Enzyme catalyzed hydrolysis of inorganic pyrophosphate. Current view of problems in characterization of PP1-phosphohydrolytic activity associated with alkaline phosphatases (EC 3.1.3.1).

A survey of the hydrolytic activity of alkaline phosphatase (EC 3.1.3.1) reveals that PP1, like phosphomonoesters, can serve as substrate in vitro. This pp1-phosphohydrolytic activity can be distinguished from PP1-phosphohydrolytic activities of inorganic pyrophosphatases (EC 3.6.1.1) and glucose-6-phosphatase (EC 3.1.3.9) by several criteria. Discrimination among these hydrolytic enzymes is possible by their dependence on variation of pH and of magnesium to PP1 ratios in the assay solutions. The true substrates and modifiers are not simply PP1 and magnesium, but the equilibrium species in mixtures of these two. The physiological significance of each of the three enzymes is not predictable from their differential efficiency as catalysts of PP1-hydrolysis in vitro.


The calcium paradox: a reaffirmation.

Perfusion of isolated rat hearts with a Ca-2+-containing medium, after a Ca2+-free perfusion period, results in an irreversible loss of electrical and mechanical activity of the heart, as well as a large release of intracellular components such as lactate dehydrogenase. These phenomena can be evoked, when hearts are perfused at a constant perfusion pressure or at a constant flow rate. Evidence is provided that recovery of the mechanical activity of the heart is not a requisite for lactate dehydrogenase release.


Mechanism of action of narcotics in the production of menstrual dysfunction in women.

The ability of morphine to block ovulation in animals prompted investigation of the frequency and mechanisms of menstrual abnormalities in women addicted to narcotic analgesics. Menstrual histories obtained from 76 former heroin addicts receiving daily methadone maintenance revealed that more than one-half of these women had experienced menstrual abnormalities while taking heroin or methadone. In order to determine the specific physiologic effects of narcotic analgesics on reproductive function, detailed endocrinologic studies were carried out in seven of these patients who complained of amenorrhea or irregular menses while receiving methadone. Four of the seven women manifested abnormalities of the control of gonadotropin secretion. Three of these four failed to exhibit cyclic gonadotropin release, as evidenced by an absence of increased levels of follicular phase follicle-stimulating hormone, midcycle gonadotropin peaks or luteal phase progesterone increments. In the fourth patient a prolonged follicular phase (30 days) of the menstrual cycle was detected. One of these four patients also had low basal gonadotropin levels and failed to exhibit luteinizing hormone increments greater than control levels in response to ethinyl estradiol (positive feedback). The remaining three women exhibited normal patterns of gonadotropin secretion during the observation period. In these women, menstrual bleeding occurred in response to withdrawal from luteal phase (10 to 20 ng/ml) progesterone levels and to exogenous ethinyl estradiol, suggesting normal uterine responsivity to progesterone and estrogen. Although not documented, it is likely that oligo-ovulation was the cause of the irregular menses in these three patients. Amenorrhea is commonly associated with methadone ingestion or heroin addiction and appears to be related to an alteration of the hypothalamic mechanisms controlling gonadotropin secretion. Tolerance to these effects of methadone may develop after chronic ingestion.


Effects of hormonal treatments which alter ovum transport on beta-adrenoceptors of the rabbit oviduct.

Beta-Adrenoceptor activity of rabbit oviductal isthmus has been determined quantitatively in vitro in tissues isolated 24 and 72 hours after human chorionic gonadotropin injection and after two hormonal treatments known to accelerate (progesterone) and retard (estrogen) ovum transport. Under conditions in which ova are located at the ampullary-isthmic junction and distal isthmus, the isthmus demonstrates a low incidence of spontaneous activity in vitro, poor response to acetylcholine, low affinity of beta-adrenoceptors for isoproterenol, and low efficacy of isoproterenol to inhibit acetylcholine-induced contractions. Under conditions in which ova are passing into the uterus, the isthmus shows the converse of these parameters. These data support the hypotheses that the oviductal isthmus functions as an adrenergic sphincter and that the actions of progesterone and estrogen in altering ovum transport rates are at least partially mediated through changes in adrenoceptors.


[Neuro-humoral interrelationships in ontogeny].

A.A. Ukhtomsky's ideas of the relationship between the electric and the humoral components of transmission of nervous impulses in thr organism, are considered.  The data obtained recently when studying synaptic transmission of excitation in the sympathetic ganglia of mammals during ontogeny are presented in corroboration of A.A. Ukhtomsky's ideas of that the humoral factors affect neural reactions changing the lability of substratum, which is manifested by the grade of polarization of cellular membrane to a certain critical extent.


PLasma glucagon levels during rapid exsanguination with and without adrenergic blockade.

In eleven dogs made hypotensive by means of rapid exsanguination over a period of forty to seventy minutes, mean glucagon rose to a peak of 516 plus or minus (S.E.) 150 pg/ml. and mean glucose to a peak of 341 plus or minus 27 mg. per 100 ml. The hyperglucagonemia induced by exsanguination was substantially reduced, although not abolished, by propranolol infusion, but was not diminished by phentolamine, suggesting that it was largely a beta-adrenergic effect. Its possible contribution to survival during shock is considered.


Effect of pH on bile salt injury to mouse gastric mucosa. A light- and electron-microscopic study.

Bile salts break the gastric mucosal barrier. To explain this, the suggestion has been made that bile salts may disrupt surface epithelial cell membranes or break the tight junctions between cells, but appropriate ultrastructural studies are lacking. We therefore instilled control and bile salt-containing solutions into the stomachs of fasted mice at pH, 1, 3, 5, AND 7. Taurocholate (pKa equals 1.8) caused mucosal injury only at pH 1, whereas glycochenodeoxycholate (pKa equals 4.2) injured the mucosa at pH 1 and 3. By electron microscopy, areas of mild mucosal injury were characterized by clumping of nuclear chromatin and loss of cytoplasmic density within surface mucous cells. The apical cell membranes and tight junctions remained intact. In areas of severe damage surface cells were ruptured but tight junctions still appeared unbroken. These studies indicate that acid pH markedly augments the damaging effects of bile salts on mouse gastric mucosa. Moreover, as an initial step in the mechanism of bile salt-induced gastric injury, the nonionized moiety of a given bile salt which exists below its pKa may be important in altering the gastric surface epithelial cell in a way which allows the ingress of bile salt and/or hydrogen ion to cause intracellular damage.


Failure of cholestyramine to prevent bile salt injury to mouse gastric mucosa.

