H2-Histamine receptor blocking agents in the Zollinger-Ellison syndrome. Experience in seven cases and implications for long-term therapy. H2-Histamine receptor blocking agents metiamide and cimetidine were assessed in seven patients with Zollinger-Ellison syndrome (serum gastrin greater than 300 microgram/ml, basal acid output greater than 15 meq/h, ratio of basal acid output to maximal acid output greater than 0.5). Intravenous or oral administration of the drugs lowered acid secretion by at least 70% in all cases. Subsequent treatment of six patients for 3 to 15 months (oral therapy) and one patient for 1 month (intravenous therapy) showed that the drugs abolished symptoms in all seven, abolished diarrhea in five, allowed ulcer healing in six, and were well tolerated without adverse effects in seven. No patient failed to respond to the drug, although one died from tumor progression and two required total gastrectomy for complex reasons. The results indicate that patients with Zollinger-Ellison syndrome can be managed medically and, in light of current mortality trends, gain little from the extra risks attending total gastrectomy.


Protective effect of cimetidine on aspirin-induced gastric mucosal damage. Aspirin alters the gastric mucosal barrier as measured by ionic flux and potential difference. The effect of cimetidine on aspirin-induced alterations in gastric mucosa was studied in five normal male volunteers. Aspirin effects were studied with and without previous treatment with cimetidine. Mean (+/- SEM) basal potential difference was -48 +/- 1 mV. After 600 mg of aspirin in 1 dl of isotonic saline, potential difference decreased in 10 min to -39 +/- 1 mV (P less than 0.001) and returned to baseline within 60 min. Control biopsies showed 2% damaged mucosal cells compared with 20% damaged at the time of maximal drop in potential difference (P less than 0.001) after aspirin. Recovery to 9% damage occurred by 60 min. In subjects pretreated with 300 mg cimetidine, potential difference rose during 1 h to -62 +/- 1 mV (P less than 0.001). After aspirin potential difference fell to -48 +/- 1 mV compared with -39 +/- 1 mV with aspirin alone (P less than 0.01) and returned to -62 +/- 1 mV at 60 min. The cimetidine-treated group showed 4% mucosal damage at the peak potential difference fall after aspirin, significantly less (P less than 0.02) than in the untreated subjects.


Sjögren-type syndrome after allogeneic bone-marrow transplantation. Four patients, treated for hematologic disorders with bone-marrow transplants from HLA-identical siblings, spontaneously complained of dry eyes 8 to 12 months after transplantation. Four allograft recipients and two recipients of autologous bone-marrow transplants were evaluated for xerophthalmia and xerostomia. Three allogeneic marrow recipients had evidence of keratoconjunctivitis sicca, and two had decreased parotid gland function. All four allograft recipients had minor salivary gland histopathology identical to that of Sjögren's syndrome. The severity of symptoms and histologic lesions corresponded with the severity of chronic graft-versus-host disease. In addition, one patient developed sclerodermatous skin changes, another had discoid lupus erythematosus, and two patients had laboratory evidence of cholestasis. None of the patients had autoantibodies but all had hypergammaglobulinemia. In contrast, none of the recipients of autologous bone marrow had clinical, laboratory, or histologic findings resembling Sjögren's syndrome.


Scleroderma, Sjögren-like syndrome, and chronic graft-versus-host disease. A patient with acute myelogenous leukemia treated with an allogeneic bone marrow transplant developed acute graft-versus-host disease manifested by severe diarrhea, hepatitis, and a cutaneous eruption. As the graft-versus-host disease progressed to the chronic phase, the patient developed marked cutaneous sclerosis and symptoms of xerophthalmia and xerostomia. Biopsy of his indurated skin showed features of both graft-versus-host disease and scleroderma. Results of Schirmer's tests, corneal fluorescent studies, parotid flow-rate testing, and a lip biopsy were consistent with Sjögren's syndrome. Possibly, activated lymphocytes may have a role in the pathogenesis of graft-versus-host disease, scleroderma, and Sjögren's syndrome.


Penicillic acid production in submerged culture. Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.


Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum. A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.


The distribution of enzymes in the epidermis of the domestic cat. With the help of enzyme histochemical methods, the distribution and activities of several oxidative and hydrolytic enzymes in the epidermis of the densely-haired domestic cat have been studied. Distinct oxidative enzyme activity could be demonstrated in all epidermal layers except the stratum corneum. Among the hydrolytic enzymes investigated, strong reactions for non-specific esterases were visible especially in the str. granulosum and the basal lamellae of the str. corneum. Positive reactions for cholinesterases could not be observed in the cat epidermis. The results are discussed in relation to corresponding investigations on the epidermis of sparsely-haired mammals, e.g. the domestic pig and man. Generally the enzyme pattern of the cat epidermis only shows limited parallels to man, especially where esterase distribution is concerned.


[Acid-base balance immediately after administration of an oral contraceptive (author's transl)]. An increase in breathing activity has often been described for the secretory phase of the menstrual cycle, for gravidity and after intramuscular application of corpus luteum and follicle hormone. The reason for this has been assumed to be a direct interaction of the hormones in the respiratory center. A shortly published paper dealing with 50 women under long-term treatment with a combination of oestrogenes and gestagenes for oral contraception leads the author to the assumption, that a metabolic acidosis is the primary reaction, followed by an increase in breathing activity for respiratory compensation. In a long-term study it is difficult to differentiate between primary and secondary effects. Therefore in the present experiments arterial blood has been analyzed for PaO2, PaCO2, pH and hemoglobin in 49 subjects on day 1 without hormone administration and on day 2 prior to (9.00 a.m.) after (11.00 a.m., 1.00 p.m., 4.00 p.m.) administration of a combination of oestrogene and gestagene. From the measured values O2-saturation, standard-bicarbonate, buffer bases and base excess have been calculated by the Thews nomogram. By comparison of the results before and after hormone administration it was shown, that the primary effect is a metabolic acidosis, partly compensated during the next 7 h by respiratory adaptation. There is no indication for a primary respiratory alkalosis.


Influence of cobra venom (CoF) and pneumococcal S III capsular polysaccharide on immunologic humoral response and visceral distribution of antigens. The immunologic response to sheep erythrocytes was compared in mice treated with Cobra venom (CoF) and with pneumococcal capsular antigen S III. Suppression of the immunologic response in both cases was attributed to the ability of both tested substances to activate the third component of complement. CoF and S III both changed the organ localization of labeled antigens capable of activating the complement system. No evidence for an alternative pathway by which S III could activate the complement system was found. On the basis of the literature data and experimental findings, the properties of CoF and S III were compared in the light of their ability to suppress the immunologic response.


Variation of human intestinal gamma-glutamyl transpeptidase in ontogenetic development. Activity of gamma-glutamyl transpeptidase in human intestines was measured against alpha-naphthylamide and 12 gamma-glutamyl amino acids and peptides as substrate. Distinctly altered activity was found to accompany ontogenetic development. The ratio of the transpeptidase activity tested against monoglutamyl substrates in the intestines of 7-month fetuses, newborns and adults was 15:1:4, whereas the ratio of gamma-glutamyl cyclotransferase activities in the same age groups was 1-0:1-2:1-6. Distinct differences were found in resistance to heating, sensitivity to L-serine plus borate, and other effectors, and electrophoretic mobility, between fetal gamma-glutamyl transpeptidase and the enzyme from adults, which supports the hypothesis of existence of two forms of the enzyme in the human intestines. The results suggest involvement of gamma-glutamyl transpeptidase in the pathomechanism of celiakia in children.


Reaction of antibody with gamma-glutamyl transpeptidase. I. Isolation of the human enzyme and inhibition of its activity by antiserum. Use of a four-step method of purification from human kidneys yielded gamma-glutamyl transpeptidase with specific activity 330 times higher than activity in the homogenate. The enzyme from human liver was also purified, and from urine two fractions of it were separated. From rabbits immunized with the enzyme from human kidney, antiserum was obtained which inhibits and precipitates the human-derived enzyme, and in a low degree the enzyme purified from bovine kidneys. This inhibition was independent of incubation time and precipitate formation, but dependent on the amount of antiserum used. A gamma-glutamyl transpeptidase complex with antibody was isolated on a column with CM-cellulose with electrophoretic mobility different from that of the native enzyme.


[Heterogenity of the cryptorchid syndrome. Study of the pituitary gonadotropin reserve in 50 prepuberal bodys]. As compared to a control group, the study of the pituitary reserve of gonadotrophins in 50, not yet puberous, cryptorchid boys, evoked that there is a heterogeneity in cryptorchism. The LH pituitary reserve may be insufficient, normal or increased; the FSH pituitary reserve may be normal or above normal. A longitudinal study of these patients, as well as the correlations with ultra-structural and histologic studies should lead to a better understanding of the significance of the hormonal abnormalities encountered in cryptorchism, and to a better appreciation of the associated testicular changes.


Cobalt-activated acylase activity in experimental toxic liver damage. Co++-activated acylase activity was studied in the blood serum and liver homogenates from rabbits poisoned with CCl4 in doses of 0-5 g/kg body weight intraperitoneally. Activity of this enzyme in serum increased on the first day after poisoning, but the rise was of short duration. Increased serum activity of the enzyme was accompanied by an increase in acylase activity in liver homogenates, which persisted to the ninth day, i.e. to the end of the observation period, when serum acylase activity returned to normal. Co++-activated acylase activity in serum was not correlated with its activity in liver homogenates. Co++-activated acylase activity was significantly correlated with AlAT and GGTP activities, but acylase and LAP were not correlated.


