gutimine and sovcaine. Hydrated compounds, analogues of acetylene amines are devoid of the ability to raise the resistance of the cellular organoids membranes to damaging factors


[Study of the induction of thrombocyte adhesiveness in the presence of heparin]. An investigation of the thrombocytes adhesion after Hellem (1960) showed that "in vitro" tests norepinephrine, epinephrine, histamine, serotonin and adenosine-diphosphate, when used in doses of 100-25,0 gamma/ml, were capable of raising the thrombocytes adhesion, while heparin administered in doses of 0.5-0.05 U/ml depressed and in a dose of 0.025 U/ml mildly activated this capacity of the thrombocytes. In the presence of heparin the ability of biogenic amines to induce the adhesion of thrombocytes decreased. Between concentration of the agents and the adhesive index of thrombocytes there exists a two-way logarithmic relationship. Intravenous administration of serotonin in a dose of 5 mg/kg and of epinephrine in an amount of 0.05 mg/kg to rabbits increased the adhesivity of thrombocytes, whereas intravenous introduction of 100 un/kg of heparin tended to greatly reduce it. On introduction of serotonin and epinephrine to rabbits against the background of heparin their ability to activate the thrombocytes adhesion diminished quite significantly. The logarithm of time lapsed from the instance of the heparin administration is linearly dependent on the  logarithms of values characterizing the adhesivity of thrombocytes. Intravenous administration of norepinephrine and histamine in a dose of 0.5 mg/kg did not cause any changes in the adhesive properties of thrombocytes.


A double-blind trial of penbutolol: a new beta-receptor blocking agent in the treatment of angina pectoris. A double-blind placebo controlled study of angina pectoris with penbutolol was undertaken in parallel groups in fifty-two patients. The duration of the study was six weeks. The dosage range for penbutolol was 8 mg to 50 mg per day. Six patients were dropped from the analysis. Seventeen patients (81%) in the penbutolol series exhibited a 50% reduction in anginal attacks, NTG consumption and subjective improvement. Significant reduction in nitrite intake was observed. Effort tolerance was improved significantly in those receiving penbutolol. Penbutolol was well-tolerated.


A trial of benorylate tablets in the symptomatic relief of osteoarthritis. The effectiveness of Benoral Tablets in controlling joint pain and stiffness in osteoarthritis was assessed over a two-week period in a multicentre general practice open study. In this trial it has been demonstrated that a correlation exists between pain relief from stiffness for patients suffering from osteoarthritis taking Benoral Tablets over this period. It was also found that patients with initially mild to moderate pain benefited most from their treatment.


Polygraphic sleep recordings before and after the administration of flunitrazepam. By means of polygraphic sleep recordings (EEG, EOG, EMG, ECG, EDG, Respirogram, Positogram), neurologic-psychologic investigation and questionnaires on subjective feeling, ten patients aged from 22 to 40 years (mean: 29 years), who suffered from irregular disturbances in falling asleep and in sleeping continuously, were examined over a period of ten consecutives nights. The first investigation night was reserved for adaptation of the patients and could therefore not be evaluated. On the following three evenings placebo (Placebo I) was given, then 2 mg of flunitrazepam for the ensuing three nights and placebo again (Placebo II) for the last three nights. Thus, 90 investigation nights could be evaluated. 1. Latency times until the patients fell asleep and up to the first deep sleep were reduced significantly by the active preparation. Latency times up to the first REM-phase were prolonged. 2. The frequency of nocturnal awakenings was lessened significantly. Thus, disturbances in sleeping continuously disappeared for the duration of the treatment. Duration of wakefulness after having woken during the night decreased considerably. 3. Duration of wakefulness during the total night was reduced considerably, deeper sleep stages, such as D (stage III) and E(stage IV) were prolonged. 4. Duration of the REM-phases was reduced slightly. This reduction was not significant according to the t-test. 5. According to these results, the preparation showed a clear effect. A placebo-effect can be excluded, since the improvements mentioned were not found when placebo was given before administration of the medication and afterwards (Placebo I and II). 6. When comparing our results to those of other authors, who described the effects of various preparations, we found that the substance flunitrazepam showed different effects.


Effect of secretin on plasma motilin in man. Graded doses of 0-1, 0-3, 0-9, and 2-7 clinical units/kg/h of pure synthetic secretin were infused over 60 minute periods in six healthy volunteers. Duodenal bicarbonate output and pH were recorded and plasma secretin and motilin levels were measured by radioimmunoassay. During the infusions plasma motilin fell in a dose dependent manner to a nadir of 35%. This fall was linearly correlated with pancreatic bicarbonate output, whereas a non-linear correlation was observed between plasma motilin and both plasma secretin level and duodenal pH. It is suggested that plasma motilin levels are decreased by secretin-induced pancreatic bicarbonate juice flow. This may be important for the control of motilin secretion initiated by duodenal acidification and the concomitant delay in gastric emptying.