Bile salts have been implicated in the pathogenesis of gastritis and gastric ulcer. Because the bile salt binding agent cholestyramine has been suggested as a possible therapy for gastric ulcer, we studied the effects of cholestyramine, in the form of Questran, on bile salt-induced injury to mouse gastric mucosa. Solutions of taurocholate or of glycochenodeoxycholate, with or without added Questran, were instilled into the stomachs of fasted mice at pH 1, 3, 5, and 7. Taurocholate damaged the mucosa only at pH 1, whereas glycochenodeoxycholate caused injury at pH 1 and 3. Questran failed to prevent mucosal damage by either bile salt. The ineffectiveness of cholestyramine to prevent injury may be due to the nonionized fraction of bile salts at pH's below their pKa's which will not be sequestered by the anion exchange resin. This phenomenon may help explain the insignificant effect of Qestran treatment in promoting healing of gastric ulcers in a previous clinical trial.


Gene duplication as a mechanism of genetic adaptation in Saccharomyces cerevisiae.

It has been shown that specific mutations of the gene that codes for the general acid monophophatase (Aphtase) of S. cerevisiae can increase the affinity of this enzyme for beta-glycerophosphate (BGP) and thereby provide this organism with the capacity to exploit extremely low concentrations of this organic phosphate (Francis and Hansche 1973). In this report two additional avenues are demonstrated to be available to this organism for increasing its capacity to exploit low concentrations of organic phosphates. One avenue is through mutations that increase the amount of Aphtase that associates with the cell wall, where it catalizes the hydrolysis of exogenous organic phosphates. The other avenue is through duplication of the gene that codes for Aphtase, doubling the amount of Aphtase synthesized.--The spontaneous duplication of the structural gene of Aphtase and the incorporation of the duplicate into this experimental population as a means of exploiting low concentrations of exogenous organic phosphates provides direct support for the first step of the mechanism through which new metabolic functions are postulated to evolve.


Isolation and study of mutants lacking a derepressible phosphatase in Chlamydomonas reinhardi.

In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa)) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of elctropherograms form WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas: in addition tothe neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.


Membranoproliferative glomerulonephritis with polyarteritis: a case report.

A case of membranoproliferative glomerulonephritis with polyarteritis is described. The patient was a 68 year old male who had the disease for about four months. A kidney biopsy specimen taken just before the patient died was studied by light, immunofluorescence, and electron microscopy. Splitting of the basement membrane, IgG, IgA, BlC, Clq, fibrinogen, and electron dense deposits were found in the glomeruli. It is considered that perhaps some cases of membranoproliferative glomerulonephritis are produced by circulating immune complexes.


Tularaemia in the rat. I. The cellular basis on host resistance to infection.

Rats infected with the live vaccine strain (LVS) of Francisella tularensis develop in vivo and in vitro evidence of cellular hypersensitivity and a concomitant state of cellular resistance to infection. They key role of sensitized lymphocytes in cellular resistance was domonstrated in transfer experiments. Using this technique, it was shown that thoracic duct lymphocytes from Francisella immune donors conferred specific antimicrobial resistance on normal recipients, whereas antiserum afforded no protection whatsoever. Further evidence for the participation of sensitized lymphocytes in the host's defence emerged from experiments in which a comparative analysis was made of the immunogenic properties of living and heat-killed LVS organisms. Rats stimulated with the living parasite developed cellular hypersensitivity and specific antibodies. Throacic duct lymphocytes obtained from such animals could immunize adoptively. By comparison, rats stimulated with a substantially larger number of dead organisms failed to develop cellular hypersensitivity and their lymphocytes were devoid of protective activity. Dead organisms, however, provoked an antibody response similar to that observed in infected rats. The development of cellular hypersensitivity in Francisella-infected rats is associated with enhanced resistance to Listeria monocytogenes. This finding accords with the results of similar studies of infection immunity to other intracellular parasites, and implies that the expression of cellular resistance to F. tularensis is a cooperative venture involving specifically sensitized lymphocytes and non-specific inflammatory cells, presumably macrophages.


Immunodeficiency in the chicken. III. Hypoplasia of bursal follicles following intravenous injection of embryos with lipopolysaccharide or allogeneic lymphocytes.

Intravenous injection of LPS or allogeneic lymphocytes resulted in impaired maturation of B cells within the bursa of chick embryos. A gradual decline in the sensitivity of embryos towards both agents was reflected by a decrease in the number and size of bursal follicles following injection between 10 and 15 days of embryonation. The hypoplasia of these follicles in embryos injected with lymphocytes on the 10th day was a more sensitive measure of graft-versus-host reaction than splenomegaly. The relevance of results with this model to the aetiology of immunodeficiency diseases has been briefly discussed.


Radiosensitization of cultured mammalian cells by 5-iodouridine.

Radiosensitization of cultured mammalian cells was studied with halogenated pyrimidines, such as 5-iodouridine or 6-chloropurine, which have been shown to promote bacterial cell lethality when combined with gamma-irradiation. When Chinese hamster cells were exposed to gamma-rays to acidic pH values and the number of colonies was scored after 6 to 11 days of incubation, many more cells were inactivated in the presence of the drug than in its absence. This may be due to radiation-induced cytotoxic iodine radicals from the reagent in the case of 5-iodouridine, because the cells were inactivated efficiently only be contact with the previously-irradiated drug solution. The toxicity of the irradiated drug solution increased remarkably when the pH shifted to acidic side. The radiosensitization and the cytotoxic effects of gamma-irradiated drug solution were not found with 6-chloropurine. This may be the first observation on the lethal effect of chemical radicals on mammalian cells, and it is concluded that radiosensitization with 5-iodouridine does not require the drug incorporation into cellular DNA, at least under the conditions adopted in the present studies.


Paradoxic aciduria in bovine metabolic alkalosis.

Cows with metabolic alkalosis secondary to abomasal displacement and other abomasal disorders were often found to excrete acidic urine. This paradoxic aciduria contradictsthe classical view that the pH of the urine may be used to estimate the acid-base status of the body. Data from bovine clinical patients with metabolic alkalosis, serum electrolyte changes, and paradoxic aciduria suggested that the balance of sodium potassium, and chloride in the body places limits on the kidneys' ability to regulate the acid-basebalance.


Effect of choleretics on biliary and urinary excretion of cholecystographic agents.