[Pathomorphology of the vessels of the microcirculatory bed of the serous membranes in systemic allergic vasculitis]. The impregnation method and micropreparation were used to study the morphological changes in vessels of the microcirculatory channel of serous membranes in systemic allergic vasculites. Inflammatory and sclerotic processes in arterioles, precapillaries, capillaries, postcapillaries, and venules were found to be accompanied by regeneration changes and complicated adaptative alteration of the intact structures of the terminal vascular bed. The paper also presents morphological data reflecting decompensation processes in local lymph and blood flow.


The gold solder, gold alloy interface.

The gold solder, gold alloy interface. With soldering the physical properties of the basis metal must be maintained and a minimum of molten metal is used. An examination of joints in 17 gauge platinized gold wire is made with 18 k gold solder following quenching immediately after flow and cooling in air; there is a widening of grain boundaries and intergranulation penetration in the latter method. A comparison with the cast gold joint is made. Some inclusions were noted.


Self-association of troponin.

Self-association of troponin. Ox muscle troponin was shown by equilibrium- and velocity-sedimentation studies to undergo concentration-dependent dissociation into its constituent subunits as well as self-association in imidazole buffers, pH 6.9. The extent of troponin association was found to be strongly dependent on ionic strength and also to exhibit a dependence on pH and temperature; under conditions physiological in regard to pH, temperature and ionic strength the extent of polymerization of troponin is considerable in 2 mg/ml solutions. The ability of polymeric troponin to bind to tropomyosin has been inferred from studies of mixtures containing actin-tropomyosin and an excess of troponin over the amount required for the normal 7:1:1 actin-tropomyosin-troponin complex. These findings should be relevant to studies of reconstituted actin-tropomyosin-troponin preparations, since they signify possible chemical as well as physical differences between the gel, paracrystalline and filamentous states of the complex that result from adoption of different preparative procedures for analogues of the native thin filament.


The state of aggregation of red deer (Cervus elaphus L.) beta-lactoglobulin preparations near neutral pH. 1. The state of aggregation of four red-deer (Cervus elaphus L.) beta-lactoglobulin preparations and a control ox beta-lactoglobulin A preparation was studied by sedimentation-equilibrium experiments at pH 6.5 and 20 degrees C. 2. Three of the deer preparations and the ox control each behaved as a monomer-dimer system, with a value of log K (where K is the association constant in litres/mol) in the range 5.4-5.5. 3. When one of these deer preparations was examined in the presence of dithiothreitol, log K appeared to decrease to 4.5.4. One deer preparation, comprising recovered material, appeared to have undergone irreversible changes and to behave like a non-equilibrating system containing monomer, dimer and trimer. 5. The sedimentation-equilibrium properties of the deer monomer was studied in 6M-guanidine hydrochloride at pH 7.0; the mol.wt. was 17600, the second virial coefficient was 3.4 x 10(-3) ml - mol - g-2, and the apparent partial specific volume 0.724 ml/g, a value indicating an appreciable decrease in volume on dissociation and denaturation.


Tritium effect in peroxidation of ehtanol by liver catalase. 1. Simultaneous determination of the rate of appearance of 3H in water from [(1R)-1-3H1] ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37 degrees C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25 degrees C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. 2. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the two rates measured in the assay. 3. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supports the view that both values are close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. 4. The constancy of the isotope effect under various conditions makes it possible to use it for interpretations in vivo. 5. It was established that beta-D-galactose dehydrogenase exhibits B-specificity towards the nicotinamide ring in NAD.


Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers. The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.


Comparison of lorazepam and diazepam as premedicants. A double-blind random study compared lorazepam with diazepam as i.m. premedicants in 84 healthy women undergoing uterine curettage. Anxiety, assessed by a self-rating test by the patient and by a trained observer, was reduced 90 min after both lorazepam (P less than 0.001) and diazepam (P less than 0.01). There was more sedation and a longer recovery time after lorazepam than after diazepam. Amnesia at 24 h after operation (lack of recall rather than lack of recognition) was greater after lorazepam. There was transient local discomfort at the site of the injection in most patients in both groups, but no serious effects. Local erythema was present in 12 patients who received lorazepam and 10 who received diazepam 90 min after the injection, disappearing after 24 h in the former group but remaining in the latter. The incidence of nausea, vomiting and headache in both groups was small and similar, but there was more restlessness and dizziness after diazepam in the early recovery period.


Epidermal nucleases. III. The ribonucleases of human epidermis. Sodium acetate and sulphuric acid extracts of human epidermis can each be separated by chromatographic techniques into three or more fractions with ribonuclease activity. Eight of these fractions were compared with respect to molecular weight, pH activity profile, polyribonucleotide hydrolysis, and activity in the presence of low levels of spermidine. Sodium acetate and sulphuric acid extracts were also prepared from callus and from psoriatic lesions and compared with extracts from normal epidermis for their response to exogenous spermidine. All eight human epidermal ribonuclease fractions studied had an apparent molecular weight of 15,000 daltons. Seven of the ribonuclease fractions were optimally active at alkaline pHs (pH 7.3-7.6 in sodium phosphate and pH 8.I in Tris-HCl) while the eighth ribonuclease was most active at pH 5.6 in a citrate-phosphate buffer. All enzymes hydrolyzed polycytidylic acid and five also hydrolyzed polyuridylic acid. None hydrolyzed polyadenylic acid. Seven of the eight ribonucleases studied exhibited greater activity in the presence of added spermidine. The extracts from psoriatic scales showed markedly elevated ribonuclease levels which could not be raised further by the addition of spermidine.


Enzymes of galactose metabolism in human hair roots. Micro-methods, making use of radioactive substrates, are described for the quantitative estimation of galactokinase and galactose-1-phosphate uridyl transferase activities in lysates of hair roots obtained from the human scalp. Enzyme assays can be carried out with fractions of one hair root. Both enzymes have been investigated with regard to stability, pH optimum and Michaelis-Menten constants. Along with similarities there were also certain differences as compared to galactokinase and galactose-1-phosphate uridyl transferase activities in other human tissues. The findings were used to optimise and standardise a radiochemical micro-assay for both enzymes in human hair root lysates, applicable to carrier detection studies in galactosaemia, an inborn error of carbohydrate metabolism. Because they can easily be obtained, hair roots are a very suitable biopsy material for both fundamental and diagnostic investigations of these enzymes.


Tobacco mosaic virus protein: sedimentation equilibrium studies of the initial stages of polymerization. The lowest stages of polymerization of tobacco mosaic virus protein were studied by means of high-speed sedimentation equilibrium experiments. Several distinct modes of polymerization were found. At pH 7.1 the expected monomer-trimer-higher polymer equilibrium was observed--very little dimer was detected at this pH. At pH 7.5, however, a strong dimerization was observed--neither monomer nor trimer was detected at this pH. An octamer appeared to be the only species present other than the dimer. When 0.01 M beta-mercaptoethanol was added to the solvent pH 7.5, the dimer was dissociated, resulting in a monomer-trimer association. The dimerization may be the basis for the larger "doubled" polymers formed by the protein at alkaline pH, while the octamer may correspond to the 8S peak frequently observed in sedimentation velocity experiments at alkaline pH. On the other hand, the monomer-trimer-higher polymer equilibrium may correspond to the single helix formed by the protein at slightly acid pH and to the combination of 4S and 20S peaks seen in sedimentation velocity experiments at slightly acid pH.


Factors affecting the adenosine triphosphate induced release of iron from transferrin. The release of iron from transferrin was investigated by incubating the diferric protein in the presence of potential iron-releasing agents. The effective chemical group appears to be pyrophosphate, which is present in blood cells as nucleoside di- and triphosphates, notably adenosine triphosphate (ATP). An alternative structure with comparable activity is represented by 2,3-diphosphoglycerate. Neither 1 mM adenosine monophosphate (AMP) nor 1 mM orthophosphate released iron from transferrin. The ATP-induced iron-releasing activity was dependent on weak acidic conditions and was sensitive to temperature and sodium chloride concentration. The rate of iron release rapidly increased as transferrin was titrated with HCl from pH 6.8 to 6.1 in the presence of 1 mM ATP and 160 mM NaCl at 20 degrees C. Iron release from transferrin without ATP was observed below pH 5.5. Ascorbate (10(-4) M) reduced Fe(III), but only after iron release from transferrin by a physiological concentration of ATP. A proposal for the mechanism of iron release from transferrin by ATP and the utilization of reduced iron by erythroid cells is described.


Gated proton conduction via the coupling factor of photophosphorylation modified by N,-N-orthophenyldimaleimide. The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient greater than 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.


Effects of dietary vitamin B-2 and vitamin E on the delta9-desaturase and catalase activities in the rat liver microsomes. The effects of dietary vitamin B-2 and vitamin E on delta9-desaturation  of stearoyl-CoA, catalase, glutathione peroxidase, superoxide dismutase and electron transport components in rat liver microsomes have been investigated. delta9-desaturase activities were decreased on diets deficient of vitamin B-2, E and supplemented with E. Among the peroxide-scavenging enzymes, only the catalase activity in microsomes correlates significantly with delta9-desaturase activity. In vitro addition of bovine catalase had no effect on microsomal delta9-desaturase activity on control diet. However, it enhanced the delta9-desaturation in microsomes on vitamin B-2-deficient diet which contained low catalase and high superoxide dismutase activities, compared to those in microsomes of control diet. It is suggested that the hydrogen peroxide-generating and -decomposing systems may play an important role on the delta9-desaturase activity in microsomes.