Jejunal mucosal gamma glutamyl transferase activity in coeliac disease. In view of data suggesting that deficiency of gamma glutamyl transferase (GGT) may be the primary abnormality in the small-intestinal mucosa of patients with coeliac disease, this enzyme was measured in mucosal biopsies from coeliac patients at different stages of treatment. Activity was only slightly reduced in only one of nine well-treated patients, suggesting that deficiency of this enzyme has no primary role in the aetiology of coeliac disease.


Correlation between manometric and pH tests for gastro-oesophageal reflux. A series of tests, including gastro-oesophageal sphincter pressure measurement, short-term pH tests, and 15-hour overnight oesophageal pH recording were applied to 42 normal subjects and 214 patients with typical reflux symptoms. The results were compared by multivariate discriminant analysis. Sphincter pressure measurements misclassified 32%, stressed provocative manoeuvres 14-5%, and the best single discriminator from the overnight pH study was time below pH 5, which misclassified 13%. However, a combination of the number of reflux episodes in 15 hours with their mean duration reduced misclassification to 8-8%. Using this function, a boundary between normal and reflux can be drawn, and the degree of abnormality can be expressed visually as well as numerically.


[Utilization of the MVA-immunization schedule from the view point of the public health service]. The world-wide smallpox eradication program sponsored by the WHO has been successful. Furthermore the legislation regarding smallpox vaccination in the Federal Republic of Germany has been given a new form. The place of the previous mass-vaccination has been taken by vaccination of individuals. Under this aspect safe procedures for vaccination are required. Any vaccination used by Public Health Service has to fulfill the following criteria: High protection against the disease without adverse side-effects or risks; it should be simple to administer and applicable under ordinary circumstances. For primary vaccination against smallpox the MVA-vaccination meets these demands entirely. It is reported about own experiences with 700 vaccinations using the attenuated virus "MVA"; complete documentation is given. Not a single complication nor any adverse side-effects have been seen. The Public Health Service has gained great experience in the field of vaccination. It is responsible for the protection of the individual as well as the public, as it is undesirable that there develops and unvaccinated group within the population. The individual claim a complete protection through vaccination which should be absolutely free of any risk.


Characteristics of the maternal lymphoid response of mice to paternal strain antigens induced by homologous pregnancy. Mice mated with H-2 incompatible males were shown to have an increased frequency of specific allocluster-forming spleen cells to paternal strain erythrocytes. The time course of the response was similar to that expected following a skin allograft. The graft-versus-host responsiveness of the maternal lymphocytes was generally elevated in the post-partum period in matings with males incompatible at either the H2 locus or non-H2 loci. The implications of these responses with respect to their different lymphocyte mediators and possible interactions are discussed with the view that a B-cell mediated immune response to paternal antigens may be contributory to foetal survival.


Identification, separation, and preliminary characterization of invertase and beta-galactosidase in Actinomyces viscosus. The initial step of disaccharide dissimilation by Actinomyces viscosus serotype 2 strain M-100 was studied. Sucrase activity was found in the 3,000 X g particulate fraction and the 37,000 X g soluble fraction of the cells, whereas lactase activity was found almost exclusively in the 37,000 X g soluble fraction. Neither sucrase nor lactase activity was appreciable in the culture liquor. Sucrose phosphorylase, alpha-glucosidase, and polysaccharide synthesis activities were not observed in the soluble cell fraction. The sucrase was identified as invertase (EC 3.2.1.26; beta-D-fructofuranoside fructohydrolase). The lactase was identified as beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase). The enzymes in the 37,000 X g soluble fraction were separable by diethylamino-ethyl-cellulose chromatography, giving one beta-galactosidase peak and one major and one minor invertase peak. Acrylamide gel electrophoresis showed different electrophoretic mobilities of the enzymes. The molecular weight of the beta-galactosidase is about 4.2 X 10(5) and that of invertase is about 8.6 X 10(4). The beta-galactosidase has a Km for lactose of about 6 mM and a pH optimum between pH 6.0 and 6.5. The major invertase component has a Km for sucrose of about 71 mM and a pH optimum between pH 5.8 and 6.3.