Two choleretic agents, taurocholate and cinchophen, were studied in the dog to determine their effect on both the biliary and urinary excretion of two related cholecystographic agents, iopanoic acid and iophenoxic acid.  Liver concentrations were determined on biopsies.  Trace amounts of radioactively labeled compounds were used for measurements.  The effect of the choleretic agent was specific for the cholecystographic compound.  Taurocholate increased both biliary and urinary excretion of iopanoic acid and decreased its intrahepatic level, but had no effect on iophenoxic acid.  Cinchophen had no effect on iopanoic acid but increased both biliary and urinary excretion of iophenoxic acid.  When examined against a pre-existing high rate of bile flow, the active choleretic increased, rather than decreased, the biliary concentration of the cholecystographic compound.  The increased excretion in both bile and urine cannot be attributed simply to the lessened concentration gradient from plasma to bile, as proposed by others.  The data are consistent with  a direct action of the choleretic agent on hepatic mechanisms, either to decrease intracellular binding or to alter the characteristics of transport.


Factitious changes in binding of oxygen to hemoglobin when based on extracellular pH in the presence of certain blood additives like radiographic contrast media.

Construction of oxygen-hemoglobin DISSOCIATION CURVES BASED ON EXTRACELLULAR PH and using blood tonometered with 5 per cent CO2, is misleading under certain experimental conditions.  These include the presence in blood of poorly penetrating non-ionic molecules like sucrose of poorly penetrating anionic aompounds like radiographic contrast materials.  False conclusions regarding the position of the oxygen-hemoglobin dissociation curve can result because of the disturbance of the normal pH gradient between plasma and red cell induced by such chemicals.


Purification and properties of leucine beta-naphthylamidase from rabbit small-intestinal mucosal cells.

Leucine beta-naphthylamidase associated with the microvilli membranes of rabbit small intestine was solubilized with papain [EC 3.4.22.2] and purified by Sephadex G-200 gel filtration, DEAE-cellulose column chromatography, passage through a column of Sepharose 4B coupled with anti-sucrase antibodies and preparative disc electrophoresis in polyacrylamide gel. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis, but a double immunodiffusion test showed the presence of a minor component which was probably denatured enzyme. The molecular weight of the purified enzyme was estimated to be 225,000 by Sephadex G-200 gel filtration and the sedimentation coefficient (S-0-20, w) was found to be 6.90S. Purified enzyme required bovine serum albumin for maximal activity, perhaps for its protection from autodigestion. It hydrolyzed, in addition to L-leucine beta-naphthylamide, various L-amino acid beta-naphthylamides and dipeptides with a free alpha-amino group, but did not hydrolyze benzoyl-L-arginine beta-naphthylamide. Therefore, the purified enzyme is an aminopeptidase. Hg-2+ and Cu-2+ ions strongly inhibited the enzyme activity, but other metal ions and EDTA showed no or only slight effect. N-Ethylmaleimide exhibited a weak inhibition. Purified enzyme had an optimal pH and Km value for leucine beta-naphthylamide similar to those of enzymes from other sources. Antibodies against the purified enzyme were raised in guinea pigs. The antibodies obtained were found by double immunodiffusion to be specific for the enzyme. They precipitated the enzyme quantitatively and partially inhibited the enzyme activity.


pH jump-induced phosphorylation of adenosine diphosphate in thylakoidal membranes.

The kinetic properties of pH jump-induced phosphorylation in thylakoidal membranes of spinach chloroplasts were investigated, and the following results were obtained. 1. The pH jump-induced P incorporation proceeded linearly with time for at least 2 sec after the start of the reaction. Phosphate was incorporated mainly into ATP. The amounts of incorporation into ADP during 0.1 and 2.0 sec were 0.02 and 0.1 mole/400 moles chl, respectively. The amounts of P incorporation into ADP and the beta-position of ATP during a 2 sec reaction were less than 5 and 0.1% of the total amount of P incorporation, respectively. Even in the absence of added ADP, ATP was formed by the pH jump, but the amount was very small, i.e., less than 1% of that in the presence of a saturating amount of ADP. Formation of ATP was not enhanced by the addition of 0.1 mM AMP, instead of ADP. 2. The dependence of the rate of ATP formation, v, induced by a pH jump from 3.85 to 8.11 on the concentrations of ADP and Pi was given by v=Vopt/[1+psi1/[ADP]) (1 + psi2/[Pi)], where the values of the constants, Vopt, psi1, and psi2 were 14--20 moles/10-6 g chl/sec, 12.5-15 muM and 11-20 mM, respectively, at 0 degrees. 3. The dependence of v on the concentration of protons was given by v=Va/[1 + psi H-a/[H+,-a])-2], and v =Vb/[1 + ([H+,-b]/psiH-b)-2], in the acidic and basic phases, respectively. The values of the constants psi H-a and psi H-b were 10-5.7 and 10-7.9 M, respectively. 4. ATP formation was initiated by adding one of the substrates, ADP or Pi, at various times after after the pH jump in the presence of the other substrate. The rate decreased logarithmically with increase in the time between the pH jump and the start of the reaction. When phosphorylation was initiated by adding Pi after the pH jump in the presence of ADP, the decay constant of v was about 0.08 sec-1, which was one-third of that observed when the order of addition of ADP and Pi was reversed.


Purification and properties of elastolytic enzyme from Flavobacterium immotum.

Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.


Interaction of aflatoxin B1 with DNA.

The interaction of aflatoxin B1 with DNA was investigated. In the presence of native DNA the absorption spectrum of the toxin showed an obvious spectral shift in the region of 300-420 nm with an isosbestic point at 376 nm. DNA-bound aflatoxin is hydrolyzed in alkaline media more easily than the free toxin. The hydrolized form has a labile structure and can decompose further. The bound toxins are easily dissociated by heat as well as by salt, and all toxin molecules are released from DNA which remains double-stranded. Aflatoxin can bind to thermally denatured DNA as well, with an accompanying spectral shift which depends on the particular preparation of denatured DNA, and there was an isosbestic point at 380 nm. The complex of toxin and denatured DNA was stabilized by salt up to 0.1 M. Thus it was concluded that aflatoxin was bound with denatured DNA in a different form from native DNA. The number of binding sites of DNA was estimated by constructing Scatchard plots based on both spectral analysis and equilibrium dialysis. However, these show no definite value but fall in the region of 0.07 to 0.013, that is one toxin molecule per 80-140 nucleotide of native DNA. Several lines of evidence suggest the possibility that aflatoxin exists in aqueous solution as aggregates. The mechanism of the binding was discussed. It is noteworthy that the number of binding sites of DNA doubles by the presence of histones.