The polymerization pattern of zinc(II)-insulin at pH 7.0. Sedimentation equilibrium experiments were conducted at pH 7.0 using solutions of bovine insulin containing 2 mol of zinc(II) ions per six base-mol of insulin. A detailed analysis of these results revealed the existence of a stable zinc-insulin hexamer together with linked polymerization reactions. Specifically these are a background polymerization of zinc-free insulin as previously described by Jeffrey et al. ((1976) Biochemistry 15, 4660--4665) and a slight tendency for the zinc-insulin hexamer to undergo indefinite self-association. Equilibrium constants governing these reactions are reported together with equations which permit calculation of the composition of the solution at any given total concentration. Comment is made on the possible biological significance of this linked polymerization pattern, and on the likely identity of the structure of the stable zinc-insulin hexamer with that previously reported from X-ray crystallographic studies.


On the molecular conformation of human haemopexin. I. Reactivity of the tyrosine and tryptophan side chains. The reactivity of the aromatic side chains of Tyr and Trp in human haemopexin were studied by chemical modifications and analysis of spectrophotometric titration curves. It has turned out that: 1. Under non-denaturing conditions the aromatic rings of Tyr resisted both acetylation and nitration. 2. Three indole groups of Trp reacted with the Koshland agent, without the native conformation of the protein being markedly affected (CD spectra). 3. Oxidation by N-bromosuccinimide split the peptide chain and the molecular conformation collapsed. 4. The Tyr residues could be placed into three classes, according to their pK values: 2 (or 1 in the haem-haemopexin complex) were normally accessible to titration, 5 were masked and the remaining 7 (or 8) were buried. 5. The spectrophotometric titration curve could not be analysed in terms of the Linderstrøm-Lang equation. The findings 1 to 3 refer to both haemopexin and its complex with haem; the spectrophotometric titration curves of the two molecules are very similar too. Consequently, the binding of haem is not associated with a profound alteration of the molecular architecture. The generally low reactivity of the side chains studied indicates that the hydrophobic peptide core of this glycoprotein is a compact one, very restricted in its contacts with the environment.


Functional properties of normal and sickle cell hemoglobins in polyethylene glycol 6000. The functional properties of human hemoglobin A and S were studied in concentrated solutions of polyethylene glycol. Polyethylene glycol solutions are frequently used as media for protein crystallization. In particular, sickle cell hemoglobin, which does not make X-ray quality crystals in high salt solutions, will form high-quality crystals in polyethylene glycol. Comparison of the functional properties of normal and sickle cell hemoglobin in polyethylene glycol show that pH, anion effects and cooperativity of ligand binding are largely unaffected by polyethylene glycol. This suggests that the crystals grown in this medium are representative of the native structure.


Biosynthesis of beta-glucans by cell-free extracts from Saccharomyces cerevisiae. Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-14C]glucose into beta-1,3-glucans which contained a significant proportion of beta-1,6-glycosidic linkages. When GDP-[U-14C]glucose was used as substrate only trace amounts of glucose were incorporated. Activity of beta-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. Beta-glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of beta-1,6-glycosidic linkages respectively. A marked decrease in the activity of beta-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.


[Light-dependent uptake of hydrogen ions in chloroplasts and chromatophores: effects of hearing, solvents and detergents]. The effects of heating, organic solvents and detergents on the light-dependent hydrogen ion uptake in chloroplasts and chromatophores and the coupled photophosphorylation were compared. It was shown that the membrane structure of the chromatophores is much more stable than that of the chloroplast thylacoids. The activation of the pH function in the chromatophores in the presence of low concentrations of diethyl ether and detergents was noted. The effects observed may be due to the changes in the physico-chemical properties of the membranes rather than to the direct effect on the photosynthetic electron transfer chain.


[Nad-dependent formate dehydrogenase from methylotrophic bacteria. Characteristics and stability of soluble and immobilized enzyme]. pH-dependency is studied of kinetic parameters of the reaction catalyzed by NAD-dependent formate dehydrogenase from methylotrophic Bacterium spl strain. Values of Km for NAD and formate, and also of maximum reaction rate are found not to change within the pH range from 6 to 9. Role of SH-groups in the development of the enzyme catalytic activity and the effect of different factors on stability of soluble and immobilized enzyme forms are investigated. Molecular weight of the enzyme (70000), extinction coefficient and catalytical constant (6 s-1) are determined.


[Photoinduced inhibition and stimulation of respiration in cells of Halobacterium halobiums kinetics, action spectra, relationship to photoinduction of deltapH]. Along with the inhibition illumination also causes the stimulation of the respiration of H. halobium R1 cells. When light is switched off photoinhibition of respiration (PIR) decays much faster (tau 1/2=12 sec) than photostimulation (PSR) (tau 1/2=60 sec). This allows the evaluation of the contribution of the each phase into the total change of the respiration rate. PIR prevails in neutral and alkaline media (at pH 6.8 the amplitude ratio of PSR/PIR=0.3). At the same conditions light induced alkalization of the medium is observed, which at high light intensities is followed by acidification. The half rise time of PIR is 0.5 divided by 0.8 sec under excitation with short light flashes at 18C and pH 6.8. When pH of the medium is reduced the rate of dark respiration decreases, PSR amplitude increases, PIR is almost not changed and light-induced alkalinization of the medium decreases. At pH 5.5 PSR prevails: at light of 10(5) erg/(cm(2).s) the ratio PSR/PIR=2. The maximum value of PIR and PSR at 18C reaches 20-30 percent of the dark respiration level. Uncouplers (CCCP, DNF) and inhibitor (DCCD) of phosphorylation suppress PIR and light induced alkalinization of the medium and significantly (5-7 times at Ph 6.8) increase PSR and light induced acidification. The action spectra show that bacteriorhodopsin is responsible for all the observed light induced changes of O2 and H+ exchange; carotinoids do not participate in sensibilization. It is suggested that photophosphorylation is necessary for PIR and that PSR is caused by the rise of internal pH due to light induced efflux of protons mediated by bacteriorhodopsin.


[Form of the tropocollagen macromolecule]. It has been shown with electron microscope that the form of the tropocollagen macro-molecule in solution is determined by hydrogen ions concentration. It is proposed that in native collagen fibrils the tropocollagen macromolecules have an unknown before form of rods with the thickened central part. Their length is about 1000 A. The known extended form of the tropocollagen macromolecule (the rod of 2800 A length) is probably transformed to the described form by the folding of the macromolecule. The origin of cross striation of the collagen fibril is discussed.


The early assessment for individualized treatment in the prune belly syndrome. Patients with prune belly syndrome present a spectrum of abnormality, both in the abdominal wall and the urinary tract. Ureteral pathology has characteristic features and the ureter may be more severely involved at the bladder end than in its upper portion. Early neonatal investigation is required to determine which patient can be treated in a conservative manner and which require neonatal reconstruction or temporary vesical or upper tract drainage.


[Effect of dipyroxime on the concentration of nicotinamide coenzymes and adenylate nucleotides in the myocardium and liver of rats poisoned with phthalophos]. Experiments were conducted on rats; in depression of blood cholinesterase activity by 68.6 percent phthalafos proved to decrease the myocardial nicotinamide coenzymes content on account of reduction in the amount of the oxidized forms. In the liver phthalafos diminished the content of oxidized and reduced forms of nicotinamide coenzymes, decreased the level of adenylic nucleotides chiefly at the expense of ATP. Diproxim prevented the changes caused by phthalafos in blood cholinesterase reactivation to 47.5 percent. It is supposed that the capacity of diproxim to normalize the oxidative processes in the cell by acting upon the nicotinamide coenzymes and adenylic nucleotides underlies its antidote action.


Ammonia absorption from the canine colon after portacaval shunt. Ammonia absorption was studied from Thirty-Vella colon loops in 6 dogs. Four underwent an end-to-side portacaval shunt and it was shown that absorption of ammonia from the colon significantly increased postoperatively. Absorption of ammonia from various solutions was also measured before and after portacaval shunt and it was shown taht absorption was increased from a high pH solution and from a solution with a high bicarbonate content and reduced from a low pH, low osmolality and high osmolality solution. Increased deposits of stainable iron were demonstrated in the livers of dogs following portacaval shunt.


Sequelae after the intravenous injection of three benzodiazepines--diazepam, lorazepam, and flunitrazepam. The occurrence of thrombosis and phlebitis after intravenous injection of 10 mg diazepam, 4 mg lorazepam, or 1-2 mg flunitrazepam was studied on the second or third and the seventh to 10th days. A significantly higher incidence occurred with all drugs on days 7 to 10 than on days 2 and 3. Painless thrombosis occurred much more often with diazepam than with the other two benzodiazepines. Its incidence was greater in small hand or arm veins than in large antecubital vessels. Lorazepam and flunitrazepam therefore have clear advantages over diazepam.


Evidence concerning the anatomical location of the dopamine stimulated adenylate cyclase in the substantia nigra. The dopamine (DA)-sensitive adenylate cyclase in the substantia nigra was assayed in rats which had been subjected to 3 different kinds of brain lesion: (1) unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle; (2) unilateral lesions of the descending strio-nigral and pallido-nigral projections; (3) total lesions of the serotoninergic raphe-nigral pathway. Lesions of the medial forebrain bundle causing 97% depletion of striatal DA, 72% depletion of nigral tyrosine hydroxylase, and no change in nigral glutamate decarboxylase (GAD), resulted in no change in basal or DA-stimulated cyclic AMP production ipsilateral to the injection. Lesions of the globus pallidus, causing 70% and 79% reductions in GAD and substance P respectively in the ipsilateral nigra, produced a reduction in basal cyclic AMP production and abolished the normal increase in cyclic AMP produced by DA on the side of the lesion. Lesions to the dorsal and median raphe nuclei did not affect the normal DA-sensitive adenylate cyclase response in the nigra. The results suggest that one of the neurotransmitter functions of DA in this brain region may be to modulate the release of psi-aminobutyric acid (GABA) or substance P from synaptic terminals afferent to the nigra.