Corneal transplantation reaction in avascular keratoconus patients due to HLA-associated immune aberration against infection. A hypothesis. HLA-B12 and HLA-B27 appeared to be characteristic of avascular keratoconus patients at risk of developing a corneal allograft reaction, which was mainly of late type and triggered by a nasopharyngeal infection. Equally incompatible grafts to patients with HLA-B5 had an uneventful course. This may be inference to the role of HLA-associated immune resistance factors, which necessitate the search for histocompatible corneal grafts also for avascular recipients bearing such indicators of risk.


Hepatic microsomal enzyme induction and its evaluation in a clinical laboratory. We tried to determine whether short-term treatment with alpha-methyldopa, quinidine, digoxin, diazepam or furosemide--drugs in common use in hospitals--is capable of stimulating the activity of hepatic microsomal drug-metabolizing enzymes. Glucaric acid (GA) excretion and serum activity of gamma-glutamyl transpeptidase (GGT) were used as indicators of hepatic microsomal enzyme activity. Increased GA excretion was found in 45% and increased serum GGT activity in 40% of the patients on drug treatment. Only 14.3% showed an increase in both indicators. The excretion of GA rose significantly in patients who received drugs for less than 10 days, as compared with those who received drugs for less than 10 days, whereas the percentage of high GGT values did not rise significantly with increased duration of treatment. The lack of correlation between serum GGT activity and GA excretion casts doubt on the value of GGT as a consistent indicator of microsomal enzyme induction. GA excretion, on the other hand, seems to be a dependable index of microsomal enzyme induction in response to short-term treatment with standard doses of several widely used drugs.


The interaction of barbiturates and analeptics. The interaction of pentobarbitone sodium with three analeptics viz. micoren, pentylenetetrazol and methedrine was studied in mice. Micoren prolonged pentobarbitone sleeping time. pentylenetetrazol shortened the sleeping time. Methedrine also shortened the sleeping time, but clonic convulsions of mild to severe intensity were noticed 45-60 minutes after the drug injection.


[Unusually high gamma-glutamyltransferase level in the semen and ist diagnostic significance]. A remarkable high activity of the enzyme gamma-glutamyltransferase (gamma-GT) is found in seminal fluid in 200 to 500 fold higher concentrations as found in blood serum. The seminal fluid gamma-GT originates expecially in the prostate gland and, to a smaller extent, in spermatozoa and secretion of testis and epididymis. Gamma-GT could be valuable to investigate the function of the prostate gland.


Aids for in vitro Mycobacterium lepraemurium investigations: estimation of oxidation-reduction potentials and pO2 with 2, 6 dichlorophenol indophenol. The partial success in cultivating a "host-dependent" microbe provided the incentive to develop methods which may aid the growth of the organism. The oxidation-reduction potential (ORP) of NC-5, an aerobic, cysteine containing medium which supports the limited in vitro growth of Mycobacterium lepraemurium, is measurable with the redox dye, 2, 6 dichlorophenol indophenol. Both cysteine and autoclaved glucose can be used to adjust to ORP. Glucose autoclaved in phosphate buffer but not in aqueous solutions reduced the dye. The dye was also reduced in glucose solutions by atmospheres containing 10% and 1%-2% pO2. With exposure to 20% pO2 the reoxidation of the dye was slow but complete. Thus, the dye in glucose solutions provides a general method for estimating pO2 above the level of anaerobiosis. Proper adjustment and monitoring of the ORP and pO2 may enhance growth.


[Microchemical investigation of alpha D-glucosidases using 4-methylumnelliferyl- and 2-naphthyl-alpha-d-glucoside (author's transl)]. In crude homogenates prepared from freeze-dried cryostate sections of various rat organs the Km and Vmax of acid and neutral alpha-glucosidase as well as the effect of the pH, substrate and enzyme concentration and the incubation time on the activity were determined fluorometrically with 4-methylumbelliferyl- and 2-naphthyl alpha-d-glucoside as substrates. On the basis of the biochemical data 2 assays were developed for the microchemical measurement of both alpha-glucosidases in groups of epithelial cells isolated from freeze dried cryostate sections of the epididymis, jejunum, ilium, liver and kidney of suckling and adult rats. The rate of hydrolysis of 2-naphthyl and 4-methylumbelliferyl alpha-d-glucoside differs moderately. However, due to the higher sensitivity of 4-methylumbelliferone the methylumbelliferyl derivative is preferable especially for the evaluation of alpha-d-glucosidases in cells with low enzyme activity.