Studies of glucose metabolism in Bacillus subtilis. I. Purification of glucose-6-phosphate dehydrogenase from the vegetative cell and its properties in comparison with the spore enzyme.

1. Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate : NADP-+ 1-oxidoreductase, EC 1.1.1.49] from vegetative cells of Bacillus subtilis was purified about 2,400-fold. The purified enzyme was shown to be homogeneous as judged by chromatographic profile, polyacrylamide gel electrophoresis and ultracentrifugation. The correspondent enzyme from spores was also partially purified. 2. The molecular weight of the vegetative cell enzyme was estimated to be 350,000 by Sepharose 6B chromatography and sedimentation equilibrium studies, and that of its subunit was 58,000 as determined by SDS gel electrophoresis, indicating that the enzyme was found to be 240,000. 3. The vegetative cell enzyme and spore enzyme have the same Km values and pH optima. The optimum pH was about 9.2 for both enzymes and the Km values for NADP-+ and glucose-6-phosphate were determined to be 6.7 X 10-minus 6 and 7.5 X 10-minus 5M, respectively. 4. The amino acid composition of the vegetative cell enzyme was examined and was compared with those of enzymes from other sources.


Purification and properties of beta-galactosidase from Aspergillus oryzae.

Beta-Galactosidase [EC 3.2.1.23] has been purified from a culture of Aspergillus oryzae by 2-propanol fractionation, column chromatography on DEAE-Sephadex A-50 and Sephadex G-200. The preparation was homogeneous on ultracentrifugation and disc electrophoresis. The enzyme showed pH optima of 4.5 with ONPG-1 as a substrate and 4.8 with lactose as a substrate. The stable pH range was from 4.0 to 9.0 and the optimum temperature was 46 degrees. The Michaelis constants were 7.2 X 10-minus 4 M with ONPG and 1.8 X 10-minus 2 M with lactose. Hg-2+, Cu-2+, N-bromosuccinimide, and sodium laurylsulfate caused marked inhibition. The apparent molecular weight was calculated to be about 105,000 by Sephadex gel filtration and sucrose density gradient centrifugation.


Solubilization of valine-sensitive acetohydroxy acid synthetase from Neurospora mitochondria.

Acetohydroxy acid synthetase [EC 4.1.3.18] of Neurospora crassa has been solubilized from a mitochondrial pellet fraction by sonic treatment in 1.0 M potassium phosphate buffer at pH 7.5 containing 10 mM MgSO4 and centrifugation at 180,000 X g for 20 min. The soluble enzyme thus obtained has a high specific activity and a high sensitivity to valine comparable to the original mitochondrial pellet suspension when the activity was determined in high concentrations of potassium phosphate.


Enzymatic studies on a cellulase system of Trichoderma viride. II. Purification and Properties of two cellulases.

Two cellulase [EC 3.2.1.4] components derived from Meicelase, a commercial crude cellulase preparation from Trichoderma viride, were purified by consecutive column chromatography, and were designated as cellulase II-A and cellulase II-B. Cellulases II-A and II-B were each homogeneous on polyacrylamide gel electrophoresis. The molecular weights of cellulases II-A and II-B were 30,000 and 43,000, respectively, on the basis of Sephadex G-100 gel filtration. Both enzymes contained 12-14% carbohydrates (as glucose). Some properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases II-A and II-B were pH 4.5-5.0 and 60 degrees, and pH 4.5-5.0 and 50 degrees, respectively. Both enzymes were stable over the range of pH 5.0-7.0 at 4 degrees for 24 hr. Cellulases II-A and II-B retained 27 and 41% of the original CM-cellulose-saccharifying activities, respectively, after heating at 100 degrees for 10 min. Both enzymes were completely inhibited by some metal ions such as 1 mM Hg-2+, and partially by 1 mM Ag-+ and Cu-2+. However, Mg-2+, Fe-2+, and several other metal ions showed no inhibition at this concentration. The hydrolysis of CM-cellulose by cellulase II-A was more random than that by cellulase II-B.


Reassembly of functionally active 50S ribosomal particles from proteins and RNAs of Escherichia coli. Dependency of 50S ribosomal reassembly on 30S subunits.

Further studies were made on the reassembly of 50S ribosomal subunits from proteins and RNAs of E. coli. The reassembled particles had high activity in poly U-directed polyphenylalanine synthesis and their sucrose sedimentation properties were similar to those of the original intact particles. Several factors affecting the reassembly were examined. The optimal pH for solubilization of ribosomal proteins was pH 9.5, and the optimal Tris concentration was 0.75 to 1.00 M. In the reassembly mixture the pH was adjusted to 8.2 A sharp optimum magnesium ion concentration of 6 to 10 mM was observed. The reassembly required 0.2 to 0.5 M KCl, the optimum concentration being 0.40 M. On incubation for 20 min a temperature of 34 and 40 degrees was necessary, 37 degrees being best. Oligonucleotides, which we previously added to the reassembly mixture were found not to be necessary for inhibition of RNase II [EC 3.1.4.20] activity remaining in the reaction was found necessary to dialyze the reassembly mixture against a buffer containing 10 mM magnesium ion after the incubation. Simultaneous reassembly of 30 and 50S subunits with time was observed, showing that 70S ribosomes were formed first and that they then dissociated into subunits. Reassembly of 50S subunits from their component proteins and RNAs was completely dependent on either 30S particles or the simultaneous reassembly of 30S subunits. Other critical factors affecting the reassembly of 50S subunits must be examined, since the reproducibility of this reassembly is only about 60%, even under the above controlled conditions.


Kinetic studies of carboxypeptidase Y. I. Kinetic parameters for the hydrolysis of synthetic substrates.

Kinetic parameters for carboxypeptidase Y [EC 3.4.12.1], characterized as a nonspecific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides. We confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed. The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin [EC 3.4.21.1]. Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid. The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described.


Kinetic studies of carboxypeptidase Y. II.  Effects of substrate and product analogs on peptidase and esterase activities.

Reversible inhibition of the peptidase and esterase activities of CPase Y [EC 3.4.12.1] was investigated with substrate and product analogs known to be inhibitors or effectors of pancreatic carboxypeptidases A or B [EC 3.4.12.2 or 3]. L-Amino acids and NH2-blocked L-Amino acids showed competitive-type inhibition, whereas their D-enantiomers caused less inhibition than the L-enantiomers, and showed non-competitive or mixed-type inhibition. Some phenylalanine analogs, e.g. beta-phenylpropionic acid and t-cinnamic acid, were also reversible inhibitors of CPase Y. The type of these inhibitors and their K1 values were generally parallel for both the peptidase and esterase activities.