The effects of postnatal hyper- and hypothyroidism on the development of D-amino acid oxidase in rat cerebellum and brain stem. Treatment of rats with propylthiouracil for the first 30 days of postnatal life drastically retards the ontogenesis of D-amino acid oxidase in the brain stem and cerebellum. There is a marked terminal deficit of D-AAO in both the brain stem (--64%) and cerebellum (--67%) at 94 days (adults) despite the near euthyroid status at this age. If initiated early enough, thyroxine replacement therapy reverses the effects of PTU on the development of D-AAO. Hyperthyroidism significantly accelerates the development of D-AAO in both brain stem and cerebellum. Nonetheless, animals treated with thyroxine the first month of life display a net deficit of cerebellar D-AAO content in adulthood. The results are discussed in terms of the localization of D-AAO in cell types especially sensitive to thyroid hormone: (1) a cell type which is among the last to derive from the external germinal zone in the developing cerebellum, and which in the adult is located adjacent to the Purkinje cell soma; and (2) mossy fiber neurons and cerebellar glomeruli.


[Comparative affinity of several standard anti-secretory agents for the intestinal cholinergic receptors of of rats and dogs]. Anticholinergic potentialities of four standard anti-secretory drugs (N-methyl-scopolammonium methylsulfate, atropine, diphemanil and prifinium) have been investigated with the help of molecular pharmacology techniques. The results gained with two different agonists (acetylcholine and carbachol) on rat and dog intestinal cholinergic receptors-jejunum and duodenum respectively-show : 1) That relative potentialities of the anticholinergic drugs (pA2) as well as those of the cholinomimetic agonists (pD2) are markedly modified according to which effector is used. 2) The N-methyl scopolammonium methylsulfate remains in any event the most potent anticholinergic drug investigated.


Sodium nitroprusside: factors which attenuate its action. Studies with the isolated gracilis muscle of the dog. In a laboratory preparation of the isolated, acutely denervated, and separately perfused canine gracilis muscle we have made the following observations: 1. At physiological pH, sodium nitroprusside significantly decreases the vascular resistance; 2. At physiological pH, cyanide significantly attenuates the effect of sodium nitroprusside; 3. In an acidaemic milieu, our data suggest that the effect of sodium nitroprusside may be attenuated. We speculate that patients who manifest resistance to the hypotensive effect of sodium nitroprusside may not normally eliminate the cyanide that is released from the biodegradation of sodium nitroprusside. They accumulate free cyanide which interferes with the action of sodium nitroprusside at the receptor level, leading to administration of more nitroprusside and setting in motion a positive feedback vicious cycle. When one is faced with the problem of an abnormal response to sodium nitroprusside in a fit patient, although many factors may be involved, we suggest that the possibility of rising blood cyanide levels and acidosis be given high priority.


Sex-limited and sex-modified genetic defects in swine--cryptorchidism. Review of published data suggests that cryptorchidism in Chester Whites and Yorkshires is caused by completely penetrant, recessive genes at two autosomal loci; in Lacombes, multifactorial modes of inheritance are more plausible. Different genetic systems control presence of the trait vs. number of sides affected; left testes are retained more often in almost all samples. Contrary to previous claims prenatal viability of homozygous males and females is normal. Genes causing cryptorchidism are unlikely to affect many other malformations in either sex, but pleiotropy is suggested to extend to economic traits such as conformation in females.


Medullary carcinoma of the human thyroid in monolayer culture: morphological and cytochemical correlations. Mechanically or enzymatically dissociated cells from three human medullary thyroid carcinomas (MTC) were grown in monolayer culture for periods up to seven months. Cultures of each tumor contained clusters of small epithelial-like cells which were readily identified by phase contrast microscopy. Immunocytochemical studies and electron microscopy showed that these cells contained abundant calcitonin and numerous secretory granules. Amine-storing mechanisms were also demonstrable in these cells by formaldehyde-induced fluorescence. Homogeneous cultures of epithelial-like cells showed no evidence of transitions into fibroblast-like cells. Addition of thyroxin to the tissue culture medium appeared to promote survival of epithelial-like cells in cultures of one tumor. The ability to morphologically recognize cultured cells with endocrine activity should facilitate establishment of human MTC lines for biochemical and physiological studies.


Placenta-like alkaline phosphatases from human osteosarcoma cells. Hormone-induced alkaline phosphatases in human osteosarcoma cells (LM) were extracted and purified. Characterization of the purified enzyme showed two distinct isoenzymes. One isoenzyme was heat labile, was homoarginine inhibited, and had the electrophoretic migration of alkaline phosphatase of human osseous origin. Immunodiffusion showed that this isoenzyme reacted positively only against anti-bone alkaline phosphatase antibodies. The second isoenzyme was heat stable, was inhibited by phenylalanie, and had the same electrophoretic migration as did alkaline phosphatase extracted from mature normal human placenta. This second isoenzyme had the same antigenicity as did the normal placental enzyme. Like the D-variant placental phenotype, this second isoenzyme was inhibited by L-leucine and ethylenediaminetetraacetic acid.


Possible sites of origin of human plasma ribonucleases as evidenced by isolation and partial characterization of ribonucleases from several human tissues. The ribonucleases (RNases) present in a number of human tissues, including heart, brain, lung, and kidney, were purified, partially characterized, and compared in their properties to the previously described RNases from human liver, spleen, pancreas, and serum. The enzymes appeared to fall into two major classes: liver-spleen type RNase and plasma-type RNase. These two types of enzymes were present in varying proportions in all tissues examined. The extent to which the tissues studied possibly contribute to serum RNase levels is discussed.


Mechanisms of hypercapneic pulmonary hypertension. The mechanisms and potential mediator of hypercapneic pulmonary hypertension are incompletely understood. We studied 18 dogs, anaesthetised and spontaneously breathing both room air and after the inhalation of a gas mixture containing 10% CO2, 20.9% O2, and 69.1% N2, to determine the role of histamine, serotonin, and acidaemia in pulmonary hypertension produced by hypercapnia. Hypercapnia increased the mean pulmonary artery pressure by 0.33 kPa (2.5 mmHg) while wedge pressure and pulmonary arteriolar resistance did not change. Cardiac output significantly increased, indicating that the pulmonary hypertensive effect of hypercapnia is mainly flow related. Neither chlorpheniramine nor methysergide had significant effects on hypercapneic pulmonary hypertension. The infusion of sodium bicarbonate corrected the pH; pulmonary artery pressure and cardiac output increased while pulmonary arteriolar resistance dropped, suggesting that the increased cardiac output masked the effect of pH on pulmonary arteriolar resistance. The lack of effect of chlorpheniramine or methysergide on pulmonary resistances indicates that the vasoconstrictive effect of increased hydrogen ion concentration which accompanies hypercapnia is attributable neither to histamine nor to serotonin release.


The innervation of the salivary gland of the tick, Boophilus microplus. Salivary of the ixodid tick Boophilus microplus Canestrini are at least partially innervated by a branch of the pedipalpal nerve. Axons containing both large granular and smaller agranular vesicles were observed within the acini associated with all types of secretory cells. A modification of the Falk-Hillarp histochemical technique was used to demonstrate discrete areas of fluorescence within the salivary acini. It is suggested that the transmitter involved with the control of salivary activities is a catecholamine and may even be dopamine.


In vivo oxidation of reduced nicotinamide-adenine dinucleotide phosphate by paraquat and diquat in rat lung. Intravenous injection of rats with 156 mumol/kg of paraquat or 140 mumol/kg of diquat produced, within 60 min, a sharp drop in the ratios of NADPH to NADP in lung. The effect persisted for a time period of at least 24 h. Exposure to 100% oxygen enhanced the toxicity of both compounds without substantially amplifying changes in the NADPH/NADP ratio. Lungs retained the capability to synthesize adenine nucleotides de novo. Electron microscopic studies showed that both paraquat and diquat damage type I alveolar cells, but only paraquat produces type II cell lesions. Although bipyridylium herbicides produce acute oxidation of NADPH in vivo, there seems not to exist a straightforward relationship between this event and cell damage.


Protein and enzyme release from human leukocytes: influence of phenothiazine derivatives. We studied the influence of chlorpromazine on the release of enzymes (beta-glucuronidase, EC 3.2.1.31; lactate dehydrogenase, EC 1.1.1.27; pyruvate kinase, 2.7.1.40) and proteins using human granulocytes isolated and maintained at 37 degrees C. Chlorpromazine had a biphasic effect on enzyme release and the inhibition of the glycolytic pathway could be demonstrated only at high concentrations of chlorpromazine, after one hour's incubation. The NAD+/NADH ratio was significantly perturbed at all the concentrations. This effect is time dependent. The action of 4 other phenothiazine derivatives made it possible to establish a relationship between their physico-chemical properties and protein release. The results are compared with those from other studies using other biological materials.


In vitro cultivation of the sporogonic stages of Plasmodium: a review. Complete and continuous in vitro development of the sporogonic stages of the malarial parasite has not yet been accomplished, although success with erythrocytic stages (falciparum malaria) and exoerythrocytic stages (avian malaria) has been achieved. This lag in progress appears to be due to several inherent differences between sporogony and these other sequences of development. The Trager-Jensen system for in vitro development of erythrocytic stages of Plasmodium falciparum results in the formation of gametocytes, although these gametocytes have not yet been shown to be functionally mature. An improvement in culture conditions, leading to the formation of infective gametocytes, would be an important advance. Culture systems for the transformation of gametocytes to ookinetes have been described, but whether this can be easily accomplished for falciparum malaria remains to be determined. The subsequent stages of sporogony, leading from oocyst differentiation to the formation of mature, infective sporozoites, have been successfully grown in short-term in vitro cultures. The entire developmental sequence, however, has been obtained only by overlapping successive stages in different cultures. This has established that all phases of sporogony are inherently capable of being supported in vitro. Further improvements may come through a better understanding of appropriate culture conditions.