Nursing education in crisis: a computer alternative. The impact of computer-assisted instruction upon the educational process is having its effect. The computer is being used in nursing education today to manage the educational environment, to instruct, to evaluate, to identify problem areas, to gather data, to manipulate data for research purposes and for continued education. As we struggle in our efforts for quality individualized education, we are minutely scrutinizing every aspect of the teaching learning process. We are not only examining how we teach but what we teach and why we are teaching it! Our deeper understanding of the process of education has helped us to delineate nursing process as content. Higher levels of instructional goals -- cognitive, affective and conative -- are resulting in education of the whole individual. The systematization of the learning process with the greater ease of accountability is leading nursing education to blaze promising new trails into the twenty-first century.


Using the computer to prepare multiple-choice examinations: a simplified system. Program TEST offers a quick, easy, and efficient way to produce objectively-scored exams from an established item bank. It is especially useful in situations where frequent testing is utilized and secretarial help is scarce. The capability of constructing a customized exam the day before its administration should make the exam more reflective of the instruction. The provision for keyed copies has been found to be a useful tool for feedback to the student after the exam. Lastly, use of the program requires little computer assistance.


Itaconate, an isocitrate lyase-directed inhibitor in Pseudomonas indigofera. Enzymes catalyzing steps from ethanol to acetyl-coenzyme A, from malate to pyruvate, and from pyruvate to glucose 6-phosphate were identified in ethanol-grown Pseudomonas indigofera. Enzymes catalyzing the catabolism of glucose to pyruvate via the Entner-Doudoroff pathway were identified in glucose-grown cells. Phosphofructokinase could not be detected in Pseudomonas indigofera. Itaconate, a potent inhibitor of isocitrate lyase, abolished growth of P. indigofera on ethanol at concentrations that had little effect upon growth on glucose. The date obtained through enzyme analyses and studies of itaconate inhibition with both extracts and toluene-treated cells suggest that itaconate selectively inhibits and reduces the specific activity of isocitrate lyase.


Chemotaxis toward amino acids by Bdellovibrio bacteriovorus. Chemotaxis toward amino acids by Bdellovibrio bacteriovorous strain UKi2 was studied by the capillary technique of Adler (J. Gen. Microbiol. 74:77-91, 1973). Chemotaxis was shown to be optimal when the capillaries were incubated at between 15 and 40 degrees C for 30 min; the optimal pH was between 7.0 and 8.2. The chemotactic response was proportional to the density of the suspension of bdellovibrios up to a density of 10(8) cells/ml. B. bacteriovorus was attracted to L-asparagine, L-cysteine, L-glutamine, glycine, L-histidine, L-lysine, and L-threonine. The possible roles of chemotaxis in the life of B. bacteriovorus are discussed.


Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation. The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo.


On the apparent inhibition of intramolecular activation of pepsinogen by pepsin substrates. Marciniszyn et al. (Marciniszyn, J., Huang, J. S. Hartsuch, J. A., Tang, J. (1976) J. Biol. Chem. 251, 7095-7102) have recently suggested an intermediate in the intramolecular activation of pepsinogen. As evidence, they showed apparent competitive inhibition of activation by globin, indication a pepsinogen-globin complex. Previous work had shown pepsinogen activation to occur very rapidly in the presence of high concentrations of hemoglobin, a very similar pepsin substrate (McPhie, P. (1974) Biochem. Biophys. Res. Commun. 56, 789-792). This contradiction has been resolved by a re-evaluation of the techniques used in the two investigations. The experimental conditions of Marciniszyn et al. Were inadequately defined to ensure denaturation of pepsin, a prerequisite of their method. A small decrease in pH, caused by the presence of extraneous protein, prevents this denaturation and leads to consistent underestimates of the rate of zymogen activation.


Circular dichroic and perturbation spectra of aromatic chromophores in rabbit tropomyosin. Topography of tyrosine residues. 1. Difference spectra of tryosyl residues obtained on denaturation of tropomycosin with urea or guanidinium chloride indicate that strong hydrophobic environments exist in the native coiled-coil state. 2. Solvent perturbation difference spectra indicate that tyrosyl residues are partially accessible to the solvent. The accessiblity decreases with increasing size of the solvent molecules. 3. Spectral pH titration of tyrosyl residues cannot provide information on the tyrosyl accessibility because conformational change accompanies the increase in pH. 4. Circular dichroism of tyrosyl and phenylalanyl residues is consistent with the effect of imposed conformational rigidity on the partially asymmetrical vibrational fine structure of the 1Lb absorption band of phenyl and benzyl chromophores; the effect is reduced by 70% on unfolding of tropomyosin.