The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin.

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.


Affinity chromatography of beta-galactosidase on controlled-pore glass derivatives.

Several controlled-pore glass (CPG) derivatives were examined as supports for the affinity chromatographic purification of the enzyme beta-galactosidase. The competitive inhibitor p-aminophenyl-beta-D-thiogalactopyranoside was coupled to an azelaic acid and a malonic acid derivative of 750-A alkylamine CPG of 80-120 mesh and to an azelaic acid derivative of 550-A alkylamine CPG of 40-80 mesh. The latter derivative exhibited particularly good load capacity and separation efficiency; however, both arm lengths were effective. Hydrophobic interactions between the arm and the enzyme contribute to the separation.


Separation of pyrimidine deoxyribooligonucleotides according to length and composition using thin-layer chromatography on deae-cellulose.

A procedure has been developed for the fractionation of pyrimidine deoxyribo-oligonucleotides, PynPn+1, according to length and composition using ascending thin-layer chromatography on DEAE-cellulose. The separation of oligonucleotides according to length (n=1-7) into individual isopliths was carried out using a linear 0-0.35 M sodium chloride gradient in 0.01 M sodium acetate buffer of pH 5.1-5.3 and 5 M urea. Then the oligonucleotides of individual isopliths were separated according to composition using the same gradient of sodium chloride in 0.01 M sodium acetate or sodium citrate buffer of pH 3.2. The procedure can be used for rapid fractionation (2-3 h) and serial determination of the frequencies of pyrimidine sequences differing in length and composition in DNAs of various origin. The procedure can be used for both preparative and analytical applications. It was employed to study the distribution of 5-methylcytosine in pyrimidine isopliths of some DNAs.


Relationship between densitometric peak area and concentration of lipid in the quantitative analysis of a lipid mixture separated by thin-layer chromatography.

A mixture of the neutral lipids squalene, cetyl stearate, triolein, oleic acid and cholesterol was separated by thin-layer chromatography and the components were measured quantitatively by photodensitometry. Several empirical expressions relating densitometric peak area (A) and lipid concentration (C) were subjected to statistical analysis, and the relationship A=k-C-B is linear in the logarithmic form log A=B-log c -0g k within 95% confidence limits on the observed results. Exponent B, representing the proportionality factor in the expression relating the relative differential changes in peak area and concentration, is constant for lipids studied on the same chromatoplate, whereas the constant k is lipid dependent. All standard measurements per plate are therefore utilized for estimating a common B value, which gives greater accuracy than in the individual cases. The plate capacity for measurements of unknown lipid concentrations may consequently be increased, as a common B value, based on a reduced number of standard measurements, may be estimated with the same accuracy as an individual B value, based upon the non-reduced number of standard measurements. Equations are given for the calculation of B and k and for the determination of unknown concentrations with appropriate confidence intervals.


Separation of dehydrogenases on polyaminomethylstyrene.

The binding of dehydrogenases, especially alcohol dehydrogenase, and other proteins to several ion exchangers and hydrophobic polymers was investigated. Quantitative parameters for the stability of the polymer-protein complexes (obtained form double reciprocal plots) indicate a high but different affinity of many proteins for polyaminomethylstyrene. The chromatography of a mixture of five dehydrogenases and human serum albumin on polyaminomethylstyrene is described.


Gas chromatographic-mass spectrometric determination of intact C3-hydroxylated benzodiazepine glucoronides in urine.

A method is described for the determination C3-hydroxylated benzodiazepine glucuronides in biological samples. Oxazepam and lorazepam glucuronides are measured as methyl esters and trimethylsilyl derivates by a gas chromatographic procedure. The applicability of the method has been tested on the urine of rats, guinea pigs, rabbits and man, receiving oxazepam orally. Oxazepam glucuronide was not found in rat urine but it was present in the urine of rabbits (5.7% of the administered dose), guinea pigs (9.5%) and man (13.4-26.9%).


Progesterone receptor of the human myometrium.

A progesterone binding protein was characterized in the cytosol of human myometrium. The receptor sedimented in the 4S region on sucrose gradients and had an equilibrium dissociation constant of 3.7 times 10-9M. Synthetic progestagens had an affinity equivalent to that of progesterone while cortisol had low affinity for the binding protein. These same progestagens showed very low binding to human CBG. In normally cycling women, the total levels of cytosol receptor were higher during the follicular phase than during the luteal phase. Subjects taking oral contraceptives and those who were pregnant had the lowest concentration of myometrial progesterone receptor. Postmenopausal women had levels of myometrial receptor lower than those of the secretory phase of the cycle. Estrogen therapy increased the receptor concentrations of the menopausal myometrium to those of the proliferative phase. The estradiol receptor levels of myometrial cytosol revealed similar changes in cycling women. When both binding proteins were measured concurrently in the same specimen, there was a significant positive correlation.


Biochemical properties of big renin extracted from human plasma.

The properties of big renin, a relatively inactive form of renin isolated from human plasma, were examined following partial purification by gel filtration. Exposure of big renin to pH 3.0-3.6, or brief incubation with trypsin or pepsin, resulted in a ten-fold increase in enzymatic activity. Activation was not effected by 4M NaCl, 6M urea, or incubation with neuraminidase. Both before and after inactivation, big renin eluted from Sephadex gel more rapidly than normal plasma renin. During polyacrylamide gel disc electrophoresis, inactive big renin migrated more slowly than either normal renin or big renin previously activated. Using sheep substrate, the enzyme kinetics of normal renin and previously activated big renin were identical, while inactive big renin possessed a higher Michaelis constant. These data indicate that big renin is closely related biochemically to normal plasma renin. As the activation of big renin results in the formation of the substance even more similar to normal renin, the possibility exists that big renin may prove to be a precursor form of normal renin.


Plasma LH and FSH response to LRH in boys with compensatory testicular hypertrophy.

The plasma LH, FSH and testosterone response to LRH was studied in 12 boys with compensatory testicular hypertrophy (CTH) and normal puberty and in a matched control group with normal testicular development. It was found, that the boys with CTH had normal basal plasma testosterone and LH concentrations; at the same time the basal plasma FSH level were significantly higher than in the control group. The response of plasma LH and FSH to LRH was markedly greater in the CTH group than it was in the control group. It is concluded, that the contralateral testicular hypertrophy which enables a normal pubertal process is the result of increased secretion of gonadotropins, mainly FSH.