Biochemical and technical considerations regarding the mass production of certain parasitic protozoa. This article summarizes the most relevant biochemical knowledge about growth factors as specific essential components of culture media, and calls attention to their significance with respect to the mass cultivation of some parasitic protozoa-e.g., Trypanosoma and Leishmania spp. and amoebae. Details of recent developments and techniques of parasite fermentation are reviewed.


Direct micromethod for colorimetry of serum ornithine carbamoyltransferase activity, with use of a linear standard curve. Determination of serum ornithine carbamoyltransferase (EC 2.1.3.3) activity can be a valuable diagnostic tool in the detection of liver diseases involving cytolytic processes. I describe a micromethod for measuring this activity in serum, in which the reaction product, citrulline, is measured colorimetrically in the incubation mixture without prior deproteinization. To eliminate the interference of serum protein precipitation, the concentration of sulfuric acid in the color reagent has been decreased, without substantial loss of sensitivity. Optimizing the conditions of citrulline determination, in which antipyrine and 2,3-butanedione monoxime are used, has resulted in a linear standard curve. The color formed by citrulline is found to be stable in room lighting and sensitive only to direct sunlight. The precision of the method is inversely correlated to serum enzyme activity, the CV varying between 4.6 and 21.1%.


Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.


Use of magnesium silicate before gas-liquid chromatography for determination of "total estrogens" in urine during pregnancy. The authors propose a simple, rapid, reproducible and reliable method for determination of "total estrogens" in urine during the last three months of pregnancy. The procedure consists of separation of free urinary estrogens, obtained after rapid hydrolysis, on a column of magnesium silicate. The estrogens are adsorbed on the column at acid pH and eluted by 1 M potassium hydroxide. Following extraction of the eluate by diethyl ether and formation of trimethylsilyl ether derivatives, the steroids are analysed by gas-liquid chromatography. This new procedure is used routinely in our laboratory, one assay being carried out in less than three hours. The results appear to be comparable to those obtained with classic methods. We wish to report the elimaination curves of "total estrogens" during normal pregnancies and their allowable limits.


Measurement of the alpha-mannosidase activities in human plasma by a differential assay. A differential assay based on their difference in thermal stability has been used to measure the acidic and true intermediate alpha-mannosidases in the plasma of cntrols and individuals homozygous or heterozygous for mannosidosis. The intermediate activity was found to be independent of age, sex or mannosidosis genotype. The acidic alpha-mannosidase did not vary significantly with age or between sexes for groups of the same age. The concentrations of acidic and intermediate alpha-mannosidase showed a positive correlation for adults but not for children. The ratio of acidic to true intermediate alpha-mannosidase might therefore be a useful secondary test for the detection of adult heterozygotes for mannosidosis.


The sympathetic system in hypertension.

The sympathetic system in hypertension. Several experimental observations accumulated during recent years have suggested an active participation of the sympathetic system in the pathogenesis and maintenance of hypertension in various experimental models of hypertension. The evaluation of sympathetic tone by various indirect means in human hypertension has also revealed that the sympathetic system plays an important role in the maintenance of hypertension in a subgroup of the human hypertensive population. The study of circulating catecholamines, which appears to be the best and most reliable indirect means to evaluate the sympathetic activity in the human, at present, has indicated that 25 to 40 per cent of patients with essential hypertension are characterized by higher basal circulating catecholamines and by a higher sympathetic reactivity in response to postural changes. These hyperadrenergic patients are also characterized by a higher heart rate, heart contractility, cardiac index and probably by higher plasma renin activity. The identification of these patients as a separate entity is desirable since it is possible that the evolution of the hypertensive disease and the response to therapy differ in this group of patients. The study of these patients could lead to a better understanding of the mechanisms underlying the pathogenesis of cardiovascular complications and to the development of more rational and efficient therapeutic approaches.


Phaeochromocytoma.

Phaeochromocytoma. Factors involved in the evaluation and care of patients with phaeochromocytoma have been discussed with respect to important considerations for the consulting or practising physician. Historical, physical, biochemical and other diagnostic procedures, as well as therapeutic manoeuvres have been adequately documented so that the clinician requiring additional information in depth may seek out the pertinent literature. Utilizing this manner of approach should significantly improve the care of patients with phaeochromocytoma in the hands of physicians who have not themselves had extensive experience with this disease. However, it must be emphasized that because of the potential gravity of this condition, if the physician feels insecure in the care of a patient or has further questions, he should not hesitate to seek expert advice which will benefit both the patient and himself.


Do stimulant drugs improve the academic performance of hyperkinetic children? A review of outcome studies. Stimulant drug studies based primarily on measures of teacher opinion have frequently concluded that these drugs improve the achievement of hyperkinetic children. However, a review of those studies using more objective measures of academic performance revealed few positive short-term or long-term drug effects on these measures. What few improvements have been noted can be readily attributed to better attention during testing. The major effect of the stimulants appears to be an improvement in classroom manageability rather than academic performance. It would seem that the stimulants are not able to influence those etiologic factors, other than overactivity and inattentiveness, which predispose hyperkinetic children toward school difficulties. Hence, since the goal of pediatric intervention with these children should be to enhance school performance as well as reducing hyperactive behavior, the two should be independently and objectively monitored. Since stimulant medications fail to improve the academic performance of most of these children, additional educational assistance must be provided.


Antiarrhythmic properties of 5-(3-tert-butylamino-2-hydroxy)propoxy-3,4-dihydrocarbostyril hydrochloride (OPC-1085), a newly synthesized, potent beta-adrenoreceptor antagonist. 1. The antiarrhythmic properties of 5-(3-tert-butylamino-2-hydroxy)propoxy-3,4-dihydrocarbostyril hydrochloride (opc-1085) were compared with those of propranolol and pindolol using various kinds of preparations for experimental arrhythmia in dogs. 2. Although OPC-1085 was the most potent drug to antagonize adrenaline-induced arrhythmia in animals anaesthetized with either pentobarbitone sodium or halothane, it was scarcely effective on ouabain-induced arrhythmia in pentobarbitone sodium anaesthetized animals. 3. When these compounds were administered intravenously to conscious dogs 24 h after two-stage ligation of the anterior descending artery, ectopic ventricular beats of coronary ligation-induced arrhythmia were reduced while regular sinus beats were simultaneously increased. 4. OPC-1085 was very effective on aconitine-induced arrhythmia in dogs anaesthetized with pentobarbitone sodium. The effective dose was similar to that of propranolol but about fifteen times less than that of pindolol. 5. It is concluded that different potencies among these beta-adrenoreceptor antagonists against various kinds of experimental arrhythmias cannot be simply deduced from any one of the following properties; beta-adrenoreceptor antagonism, intrinsic myocardial stimulation, local anaesthetic and so-called quinidine-like effects.


A model for evaluation of antianxiety drugs with the use of experimentally induced stress: Comparison of nabilone and diazepam. Volunteer subjects were used to compare a potential antianxiety drug (nabilone, 2-mg single doses) with a standard drug (diazepam, 5-mg single doses). A double-masked design with placebo control was used. Volunteer subjects were selected on the basis of high levels of train anxiety and were tested by two anxiety-inducing procedures--the mirror drawing test and the Stroop color-word test. Anxiety induced by the experimental procedure was alleviated by diazepam and, to a lesser extent, by nabilone. Since doses of the two drugs may not have been equivalent, or the time courses identical, conclusions about their relative efficacy must be guarded. The experimental model is unusual in that antianxiety drugs can be tested in volunteer subjects for true antianxiety effects rather than for side effects, such as cognitive or motor impairment, sleepiness, or other signs of central nervous system depression.


Intracellular pH and bicarbonate concentration as determined in biopsy samples from the quadriceps muscle of man at rest. 1. A method for measuring intracellular pH and bicarbonate concentration of human muscle is described. 2. Muscle biopsies from the quadriceps muscle of 13 healthy subjects at rest were analysed for acid-labile carbon dioxide and volume of extra- and intra-cellular water. Extracellular water volume was estimated from the chloride content and intracellular water volume from the potassium content, or alternatively derived from the sample weight. 3. The measured total carbon dioxide content in muscle was 9-84+/-1-39 mmol/kg. 4. Assuming a normal membrane potential (88 mV) and PCO2 of muscle equal to venous blood, calculated intracellular pH was 7-00+/-0-06 and intracellular bicarbonate concentration was 10-2+/-1-2 mmol/l of water.


Urine electrolyte response to 18-hydroxy-11-deoxycorticosterone in normal man. 1. To assess whether the adrenal corticosteroid 18-hydroxy-11-deoxycorticosterone [18-(OH)-DOC] affects urine electrolyte excretion in normal man, seven male volunteers received 120 microgram (353 nmol) intravenously in 1 h. This was compared with glucose (50 g/l; control) and aldosterone (80 microgram, 222 nmol) infusions in the same subjects. 2. A definite though weak antinatriuretic response to 18-(OH)DOC was observed, whereas urine potassium excretion was not altered. Aldosterone increased urine potassium excretion and reduced sodium output. Urine pH was lowered by both corticosteroids, aldosterone in general having a more marked effect. Urine volume was not altered by 18-(OH)DOC. 3. Plasma concentrations of 18-(OH)DOC and aldosterone rose approximately tenfold during their respective infusions. Compared with that of aldosterone, the metabolic clearance rate of 18-(OH)DOC was slower andits plasma half-life was longer. 4. We have been able to demonstrate that 18-(OH)DOC has a definite, albeit weak antinatriuretic action in normal man, but whether or not this corticosteroid is capable of elevating the blood pressure in man remains to be shown.