Purification and properties of tRNA(adenine-1)-methyltransferase from rat liver. An S-adenosylmethionine-dependent tRNA(adenine-1)-methyltransferase has been purified 8,000-fold from rat liver. This preparation gives a single band on polyacrylamide gel electrophoresis and is stable in long term storage. The enzyme has a molecular weight of approximately 95,000. The single methylating capacity of this adenine-1 methyltransferase, using Escherichia coli tRNA2Glu, is methylation of the invariant adenine in the GTpsiC loop. The methylation reaction is dependent on added cation with 20 to 40 mM putrescine being most effective. The Km for S-adenosylmethionine was found to be 0.3 micron, while the Ki for the product inhibitor S-adenosylhomocysteine was 0.85 micron. The Km for tRNAMetf is 12 nM while that for tRNAGlu2 is 33 nM.


PZ-peptidase from chick embryos. Purification, properties, and action on collagen peptides. PZ-peptidase is an endopeptidase that cleaves the synthetic substrate developed for clostridial collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide). The peptidase has been purified to homogeneity from chicken embryos. The enzyme has a pH optimum of 7.5 to 8.5, and isoelectric point of 5.0, and a molecular weight of 77,000. The kinetic parameters at pH 8 and 37 degrees are: Km = 2 X 10(-4) M and Vmax = 4.2 mumol/min/mg of protein. The enzyme is inhibited by p-hydroxymercuribenzoate (100%), N-ethylmaleimide (60%), and chelating agents (40 to 60%). Maximum activity is attained in the presence of reducing agents and Ca2+, Sr2+, or Mg2+. The peptidase has no detectable action on casein, serum albumin, collagen, collagen alpha chains, various collagen peptides (alpha1)(I)-CB2, alpha1(I)-CB3, alpha1(I)-CB4), (Gly-Pro-Pro)10, or (Gly-Pro-Pro)5. It does catalyze the hydrolysis of the Hyp--Gly bond in the 17-residue collagen peptide alpha1(II)-CB6-C2 and it partially digested a mixture of collagen peptides of molecular weight 350 to 2500. A role of this peptidase in collagen breakdown appears to be restricted to a late stage when degradation products would fall in the range of 5 to 30 residues.


Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen. The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.


Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase. We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.


Role of coenzyme in aminotransferase turnover. The role of coenzyme in determining intracellular contnet of pyridoxal enzymes was assessed by analyzing effects of pyridoxine deficiency on the rapidly degraded, readily dissociable tyrosine aminotransferase (EC 2.6.1.5) and the slowly degraded, nondissociable alanine aminotransferase (EC 2.6.1.2) of rat liver. Synthesis of the tyrosine enzyme was reduced, leading to a decreased amount of this enzyme, much of which was present as active apoenzyme. Synthesis of alanine aminotransferase was unchanged but much of this enzyme was present as an inactive apoenzyme which retained immunological reactivity. Degradation rates of both enzymes (t1/2 about 1.5 h, tyrosine aminotransferase; about 3 days, alanine aminotransferase) were not changed in pyridoxine deficiency. Hence, interaction with coenzyme is not a significant determinant in intracellular degradation of these aminotransferases. Coenzymes dissociation and intracellular stability probably reflect structural features of the proteins which determine both properties.


In vitro reconstitution of demolybdosulfite oxidase by molybdate. Reconstitution of purified demolybdosulfite oxidase from rat liver has been achieved using inorganic molybdate as the source of molybdenum. The activation process has a pH optimum of 7.4 and is dependent on concentrations of molybdate and demolybdoenzyme. The reaction is inhibited by high concentrations of anions and by reduction of the demolybdoenzyme and requires incubation temperatures higher than 30 degrees. A reconstitution mechanism involving loss of tungsten and concomitant replacement with molybdenum in those demolybdo molecules which contain tungsten is supported by the following observations: (a) the extent of activation achieved by molybdate corresponds to the proportion of molecules in the preparation which contain tungsten. (b) Incubation of the demolybdoenzyme preparation at 37 degrees in the absence of molybdate results in progressive and concentration-dependent loss of ability to be reconstituted by molybdate and a corresponding but more rapid loss of tungsten from the enzyme. The reconstituted enzyme displays the molybdenum EPR signal characteristic of native enzyme and is inactivated by incubation at 42 degrees in a manner identical to native sulfite oxidase.


Lacerations of the flexor hallucis longus in the young athlete. Of ten patients with lacerations of the flexor hallucis longus tendon, nine were athletically inclined. In four, the laceration was not repaired and no disability was evident. A functioning flexor hallucis longus, therefore, does not seem to be essential for good push-off and balance in running sports. If both the flexor hallucis brevis and the flexor hallucis longus are lacerated and reconstitution of the longus is not possible, the brevis should be repaired, suturing the distal segment of the longus to brevis to prevent hyperextension deformity of the metatarsophalangeal joint. Hypersensitivity of the scar due to associated nerve injury is a frequent complication associated with laceration of the flexor hallucis longus.


Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase. Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.


Modulation of in vitro erythropoiesis. The influence of beta-adrenergic agonists on erythroid colony formation. Canine marrow erythroid colony growth is enhanced by agents linked to the adenyl cyclase/cyclic AMP (cAMP) system, including cAMP, a phosphodieterase inhibitor (RO-20-1724), cholera enterotoxin, and beta-adrenergic agonists. The adrenergic effect is mediated by receptors having beta2-subspecificity. These receptors are distinct from putative receptors for erythropoietin and those acted upon by cholera enterotoxin. In addition, the population of cells most responsive to beta-agonists is distinct from the majority of erythropoientin-responsive cells, perhaps representing a subpopulation of this class of cell. This demonstration of an adenyl cyclase-linked mechanism regulating mammalian erythroid colony growth provides a model for the modulation by other hormones or small molecules of in vitro and, perhaps, in vivo erythropoiesis.


Effect of ascorbic acid on arylsulfatase activities and sulfated proteoglycan metabolism in chondrocyte cultures. A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglycans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue. Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate. The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibiton. Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred. These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix.


Studies on the mechanism of ristocetin-induced platelet agglutination. Effects of structural modification of ristocetin and vancomycin. The mechanism by which ristocetin induces platelet agglutination in the presence of the von Willebrand factor was studied by chemically altering ristocetin and a similar antibiotic, vancomycin, by reaction with a water-soluble carbodiimide in the presence of glycine methyl ester at pH 4.75. Altering ristocetin's phenolic groups (which are thought to be important in its peptide-binding properties) resulted in a loss of both platelet-agglutinating and antibiotic activities. Restoring the phenolic groups with hydroxylamine restored both activities. Vancomycin has antibiotic and peptide-binding properties similar to ristocetin's, but differs structurally in having a free carboxyl group and thus a less positive charge at neutral pH. It does not induce platelet agglutination and actually inhibits ristocetin-induced agglutination. Reacting vancomycin with the water-soluble carbodiimide resulted in alteration of phenolic groups and permanent conversion of the carboxyl to a neutral derivative. Restoring the phenolic groups with hydroxylamine (but leaving the carboxyl neutralized) produced a compound with charge properties similar to ristocetin's which induced platelet agglutination as ristocetin does. These data suggest both a binding requirement (mediated through phenolic groups) and a strong positive charge requirement for ristocetin-induced agglutination. The data are consistent with a model wherein positively charged ristocetin binds, via its phenolic groups, to sites on the platelet surface and reduces the platelet's negative charge. This could reduce the electrostatic repulsion between platelets and/or between platelets and the negatively charged von Willebrand factor, and permit the macromolecular von Willebrand factor to cause agglutination by bridging between platelets.


Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. In normal plasma, the ratio of the procoagulant activity of factor VIII (VIII(AHF)) to that of the von Willebrand factor activity (ristocetin cofactor, VIII(VWF)) or factor VIII antigen (VIII(AGN)) is approximately 1, but ratios > 1 (e.g., VIII(AHF) > VIII(VWF) or VIII(AGN)) may be observed in some patients with von Willebrand's disease and in the "late" posttransfusion plasmas of patients with this disorder. The lability of VIII(AHF) was studied by incubating plasma, diluted 1:10 in imidazole buffer pH 7.1, for 6 h at 37 degrees C. With normal plasmas, 77+/-12% (SD) of the original VIII(AHF) activity remained after incubation. VIII(AHF) was labile (e.g., 35-55% residual activity) in the "late" posttransfusion plasmas (VIII(AHF) >> VIII(VWF)) of a patient with von Willebrand's disease, but not in the "early" posttransfusion plasmas (VIII(AHF) approximately VIII(VWF)). VIII(AHF) was also labile in the (base-line) plasmas of three patients with von Willebrand's disease in whom the ratios of VIII(AHF) to VIII(VWF) were 4.4 to 8.1, but not in the plasmas of four other patients in whom the ratio was approximately 1. The electrophoretic mobility of factor VIII antigen was increased in two of the three patients with labile VIII(AHF). In both of these patients, and in the late posttransfusion plasmas, labile VIII(AHF) activity could be stabilized by the addition of purified von Willebrand factor (lacking VIII(AHF) activity) or by hemophilic plasma, but not by plasmas of patients with severe von Willebrand's disease. Thus, VIII(VWF) may serve to stabilize VIII(AHF) and this might explain the posttransfusion findings in von Willebrand's disease.