Diazepam and meperidine on arterial blood gases in patients with chronic obstructive pulmonary disease.

Arterial blood gases were not significantly altered by 0.15 mg/kg intravenous dose of diazepam in patients with chronic obstructive lung disease. The PalphaO-2 was significantly reduced and the P-alphaCO-2 increased after administration of 1.5 mg/kg meperidine intravenously and the combination of 1.5 mg/kg meperidine with 0.15 mg/kg diazepam. Diazepam did not, however, significantly increase the ventilatory depression induced by meperidine. Therefore, 0.15 mg/kg diazepam intravenously causes no ventilatory depression by meperidine in patients with chronic obstructive lung diseases.


Capillary pH and blood gas determinations in asthmatic children.

Earlobe capillary pH and blood gases in asthmatic children were compared to arterial values. Hyperemia of the earlobe was produced by applying thurfyl nicotinate (Trafuril). Approximately 15 min later capillary blood was obtained simultaneously with a radial arterial sample. Earlobe capillary PO2, PO2, and pH correlated well with arterial values. This technique is simple, safe, and appears to be a satisfactory substitute for arterial blood in normotensive asthmatic children.


A new colimetric assay for monoamine oxidase in serum and its clinical application.

A new colimetric assay for serum monoamine oxidase and clinical findings based on this method are described. This rapid, precise procedure employs 1-[(4-aminomethylphenyl)azol]-2-naphthol as a substrate. The enzymatically generated 1-[(4-formyl phenyl)azo]-2-naphthol is extracted into cyclohexane to form a colored solution whose absorbance is determined at 500 nm. The method proved to be extremely simple, and the results obtained correlated well with those given by the standard method of McEwen.


Effect of hemodialysis on factors influencing oxygen transport.

Ten patients underwent 4 study hemodialyses, one with standard dialysis conditions, one with an isophosphate dialysate, one with simultaneous ammonium chloride loading, and other, after pretreatment, with sodium bicarbonate. Measurement of hemoglobin oxygen affinity (P-50), erythrocyte 2,3-DPG, blood-gasses, and serum chemistries revealed biochemically effective hemodialyses and slight changes in oxygen transport parameters. The P-50 (in vivo) values decreased slightly but significantly (p greater than 0.05) with dialysis. When corrected to pH 7.4, eliminating the Bohr effect, P-50 increased (p greater than 0.05). With unmodified dialysis elevated values of 2,3-DPG (in comparison to normal) decreased, a change that did not correlate with delta-p-50, delta-serum phosphate, or delta-serum creatinine. With standard and isophosphate dialyses Po-2 decreased significantly. The decrease correlated with delta-hydrogen ion concentration and did not occur with dialyses designed to maintain pH constant. Thus, hemodialysis influences many factors that affect oxygen transport in different and counterbalancing directions. These changes are not totally explained by alterations in 2,3-DPG, pH or serum phosphate. Maintenance of acidosis or hyperphosphatemia during dialysis is not recommended.


Alkaline phosphatase isozymes of Xenopus laevis embryos and tissues.

Alkaline phosphatase was obtained by treating embryos of Xenopus laevis with n-butanol at different developmental stages from gastrula to tadpole; the enzyme was also obtained from adult kidney, liver, and intestinal mucosa. Purification was carried out by gel filtration and polyacrylamide gel electrophoresis. The enzyme activity is chromatographically spearated into two peaks, with molecular weights of approximately 200,000 and 400,000. Alternatively, two groups may be characterized on the basis of their electrophoretic mobilities, which correspond to the different molecular weight classes. Effects of pH, temperature, inhibitors, and substrate concentration were studied. The kinetic and physical properties of the two alkaline phosphatase isozymes are similar, and are comparable to the properties reported for this enzyme from other vertebrates. Alkaline phosphatase activity increased sharply at the gastrula stage and reached a plateau at the late tailbud stage. During this period there was an 18-fold increase in activity.


Composition of fluid from the notochordal canal of the coelacanth, Latimeria chalumnae.

Fluid from the notochordal canal of the coelacanth, Latimeria chalumnae, was analyzed for major inorganic and organic constituents and compared with blood serum from the same fish. Significantly or suggestively lower levels of sodium, magnesium, calcium, bicarbonate, sulfate, total carbohydrates, glucose, lactate, cholesterol, bound phosphate and total proteins were found in notochordal fluid than in serum, whereas potassium, chloride, urea, trimethylamine oxide, and total free amino acids were higher and inorganic phosphorus essentially identical. Osmolarity of notochordal fluid (1058 mOsm) exceeds that of serum (942 mOsm). A whitish precipitate in the fluid consisted of a matrix of fibers 100 A in diameter and of indefinite length. It resembled a sialoglycoprotein in composition and was stabilized by disulfide bonds. The fluid contained cellular debris.


Effects of diets containing dried whey on the lactase activity of the small intestinal mucosa and the contents of the small intestine and cecum of the pig.

Two studies were conducted to establish the effects of dietary lactose supplied from dried whey on the lactase activity in the contents of the small intestine and cecum as well as the mucosa of the small intestine. In the first study, feeding 0, 10, 25, or 40% dried whey for 120 days did not alter the mucosal lactase specific activity (mumoles galactose released/mg protein) in the duodenum, upper jejunum, or lower ileum. In the second study, total lactase activity in the small intestine was estimated from the total activity found in 10-cm segments of each meter of small intestine. Feeding a diet containing 40% whey continuously from weaning at 5 weeks of age or from 12 weeks of age did not alter the total lactase activity of pigs killed at 21 weeks of age. Dietary lactose tended to increase the lactase activity in the contents of the cecum and small intestine, but the increases were not always statistically significant. In the second study, Chester White pigs had approximately threefold greater mucosal lactase activities than that of Hampshires. These two breeds may be suitable models for detailed studies into the relationships between lactase deficiency and lactose intolerance.


Effects of a human plasma fraction on leucocyte migration into inflammatory exudates.

A fraction prepared from normal human plasma inhibits the migration of polymorphonuclear and mononuclear leucocytes into inflammatory exudates produced by the intrapleural injection of carrageeman or turpentine by the subcutaneous implantation of polyvinyl sponges in the rat. The mechanism of the effect does not involve complement depletion.


Effect of pregnenolone-16alpha-carbonitrile (PCN) on drug response in man.