Drug kinetics and artificial kidneys.

Drug kinetics and artificial kidneys. The factors affecting solute movement across the membrane during haemodialysis are well understood. Likewise the mathematics of drug kinetics are well described. However, studies of the effects of artificial kidneys on drug kinetics have often been limited by a lack of attention to proper methods of calculating solute clearance by the artificial kidney.


Haemodynamic observations with KO. 1366 (bunitrolol), a new beta-adrenergic blocking agent. The haemodynamic effects of a new beta-adrenergic blocking agent KO.1366 (bunitrolol) were assessed in 10 males admitted to hospital for investigation of chest pain. Measurements were made at rest, during atrial pacing at 100 beats/min, and during hand grip exercise, before and afterintravenous administration of KO.1366 at a dosage of 0.05 mg/kg body weight. There was a 12% (p less than 0.01) slowing in resting heart rate and alpha 4% (p less than 0.05) slowing in exercise heart rate after drug administration. Resting left ventricular end diastolic pressure rose by 2.2 mm Hg (p less than 0.01) following the drug, but there was no significant change during pacing or exercise. Left ventricular systolic pressure and its first derivative did not change significantly. Cardiac output rose slightly, and stroke volume at rest and during exercise showed a considerable increase. In the dosage used, KO.1366 has an important chronotropic effect on the heart without causing significant myocardial depression.


Lack of effect of acid-base alterations on growth hormone secretion in man. In order to study the possible effect of acid-base alterations on growth hormone (GH) secretion in man the exercise-induced GH secretion was compared with the tentative GH release resulting from the acidosis due to hyperlactacidemia after the infusion of fructose. Submaximal exercise on bicycle ergometer resulted in a significant increase of plasma GH and lactate and in acid-base alterations. The infusion of 60 g of fructose during 60 min was without effect on plasma GH level in spite of the increase of serum lactate and slight acid-base alterations. It is concluded that no causal relationship may be found between the acid-base alterations and the increase of plasma GH during physical exercise.


Enzymes of ammonia detoxication after portacaval shunt in the rat. II. Enzymes of glutamate metabolism. Besides the synthesis of urea, ammonia detoxication at high concentrations can also be effected through enzyme reactions involved in glutamic acid metabolism. These mechanisms are also operative in extrahepatic tissues. Hyperammonemia is also found in the animal model of the portacaval shunt (PCS) rat. This model was chosen to study the activities of glutamate dehydrogenase, glutamine synthetase and glutaminase I in liver, brain and kidney 10, 20 and 30 days after PCS. In brain and kidney ammonia is detoxified mainly by the glutamate dehydrogenase and glutamine synthetase reactions whereas in the liver these enzyme reactions play a minor role.


Purification and characterization of two aldehyde dehydrogenases from Pseudomonas aeruginosa. Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin-assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20% (spec. act. with butanal 0.33 kat/kg) was NAD-dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 kat/kg) was NADP-dependent. It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are 'constitutive' and their physiological function is uncertain. When the bacteria were grown on n-paraffin a new membrane-bound NAD-dependent aldehyde dehydrogenase activity was produced.


Inhibitory activity of terfenadine on histamine-induced skin wheals in man. The inhibitory effect of orally administered terfenadine on the area of histamine-induced skin wheals was studied by single dose and multiple dose trials in 12 normal male volunteers. Single doses of 20, 60 and 200 mg of terfenadine produced dose-dependent decreases in histamine wheal area that reached a maximum by the fourth hour after dosing. The 60 and 200 mg doses blocked almost 90% of histamine whealing, and significant reduction of the wheal area persisted for 8 h. During the multiple dose trial histamine whealing was markedly inhibited after the fifth and sixth dose of terfenadine 20, 40 or 60 mg every 8 h and of 60 mg every 12 h. On the last three dosage schedules inhibition persisted for at least 12 h after the final dose. Inhibition of histamine-induced skin whealing appears to be a quantitative index of the time course of histamine H1-receptor antagonist action.


The effect of diphenhydramine alone and in combination with ethanol on histamine skin response and mental performance. The effects of diphenhydramine and diphenhydramine plus ethanol on response to intradermal histamine and on mental performance were assessed in twelve male volunteers. A significant impairment of histamine skin response was found with diphenhydramine. This response was unaffected by ethanol. Ethanol improved performance with a tracking test compared with diphenhydramine alone, the effect was not potentiated by the combination. None of the treatments had a significant effect on a digit symbol substitution test. Co-administration of ethanol and diphenhydramine caused greater impairment of performance in a serial seven subtraction test than diphenhydramine alone. There was no correlation between central and peripheral effects of the antihistamine.


Double blind study of the effect of glafenine (Glifanan) on oral anticoagulant therapy with phenprocoumon (Marcumar). The interaction between phenprocoumon (Marcumar) and glafenine (Glifanan) was investigated in a double blind study of twenty patients receiving long term treatment with phenprocoumon. Thrombotesttime (TT) values had been stable for more than three months before the study. Patients taking glafenine showed a significant increase in TT during the second and third week of the trial (P less than 0.05) compared with the placebo group. tthe increase in TT was not significant in the fourth week. The average concentrations of phenprocoumon were similar in both groups, which suggests that displacement of the drug from binding was not important. Concentrations of clotting factors II, VII and X showed a decrease in all patients at the time of the maximum TT values. A possible explanation for this interaction is discussed, but the mechanism remains uncertain.


Secondary IgG responses to type 3 pneumococcal polysaccharide. III. T cell requirement for development of B memory cells. Mice primed with a thymus-dependent form of Type 3 pneumococcal polysaccharide (S3), i.e. S3 coupled to erythrocytes (S3-RBC) produces S3-specific IgG antibody after secondary challenge with S3-RBC. When mice are depleted of T cells by treatment with anti-lymphocyte serum (ALS) at the time of priming, no IgG antibody is produced after secondary challenge. In order to determine the cellular basis for this phenomenon, various combinations of T and/or B cells from ALS-treated or normal primed mice were transferred to irradiated recipients prior to secondary challenge with S3-RBC. The results indicated that T cells were required at the time of priming with S3-RBC in order to (a) prevent the induction of tolerance in S3-specific B cells in mice primed with high doses of S3-RBC, and (b) induced differentiation of IgG-producing B cell precursors to Bgamma memory cells in mice primed with low doses of antigen.


Receptor affinity and pharmacological potency of a series of narcotic analgesic, anti-diarrheal and neuroleptic drugs. A series of 26 drugs was tested for in vitro binding to opiate receptors in the presence and absence of 0.1 M NaCl. The results were correlated with assays for in vivo pharmacological potency. Highly significant correlation was found between binding in the presence and absence of sodium ions and analgesic potency. For 10 drugs tested for anti-diarrheal potency significant correlation was observed with binding to brain opiate receptors when binding was carried out in sodium-containing medium. These data add support to the hypothesis that stereospecific opiate binding sites are pharmacological receptors which mediate analgesia and anti-diarrheal action. We found that neuroleptics can bind to opiate receptors with affinities in the micromolar range, in agreement with reports by others. The anti-diarrheal compound loperamide exhibits no significant central opiate effects but binds to opiate receptors from brain in vitro with high affinity. Evidency is presented suggesting that the lack of specific analgesic effect is the result of poor penetration through the blood--brain barrier. Our results lend further support to the similarity of opiate receptors in the brain and in the intestinal tract.


Characterization of the adrenergic activity of carbuterol (SK&F 40383-A). Carbuterol is a beta-adrenergic bronchodilator with selectivity for bronchial smooth muscle relative to cardiac and vascular tissues of several species including man. The present studies were undertaken to further characterize its adrenergic profile. In vitro studies demonstrated that carbuterol was a direct acting beta-adrenergic agonist, not dependent on endogenous catecholamine release, and was devoid of alpha-adrenergic agonist activity. The activity of the racemate was shown to reside primarily in the l-enantiomer. Carbuterol inhibited immunologically induced release of histamine and slow reacting substance of anaphylaxis from passively sensitized fragmented rhesus monkey lung and also inhibited passive cutaneous anaphylaxis in rats. The relatively weak stimulant activity of carbuterol on beta1 receptors mediating both rate and force of contraction was confirmed in anesthetized open-chest dogs. In the anesthetized cat, carbuterol was significantly less potent than isoproterenol in decreasing diastolic blood pressure, increasing heart rate, and decreasing the tension and degree of fusion of incomplete tetanic contraction of the soleus muscle.


Transmitter release and adrenergic mechanical responses in the heart: effect of papaverine and imidazole. Chronotropic and inotropic responses were elicited in isolated rabbit hearts by stimulation of the sympathetic nerves or by infusion of noradrenaline or adrenaline and the effects of papaverine and imidazole (10(-7)-10(-6) M) on these responses were studied. The outflow of noradrenaline induced by sympathetic nerve stimulation was assayed in the absence and in the presence of papaverine and imidazole (10(-7)-5 X 10(-7) M). Papaverine increased the outflow of transmitter during nerve stimulation by 45% and potentiated both the chronotropic and inotropic responses induced by nerve stimulation and those induced by infusion of catecholamines. Imidazole inhibited the outflow of transmitter during nerve stimulation by 33%. The data indicate that the "second messenger" cyclic AMP is active in more than one step in adrenergic neurotransmission and receptor activation in the heart. Furthermore, tissue cyclic AMP seems to be involved not only in the inotropy induced by circulating catecholamines but also in the more "physiological" inotropy elicited by sympathetic nerve stimulation.