Central neurotransmitter substances and aging: a review. Available evidence suggests that age is an important determinant of the levels of neurotransmitters and their associated enzymes and metabolites in some regions of the brain. Alterations at the synaptic level, or selective cell death in the brain, or both, may be implicated in progressive loss of function, behavioral changes and the onset of age-related diseases. Stimulation of hypothalamic neuroendocrine transducer cells by agents that alter neurotransmitter metabolism might provide some measure of control of the process of aging.


The hydrolysis of Zectran in buffered and natural waters. The analyses and rates of hydrolysis of Zectran, 4-dimethylamino-3,5-xylyl methylcarbamate are reported in aqueous media. These rates were measured in phosphate buffered, distilled water and in natural waters where tempteratures of 10 degrees, 20 degrees, and 28 degrees C. and pH values of 5.94, 7.00, and 8.42 were used. Intial concentrations of Zectran exceeded 60 mg/l (0.27 mMol 1(-1), whereas in the natural waters initial concentrations were 30 mg/l (0.135 mMol 1(-1). This study shows that the persistance of Zechtran in natural waters is, in large part, dependent upon the pH value and the temperature.


Immunofixation after electrofocusing: improved method for specific detection of serum proteins with determination of isoelectric points. I. Immunofixation print technique for detection of alpha-1-protease inhibitor. An improved method is described for direct localization of human serum proteins in polyacrylamide gel with simultaneous determination of their isoelectric points (pI). The technique employs isoelectric focusing in thin-layer polyacrylamide gels to separate the serum proteins and the pH gradient is read at 4 degrees C with a dual-membrane surface microelectrode. Subsequently, the desired proteins are localized by immunofixation in the gel or by immunofixation-printing onto cellulose acetate strips soaked in specific antiserum. No sectioning of the electrofocused gel is necessary, and the entire technique can be completed in less than 14 h. When this method is applied to the detection of the genetic variants of alpha-1-antitrypsin (alpha-1-protease inhibitor) (A1Pi system), the results indicate that it can be used to specifically localize serum proteins whose pI's differ by as little as 0.01 pH units. The resolution afforded is especially evident in the analysis of A1Pi M variants.


Autonomic neuroeffector junctions--reflex vasodilatation of the skin. A general model of the autonomic neuroeffector junction is proposed. In this model, emphasis is placed on the muscle effector bundle with electrotonic coupling between individual cells via gap junctions (or nexuses) and en passage release of transmitter from autonomic nerve varicosities. This release results in transmission to effector cells across junctional clefts ranging from about 20 nm in the vas deferens and iris to as much as 2000 nm in some large arteries. The ultrastructural identification of different autonomic nerve types is described. Current theories on the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized. Some speculations are made about the possible involvement of purinergic nerves in the innervation of vessels and mast cells in the skin, and whether this involvement results in a functional link between ATP, histamine, bradykinin, and prostaglandin in cutaneous vasodilatation. Another possibility considered as the basis for this reflex is the release of substance P from sensory (pain) nerve collaterals in the skin.


The Haarscheibe.

The Haarscheibe. The Haarscheibe is a specialized spot of epidermis containing many Merkel cell-neurite complexes. It is a highly sensitive, slowly adapting, modality-specific touch receptor occurring in all mammals. Its exact role in signaling sensation in human beings is undetermined, and the function of Merkel cells, with their distinctive cytoplasmic granules, in transducing mechanical force into neural action potentials remains to be determined.


Factors which influence blood platelet migration. Migration of human blood platelets in vitro was investigated by a modification of the capillary-tube migration chamber technique used to study the migration inhibition factor of macrophages. Platelets were packed in capillary tubes and incubated in autologous platelet-free plasma (PFP). The extent of migration was quantified by planimetry (measurement of the area of platelet migration visible by stereomicroscopy). Among the various anticoagulants employed, sodium citrate was most suitable for studying platelet migration. Optimal migration occured at 22 degrees to 37 degrees C and pH 7.2 to 7.4. Migration was inhibited by metabolic inhibitors such as iodoacetic acid, sodium fluoride, and 2,4-dinitrophenol, and inhibition was proportional to the dose of the agent added to the incubation medium. Mobility was also inhibited by cytochalasin B, which disrupts cellular microfilaments, at 1 microgram/ml PFP, but not by colchicine, a microtubule inhibitor, even at 40 microgram/ml of PFP. Light and electron microscopy showed that this inhibition was not ascribable to platelet clumping. These observations suggest that platelet mobility is an active process. The possible significance of platelet migration in hemostasis is discussed.