Pregnenolone-16alpha-carbonitrile (PCN), which alters drug responses and induces hepatic microsomal drugs-metabolizing enzymes in experimental animals, is well-tolerated in man. There were no marked changes in the concentrations of blood sugar and urea; marked changes in the concentrations of blood sugar and urea; bromsulphophthalein (BSP) clearance; serum alkaline phosphatase activity; hematocrit, erythrocyte and leukocyte counts; as well as in ecg patterns. When the steroid was given by mouth for 4 days to patients treated with electroshock for psychiatric disorders, it decreased suxamethonium-induced skeletal muscle fasciculation and shortened thiopentone as well as propanidid anaesthesia. Breathing was resumed more rapidly than in control subjects. The protective effect of PCN is probably mediated through induction of activation or hepatic drug-metabolizing enzymes, or both.


The solubilization of some local anaesthetic ester of p-aminobenzoic acid by lysophosphatidylcholine.

The solubilization by lysophosphatidylcholine (LPC) of three n-alkyl esters of p-aminobenzoic acid has been studied. These esters have a local anaesthetic action. Quantitative studies show that the amount of compound solubilized is proportional to the LPC concentration and that solubilization increases in the order ethyl, n-propyl and n-butyl ester. 100MHz nmr studies indicate that the local anaesthetic esters are solubilized in the hydrocarbon interior of the LPC.


Narcotic agonist and antagonist potencies of a homologous series of N-alkyl-norketobemidones measured by the guinea-pig ileum and mouse vas deferens methods.

The narcotic agonist and antagonist potencies of the series of N-alkyl-norketobemidones from norketobemidone to decylnorketo-bemidone have been determined. The values obtained in the electrically stimulated preparations of the guinea-pig ileum and the mouse vas deferens are closely correlated. The agonist potencies observed in the guinea-pig ileum agree well with those found in the mouse hot-plate test (Oh-ishi & May) and those obtained by determining the inhibition of naloxone binding in brain homogenates (Wilson, Rogers, Pert & Snyder). The antagonist potencies in the guinea-pig ileum and, to a lesser extent, those in the mouse vas deferens agree with the values obtained in the morphine-dependent monkey.


Some effects of pilocarpine and acetylcholine on the responses of the guinea-pig vas deferens to hypogastric nerve stimulation and transmural stimulation and applied (minus)-noradrenaline.

Enhancement by acetylcholine and by pilocarpine of the contractile responses of the guinea-pig isolated vas deferens to hypogastric nerve and transmural stimulation and to exogenous (minus)-noradrenaline was examined. At high concentration (600 nM) acetylcholine enhanced responses in the order:hypogastric nerve greater than transmural greater than exogenous (minus)-noradrenaline. Pilocarpine produced a similar maximal degree of enchancement with all three stimuli. All these effects were reversibly inhibited by 1-4 mum atropine, a concentration which alone did not affected responses to the stimuli. Pilocarpine (50-400 muM) gave a concentration-related inhibition of the enhancement of responses to hypogastric nerve stimulation by 600 nM acetylcholine.


Generation of prostaglandin E-like material by the guinea-pig trachea contracted by histamine.

Challenges of the superfused (1 ml min-1) trachea with histamine (100-200 mug) result in the release of prostaglandin E-like material (3-25 ng in terms of prostaglandin E2) but no prostaglandin F-like activity has been detected in the superfusate. This release is blocked by indomethacin (1mug ml-1) and then the contractile action of histamine is enhanced. It is concluded that the release of a prostaglandin E-like material by histamine from tracheal smooth muscles is a self-defensive mechanism protecting against the strong constriction of airways. The maximal relaxation of trachea by isoprenaline (50-500 mug) is not accompanied by the release of a prostaglandin-like material.


The pharmacology of Avena sativa.

The pharmacology of Avena sativa has been investigated in laboratory animals following a report that tincture of Avena sativa reduced the craying for cigarettes in man. The tincture, evaporated to dryness, craving for cigarettes in man. The tincture, evaporated to dryness, re-constituted in an equal volume of water and administered by stomach tube or intraperitoneal injection, antagonized the antinociceptive effect of morphine in two separate test (hot-plate and tail flick). Compared with animals made depedent on morphine alone, mice pretreated with repeated injections of morphine plus extract passed a smaller number of stools and tended to jump less after administration of nalorphine. The pressor response to intravenously administered nicotine in urethane-anaesthetized rats was also antagonized by prior administration of Avena sativa. However, the aqueous extract prepared from the tincture did not affect the seizure threshold to bemegride or nicotine or the sleeping time induced by barbitone sodium.


The pharmacology of 14-hydroxyazidomorphine.

6-Deoxy-6-dihydroazido-14-hydroxyisomorphine (14-hydroxyazidomorphine) was synthesized, its analgesic potency in mice and rats, its antitussive effect in rats and its dependence liability in mice, rats and monkeys were studied. Azidomorphine the 14-nonhydroxylated parent molecule, morphine, hydromorphone and oxymorphone were used for comparison. 14-Hdroxyazidomorphine proved to be as potent an analgesic as azidomorphine, even more potent as an antitussive, and showed the same low tolerance and dependence capacity. It was 11-6 times less toxic than azidomorphine in mice and 6-5 times less toxic in rats.


Emulsion stabilization by non-ionic surfactants: the relevance of surfactant cloud point.

The effect of various additives, electrolytes and non-electrolytes, on the cloud point of non-ionic surfactants has been studied. Additives which salt-out the polyoxyethylene chains of the surfactants cause decreased stability of oil-in-water emulsions by decreasing the true hydrophile-lipophile balance (HLB) of the surfactant; additives such as sodium iodide and propanol salt-in the non-ionic surfactants and result in an increase in the effective or true HLB of the system. The latter additives do not increase the hydration of the polyoxyethylene chains but their effect must be on the structure of water so that the heat of hydration of the chains is altered. Experiments with free films of the aqueous surfactant (Brij 96) show that thinning rates are markedly affected by the additives, but there is little effect on the equilibrium thickness of the films (ca 11 nm). Nonetheless the thickness at the transition from thick film to equilibrium black film decreases with increasing cloud point of the solution indicating increased stability. The importance of structure formation in the liquid film separating the emulsion globules was demonstrated.


Aggregation of antihistamines in aqueous solution. The effect of electrolyte on the miscellar properties of some diphenylmethane derivatives.