Plasma cyclic GMP: response to cholinergic agents. S.c. injections of cholinergic agents, carbachol, methacholine and bethanechol, into fasted rats caused rapid increases in the plasma concentration of cyclic GMP, with a sharp peak at 5--10 min after the injection. Acetylcholine gave rise to a rapid accumulation of cyclic GMP in plasma only when administered together with physostigmine which produced only a slight, if any, potentiation of the action of the cholinesterase-resistant choline esters. Cyclic AMP also increased after these drugs, but only subsequently to the rise of cyclic GMP; the primary action of the cholinergic drugs appeared to be the increase in cyclic GMP. Atropine was effective not only in abolishing the increase in plasma cyclic GMP induced by cholinergic drugs but also in lowering the baseline level of cyclic GMP. It was concluded that the plasma concentration of cyclic GMP could serve as a good parameter of cholinergic activity in rats.


Narcotic cueing properties of intraventricularly administered sufentanil, fentanyl, morphine and met-enkephalin. The narcotic cueing activity of sufentanil, fentanyl, morphine and met-enkephalin was studied upon their injection into the lateral brain ventricle of the rat. Comparative studies on the analgesic activity of the three narcotics support a close correlation between the narcotic cueing and the analgesic activity of narcotic drugs.


Tritiated thymidine incorporation and cell-mediated lympholysis as correlates of acute graft-versus-host reaction. The graft-versus-host (GVH) reaction remains a serious consequence of administration of allogeneic immunocompetent cells to an immunosuppressed host even if donors and recipients are matched for major histocompatibility loci. This report describes a murine model for acute GVH reactions. Spleen cells from C3H/He (H-2k) mice, after intravenous injection of BALB/c (H-2d) spleen cells, were specifically cytotoxic for C3H target cells in vitro 4 days after irradiation and reconstitution. The cells in the recipients apparently are of donor genotype. The spleen cells exhibited rapid proliferation in vitro as measured by the uptake of 3H-TdR. The in vitro proliferation was distinguished from erythropoiesis by an assay of 59Fe incorporation. The kinetics of the in vitro incorporation of 3H-TdR and the in vivo uptake of 59Fe are reported.


Oxatomide, a new orally active drug which inhibits both the release and the effects of allergic mediators. Oxatomide is a new potent inhibitor of anaphylactic and allergic reactions. After oral administration, the compound both inhibits the release of endogenous histamine and prevents the effects of exogensous histamine, at comparable doses. The combination of these effects appears to be the basis of the effectiveness of oxatomide in allergic reactions and may lead to clinical application different from classical antihistaminics and from cromoglycate.


The effect of temperature on sperm motility and viability. Semen specimens from fertile prevasectomy patients maintained at 4 degrees, 20 degrees, and 37 degrees C were evaluated at 3, 6, 12, and 18 hours after collection. Sperm viability, assessed by eosin-nigrosin stain, and motility decreased with time at 20 degrees and 37 degrees C, but at a significantly higher rate at 37 degrees C (where the motility was halved by 12 hours). The slope of the decrease in viability closely paralleled that of the motility except at 4 degrees C, where motility was nearly absent at 6 hours but viability was retained through 18 hours. Bacterial counts rose markedly and the pH fell at 37 degrees C, which may explain the decrease in motility and viability. It is clear from this study that semen should be kept at room temperature (20 degrees C) and not at 37 degrees C if there is to be any delay in its analysis, or a falsely lowered motility will result.


[The renin-aldosterone system in essential hypertension--hypotensive action of beta adrenergic blocking agents and variation of the renin-aldosterone system (author's transl)]. The effect of beta adrenergic blocking agents on the renin release from the kidney and its possible role in the hypotensive effect of these agents were studied in patients with essential hypertension. Oxprenolol induced a significant decrease in systolic blood pressure and PRA, but the correlation between the decrease in blood pressure and the decrease in PRA was not found. When the effect of carteolol, another beta adrenergic blocking agent, was studied, a decrease in blood pressure was obtained, but there was a rise in PRA. These observations suggest that the hypotensive action of beta adrenergic blocking agents does not result from their effects on PRA.


pH effect on the percutaneous penetration of lignocaine hydrochloride. (1) The percutaneous penetration of lignocaine hydrochloride is affected in vitro by the pH; alkalinity increased the portion of the unionized drug which permeated through the lipoid outer skin membrane of guinea pigs. (2) The amount of lignocaine accumulated on the dermal side of the diffusion cell containing isotonic phosphate buffer (pH 7.4) was directly proportional to the initial concentration of the applied drug at the alkaline pH. (3) The dermal transfer rates of lignocaine into the isotonic phosphate buffer (pH 7.4) of the diffusion cell decreased with the increase in the pH of the initially applied solution on the epidermal side of the diffusion cell; the simultaneous cutaneous penetration of the alkaline buffer promoted retention of the unionized drug in the dermis. (4) Percutaneous penetration of lignocaine hydrochloride represents a dual-stage process involving dissimilar rates of clearance into cutaneous tissue and transfer from dermis to body fluids. Variations in the alkaline pH of lignocaine hydrochloride solution appear to govern the rate-limiting factor of the total percutaneous penetration; the pharmacologic action of lignocaine may thus be localized.


Biological and immunological characterization of human luteinizing hormone: I. Biological profile in pituitary and plasma samples after electrofocusing. Pituitary and plasma pools from postmenopausal women and plasma pools from women at midcycle were fractionated by electrofocusing in sucrose density gradients. The biological LH activity was determined in each of the electrofocusing fractions by the use of an in vitro bioassay method. A heterogeneous profile of LH activity was found in both pituitary and plasma samples with a large proportion present within the pH range 6.5-10. In a total of 11 electrofocusing runs 7 main regions of high LH activity were found within this range with mean pI values (+/- SD) of 6.75 +/- 0.08 (n = 6), 7.33 +/- 0.08 (11), 7.80 +/- 0.09 (11), 8.23 +/- 0.10 (11), 8.81 +/- 0.04 (7), 9.17 +/- 0.05 (6) and 9.55 (2). A significantly higher proportion of LH activity was found in the midcycle plasma samples (36%) in the pH regions with mean pI values of 8.81, 9.17 and 9.55 than in postmenopausal plasma (7%) and pituitary extracts (5%). This indicates that the profile of biologically active LH in women in the fertile age is different from that present in postmenopausal women. By detailed fractionation based on narrow pH range studies and the refocusing of specific peak fractions it was shown that each of the regions studied consisted of several peaks of LH activity indicating the presence of a large number of molecular species exhibiting varying degrees of LH activity. The relative proportions of these species showed considerable differences between sources but also between samples from the same source.


Comparative studies on the metabolism of 2-(tetrahydrofuryl)-5-fluorouracil and 5-fluorouracil. 5=Fluorouracil inhibited DNA synthesis markedly using various DNA precursors such as deoxyuridine, orotic acid, uracil, and uridine except for thymidine. 2-(Tetrahydrofury)-5-fluorouracil (FT-207) did not inhibit DNA synthesis with any of the precursors tested. The metabolisms of 5-fluorouracil and FT-207 in mice and rats were studied. When administered intravenously 5-fluorouracil was rapidly degraded to fluoro-beta-alanine in both mice and rats, while at most 70% of FT-207 was slowly degraded after a prolonged period. The metabolites of FT-207 were found to be 5-fluorouracil and fluoro-beta-alanine in both species of animals. In vitro degradation of FT-207 into 5-fluorouracil was observed mainly in the microsomal fraction in the presence of NADPH. This result suggested that microsomal electron-transport system was concerned with the degradation of FT-207.


Electron spin resonance study on the mode of generation of free radicals of daunomycin, adriamycin, and carboquone in NAD(P)H-microsome system. The mode of generation of free radicals of daunomycin, adriamycin, and carboquone in the NADPH-rat liver microsome system was studied at room temperature by electron spin resonance (ESR) spectroscopy. ESR signals of all these quinoid anticancer chemicals were detected when dissolved oxygen in the reaction mixture was consumed since the radicals are easilyaut oxidizable. All the radicals had an appreciable lifetime under anaerobic conditions. However, there were differences in the mode of their generation between daunomycin and adriamycin, on the one hand, and carboquone, on the other, with respect to the lag time and the effect of the amount of chemicals, pH of the medium, kind of electron donors, NADPH and NADH, and the presence of excess of DNA. Especially, ESR signal reappeared after the first signal had decreased considerably, in the case of daunomycin and adriamycin but not in carboquone. Intact Ehrlich ascites tumor cells also gave rise to an ESR signal of adriamycin and carboquone, but the former signal was prevented from appearing in the presence of glucose.


Establishment of a clonal strain of hepatoma cells derived from Morris hepatoma 8999. A new line of tissue culture cells derived from a slow-growing hepatoma 8999 was established and named 8999C. The isolation method, growth pattern, and morphology of the 8999C cells are described. Several hepatic enzyme activities in 8999C cells were compared to those in the original hepatoma 8999. The ornithine aminotransferase (EC 2.6.1.13), tyrosine aminotransferase (EC 2.6.1.5), and arginase (EC 3.5.3.1) activities in the 8999C cells were one-third, one-tenth, and one-hundredth of those of the respective activities in the original hepatoma 8999, and mitochondrial serine protease, which has much higher activity in hepatoma 8999 than in normal liver cells, was not detected in 8999C cells. Tyrosine aminotransferase in 8999C cells was induced by dexamethasone but not by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate or insulin. Unlike in hepatoma 8999, glucocorticoid did not induce arginase in 8999C cells.