Intestinal absorption of alpha-tocopherol in the unanesthetized rat. The influence of luminal constituents on the absorptive process. 3H-alpha-tocopherol intestinal absorption was studied in the unanesthetized rat. The rate of alpha-tocopherol absorption remained linear over a wide range of concentrations (4 nM to 400 micrometer). Increasing the sodium taurocholate concentration in the micellar infusate up to 15 mM did not increase the rate of absorption of the vitamin. Addition of long-chain fatty acids to the micellar infusate decreased the absorption rate of the vitamin (p less than 0.05). The decrease was most significant (p less than 0.01) following the addition of the polyunsaturated linolenic (C18:3) acid. Increasing the hydrogen ion concentration in the perfusate increased the absorption rate of alpha-tocopherol. The present experiments in vivo support the conclusions drawn from in vitro uptake experiments which indicated that alpha-tocopherol is absorbed by a passive diffusion process. These experiments indicate that micellar expansion with polyunsaturated fatty acids interferes with the absorption of alpha-tocopherol and may result in deficiency of the vitamin.


The defect of uric acid metabolism in Eck-fistula rats. Because of the discovery of uric acid urolithiasis in rats after end-to-side portacaval anastomosis (PCA), uric acid metabolism was studied in these animals and in appropriate controls. Hyperuricemia and hyperuricosuria were observed in all experimental rats. The fraction of purine catabolites excreted in the urine as uric acid increased from an average of 4.8% to 15.3%. If 14C-uric specifically labeled at position 6 (6-14C-ua) was infused intravenously and the exhalation of 14CO2 was used to calculate a hepatic uric acid clearance, it decreased from 2.14 to 0.97 ml/min/100 gm despite a normal content of hepatic uricase activity as measured in liver homogenates. The fraction of the filtered amount of uric acid excreted in the urine increased from an average of 11% to 30%. Increased supersaturation of the urine with uric acid after PCA may be expected to contribute to the formation of uric acid urolithiasis. This investigation defines a hepatic and renal functional defect in uric acid metabolism which occurs as a result of the PCA.


Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts. BALB/c mice were treated with fractionated high dose (3,400 rads) total lymphoid irradiation (TLI), and given semiallogeneic (BALB/c x C57BL/Ka) or allogeneic (C57BL/Ka) bone marrow and/or skin allografts. TLI alone prolonged the mean survival time (m.s.t.) of C57BL/Ka skin grafts to 49.1 days (control, 10.7 days). Shielding of the thymus during TLI produced only a slight increase in graft survival (m.s.t., 19 days). TLI combined with splenectomy was no more effective than TLI alone. Infusion of 10(7) semiallogeneic or allogeneic bone marrow cells after TLI produced stable chimeras in 7/8 and 8/15 recipients, respectively. Chimeras were specifically tolerant to donor tissues, since C57BL/Ka skin grafts were accepted for more than 250 days, but third-party (C3H/He) skin grafts were rejected rapidly. In addition, chimeric lymphocytes responded to C3H/He and C3H. Q but not to C57BL/Ka cells in the one-way mixed leukocyte reactions. BALB/c C57BL/Ka chimeras showed no clinical evidence of graft vs. host disease. These findings may have application of clinical organ transplantation, since (a) the recipient treatment (TLI) has already been shown to be safe in humans, (b) donors and recipients can be completely allogeneic, and (c) bone marrow and skin graft survival was permanent (greater than 250 days).


The phosphoenol-pyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenol-pyruvate carboxykinase. The properties of pyruvate kinase (PK) and phosphoenol pyruvate carboxykinase (PEP CK), two enzymes that determine the preferrential accumulation of either succinate or lactate as endproducts of carbohydrate metabolism, are described in adult Hymenolepis diminuta. PK activity at Vmax and Km levels of PEP was unaffected by ATP, alanine, FDP4, OR H+ ions, but was inhibited by 50% at 6.3 mM L-lactate and 30 mM HCO3. The addition of 30 mM HCO3 increased the Km(PEP) by 6-fold but did not alter the Vmax. The inhibition of PK by HCO3 cannot be explained entirely by an effect of ionic strength, but probably represents a specific modulator-enzyme interaction. Under similar conditions PEP CK was maximally activated. Although L-lactate inhibited PEP CK (Ki(lac) = 1.8 mM), this effector may play a minor role in regulation of PEP flux. These results implicate the poise of the HCO3-:CO2 system as a major determiner  of endproduct accumulation in H. diminuta.


The biosynthesis of a choline nucleotide by a cell-free extract from Streptococcus pneumoniae. Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.