Problems related to the use of serum and trypsin in the growth of monkey kidney cells. A function of serum in the growth medium for primary monkey kidney cells has been shown to be inhibition of proteolytic enzymes. Serum inactivates the residual trypsin remaining from enzymatic digestion of the kidneys and the proteolytic enzymes subsequently synthesized by the cells. Freshly trypsinized cells could be grown to monolayers in the absence of serum provided that they were repeatedly washed to remove residual trypsin. In the absence of serum, cell growth ceased on the 4-5th day after initiation of the culture, at which time the culture fluids became active proteolytically. When the 5th day fluids were replaced with fresh serum-free medium, cell growth was accelerated and a monolayer was attained by the 7th day. If cells were grown in the absence of whole serum but in the presence of medium containing alpha globulins or fetuin which inhibit both trypsin and cell proteases, such cultures grew as well as cultures containing serum. The sterilization of trypsin for use in digestion of tissues and cell cultures poses a serious problem. After filtration through 0.22 micron filters, trypsin preparations may still contain adventitious viruses, mycoplasma and minute forms of pseudomonas and other bacteria or bacteria-produced toxins, which pass the membrane pores. A process of purifying and sterilizing trypsin without deleteriously affecting its proteolytic activity is described.


Specificity of cleavage by restriction nuclease from Bacillus subtilis. The restriction nuclease from B. subtilis (Bsu) which cleaves in the middle of the tetra-nucleotide sequence 5'-GGCC-3' 3'-CCGG-5' has been found to decrease its substrate specificity at high nuclease concentrations. There are special conditions, high pH, low ionic strength, and high glycerol content, which strongly enhance splitting with decreased specificity and also lead to splitting of single-stranded DNA. By sequence analyses it is shown that the reduction in specificity of Bsu corresponds to cleavage predominantly at 5'-GC-3' 3'-CG-5' sequences. No comparable change in specificity has been observed in a restriction nuclease from Haemophilus aegyptius (HaeIII), and isoschizomer of Bsu.


Effects of dihydroergotoxine mesylate on aging neurons in vitro. C1300 mouse neuroblastoma cells gradually accumulate lipofuscin-like pigment when they are maintained in culture. Pigment was demonstrated by positive straining for acid phosphatase and with periodic acid-Schiff stain. Pigment was formation in cells was reduced by exposure of the cells to lower doses of dihydroergotoxine mesylate which also induced neurite formation and increased protein synthesis. Since lipofuscin appears to originate as a result of wear and tear within the cells, the drug probably exerts its beneficial effects by reducing the rate of intracellular wear and tear associated with aging.


Binding interactions of ergot alkaloids with monoaminergic receptors in the brain. The interactions of ergot alkaloids and of other drugs with dopamine (DA) and alpha-adrenergic receptors were investigated. The tested ergot alkaloids inhibit synaptosomal tyrosine hydroxylase activity and reverse the apomorphine-elicited inhibition of synaptosomal tyrosine hydroxylase activity. Thus, ergot alkaloids interact as both agonists and antagonists with the presynaptic DA receptors. Ergot alkaloids also compete effectively for the binding of 3H-DA and 3H-haloperidol to bovine striatal membranes. These results show that these drugs are mixed agonist-antagonists with respect to the postsynaptic DA receptors. To determine the effects of ergot alkaloids and of neuroleptics on the alpha-adrenergic receptors in the CNS, we have measured their effects on the binding of 3H-dihydroergocryptine and 3H-WB-4101 to cerebral cortical membranes. The displacing potencies of the tested ergot alkaloids and of the neuroleptics indicated that they have a high affinity for the alpha-adrenoreceptors in the CNS. The mechanisms underlying the therapeutic efficacy of mixed agonist-antigonists of DA and alpha-adrenergic receptors in Parkinson's disease and in geriatric disorders were considered.


[Acute myocardial infarct without pathological CK-MB isoenzyme activities]. Serum CK-MB is regarded as a specific indicator for myocardial diseases. Total CK and the isoenzyme CK-MB were measured in 69 patients with myocardial infarction. In 5 of the cases no pathological CK-MB-activities have been seen. Nevertheless, the clinical importance of CK-MB determination as a differential diagnostic aid remains.


Effects of culture milieus on the development of mouse blastocysts in vitro. Various culture milieus were examined for their support of mouse blastocyst development. Two important variables were the time at which human cord serum was added to the medium and the concentration of amino acids. In the best medium. Eagle's Minimum Essential Medium (fortified with six times the usual amino-acid concentration plus 20 percent fetal bovine serum, replaced after 48 hr with human cord serum), 83 percent of the blastocysts shed the zona pellucida, 58 percent developed to the early egg cylinder stage, 42 percent to the advanced egg cylinder stage and 22 percent attained the primitive streak stage after 6 to 8 days of culture.


Demonstration and characterization of a serum factor produced by activated T cells. Spleen rosette forming cells (RFC) from adult thymectomized mice have a low sensitivity to inhibition by anitheta serum (AOS) and azathioprine (AZ) in comparison with normal spleen or thymus RFC. Thymus extracts and normal mouse serum (but not spleen extracts or thymectomized mouse serum) correct this abnormality after a 30 min in vitro incubation with spleen cells. We report here the existence of a serum factor produced in allogeneic reactions with the same activity on rosettes as thymic factor (TF). This 'allogeneic' factor (AF) is detectable in mice undergoing a graft versus host reaction (GVHR), rejecting skin allografts or allogeneic cells or responding to thymus-dependent antigens such as heterologous red blood cells or BSA. The T-cell origin of AF is indicated by AF presence in nude mice submitted to the same allogeneic stimuli as listed above and in normal mice injected with PVP or LPS. AF is distinct from the thymic factor as shown by differences in electric charge. Moreover, in contrast with TF there is no specific high molecular weight inhibitor of AF. Preliminary biochemical studies indicate that AF is probably a peptide of low molecular weight (greater than 5000 daltons). Its target cell is probably a T-cell precursor.


Changes in the microflora and physiology of the anterior intestinal tract of pigs weaned at 2 days, with special reference to the pathogenesis of diarrhea. The gastrointestinal microflora and gastric physiology of piglets weaned at 2 days was compared with that of piglets allowed to continue sucking the sow. Although there was a significantly higher count of Escherichia coli in the stomach, duodenum, and jejunum of the early-weaned compared with sow-reared pigs, these differences were not detectable in samples from the ileum. There were no quantitative differences in lactobacilli and in streptococci between the two treatments. Lactobacillus fermentum, L. acidophilus, Streptococcus salivarius, S. bovis, and related biotypes were isolated from both groups of pigs. L. fermentum and S. salivarius were isolated more frequently from sow-reared piglets. The weight of digesta in the stomach was greater in weaned than in sucking pigs and was even greater in scouring weaned pigs, suggesting that in scouring pigs there may be gastric stasis. The gastric pH was higher in the weaned pigs at 4 days of age, but gradually decreased up to 10 days, during which time the lactic acid concentration rose. In weaned pigs there was a highly significant negative correlation between pH and lactic acid concentration in the stomach digesta, and also a positive correlation between pH and number of E. coli. These correlations suggest that lactic acid, from bacterial fermentation, is the major component in the regulation of gastric pH in weaned pigs. Three of twenty sucking pigs, but none of the weaned pigs, were secreting HCl (chloride concentration > 3 mg/g, pH < 3.5). In sucking pigs there was an inverse relationship between the chloride and lactic acid concentrations in the digesta. In weaned scouring pigs there was a nonsignificant increase in pepsin concentration in the stomach tissue. There was a threefold increase in the total proteolytic activity of the stomach tissue.


Complement-fixing antibody response in pneumococcal pneumonia. Previous studies of complement-fixing antibodies to pneumococcal capsular polysaccharides in humans have yielded conflicting results. We studied 65 sera from 25 patients with pneumococcal pneumonia, using both fresh sera and heat-inactivated sera with added human complement. Only 4 of the 25 patients developed detectable levels of complement-fixing anticapsular antibody. Of the 25 patients, 22 developed detectable levels of hemagglutinating anticapsular antibody, indicating that they were able to develop an immunological response during the infection. Most of the antibody detected by hemagglutination was sensitive to 2-mercaptoethanol, but some 2-mercaptoethanol-resistant antibody was also detected. In studies with rabbit antiserum, the complement fixation test was found to be as sensitive as the hemagglutination test for detection of anticapsular antibody. It is not clear why detectable levels of complement-fixing antibody do not develop more often in patients with pneumococcal pneumonia. Studies of purified anticapsular antibody would be of interest to determine whether or not these antibodies are restricted to immunoglobulin subclasses having a limited capacity to fix complement.


Fluoride uptake by Streptococcus mutans 6715. The short-term kinetics of fluoride uptake by cells from 20- to 22-h cultures of Streptococcus mutans strain 6715 were studied using rapid filtration and centrifugation techniques. Saline-suspended organisms were diluted with fluoride-containing solutions buffered at four different pH values (2.0, 4.0, 5.5, and 8.2). Fluoride disappearance from the medium was inversely related to pH and to the duration of the exposure at any given pH. The uptake was rapid and extensive at the lower pH values and decreased as the pH increased. Media fluoride concentrations subsequently increased; i.e., fluoride was released from the cells. The presence of glucose, cyanide, or iodoacetate did not influence the results. However, preincubation of the cells in fluoride-free buffers, followed by the addition of fluoride, reduced fluoride uptake markedly. Cell-to-media pH gradients were determined by the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione. Fluoride uptake was found to be a function of the magnitude of the pH gradient (P less than 0.001). It is hypothesized that fluoride uptake occurs by the diffusion of hydrogen fluoride and the subsequent trapping of ionic fluoride.