The effect of sodium chloride on the micellar properties of the antihistamines, dephenhydramine hydrochloride, bromodiphenhydramine hydrochloride, chlorcyclizine hydrochloride and diphenylpyraline hydrochloride in aqueous solution has been investigated by light scattering and viscometric methods. The drugs behaved as typical ionic surfactants showing an increase in aggregation number and decrease in critical micelle concentration as the electrolyte concentration was increased over the range 0.05 to 0.154 mol kg-minus1. A linear relation between log critical micelle concentration and log counterion concentration was established, from which values of the degree of ionization and the free energy of micellization were calculated. The intrinsic viscosity was decreased by the addition of electrolyte and this has been attributed to a decrease in micellar hydration due to a removal of hydrogen-bonded water.


The evaluation of a column-type dissolution apparatus.

An improved column-type dissolution apparatus is described. The column of the apparatus is built from standard screw-cap connected "Sovirel" glass tubes which should enable inter-apparatus variability to be reduced. The main problem with this type of device is the build up to back pressure as fragments from the disintegrating solid dosage form progressively block the filtration system, itself necessary to stop the solution process and present the solution for analysis. A simple type of light-activated switch is therefore used which works across the indicator float of a flow meter to drive a motor connected to a needle valve. As the float falls the motor is switched on to open the valve and increase the flow rate until the float rises again and causes the motor to switch off. Observation on the dissolution characteristic of dyed lactose tablets and the injection of dye solutions into the flowing stream of liquid around tablets showed that there was back-flow of solution at Reynolds Numbers below 10 but visible instability to flow at Re greater than 100. Over a range of flow rates between Re 10-70 the mass of drug released at a given time was a direct function of flow rate for a film coated product but an inverse function for a sugar coated tablet. The results are discussed in relation to the main problems which can be experienced with a column-type dissolution apparatus.


Microiontophoretic studies of the effects of false transmitter candidates and amphetamine on cerebellar Purkinje cells.

The effects of microiontophoretic applications of equivalent doses (ejection times and currents) of noradrenaline, amphetamine, octopamine and p-hydroxynorephedrine on the spontaneous firing of Purkinje and unidentified cells in the cerebellum of rats were examined. In addition, the effects of amphetamine of Purkinje cells were examined in animals pretreated with the tyrosine hydroxylase inhibitor, alpha-methyltyrosine (alpha-MpT) or with a combination of reserpine plus alpha-MpT. The results indicate that the "false transmitters" are weak agonists when compared to noradrenaline in inhibiting the firing of Purkinje cells. The results of the iontophoretic studies with amphetamine are not consistent with a pre-synaptic releasing effect by amphetamine at noradrenergic synapses in the cns since the efficacy of amphetamine on Purkinje cells was unaltered after pretreatment with alpha-MpT or alpha-MpT plus reserpine.


Intra- and extraneuronal formation of the two major noradrenaline metabolites in the cns of rats.

The two major noradrenaline metabolites in the rat brain, total 3-methoxy-4-hydroxyphenylglycol (MOPEG) and total 3,4-dihydroxyphenylglycol (DOPEG), were measured by a new analytical procedure after intraventricular (i.v.t.) injection of (3-H) noradrenaline or (3-H) dopamine. I.v.t. injection of (3-H) noradrenaline to rats with a 6-hydroxydopamine-induced destruction of nerve terminals in the central nervous system, resulted in an increased accumulation of 3-H-MOPEG compared to 3-H-DOPEG. On the contrary, reserpine induced a greater increase in 3-H-DOPEG than in 3-H-MOPEG accumulation when these two metabolites were formed from (3-H) noradrenaline synthesized in vivo by i.v.t. injection of (3-H) dopamine. These results indicate that the formation of DOPEG and MOPEG occur with some preference intra- and extraneuronally, respectively. The results, however, also show that these two formations sites should not be considered as specific.


Neuromuscular actions of edrophonium in the lateral segmental tail muscle of the rat in vivo.

The actions of edrophonium on neuromuscular transmission in vivo have been studied using the lateral segmental tail muscles of the rat. The drug produced an increase in amplitude of the miniature endplate potentials (m.e.p.p.s) without an effect on their frequency and increased the amplitude of the end-plate potentials (e.p.p.s) without increasing quantal release of transmitter. It is concluded that the anticholinesterase action of this drug in vivo facilitates neuromuscular transmission, which confirms many of the findings from in vitro studies.


A simultaneous determination of acetylsalicylic acid, salicylic acid and salicylamide in plasma by gas liquid chromatography.

A novel method for the simultaneous determination of acetylsalicylic acid, salicylic acid and salicylamide in biological fluids by gas liquid chromatography is described. The assay has been used to determine the plasma concentration of salicylates in 10 volunteers after oral ingestion of three commercially available aspirin-containing formulations. No difficulty was encountered in determining low concentrations of acetylsalicylic acid in the presence of higher concentrations of salicylic acid. The in vivo plasma half life of acetylsalicylic acid in man was found to be 15.5 min.


The gas chromatographic determination of atenolol in biological samples.

Methods are described for the gas chromatographic determination of the B-adrenoceptor blocking agent atenolol (ICI 66,082 Tenormin) in whole blood, serum, tissue and urine. The method involves the extraction of the free base from the prepared biological sample into an organic solvent mixture. Further purification is done by backextracting the base into dilute acid from which, after basification it is extracted again into organic solvent. Any atenolol in the residue remaining after solvent evaporation is converted into a heptafluorobutyrate derivative ready for gas chromatography. Use of an electron-capture detector enables concentrations as low as 0.01 mug ml-minus 1 body fluid and 0.04 mug ml-minus 1 tissue to be measured.


A rapid gas-liquid chromatographic estimation of nicotine in biological fluids.

A rapid gas-liquid chromatographic method for the estimation of nicotine in plasma is described. Nicotine is extracted from alkalinized plasma into diethyl ether. This is then concentrated by evaporation and, after an acid back extraction is re-extracted into n-heptane (nitrogen detector) or dichloromethane (flame ionization detector) before injection onto the gas chromatography. Thirty samples a day can be analysed by this method which enables concentrations of 0.1 ng ml- minus 1 nicotine to be measured. It is thus possible to measure nicotine in plasma and urine samples from non-smokers.


Hydrolysis of fatty acid esters of acetaminophen in buffered pancreatic lipase systmes I.

A series of fatty acid esters of acetaminophen were prepared beginning with acetate, the propionate, and all even-numbered fatty acids and going through the octadecanoate. The enzymatic hydrolysis of all derivatives was studied in vitro with varying amounts of lipase assed to the hydrolysis mixtures. Under the conditions of the in vitro hydrolysis, it was observed that all derivatives were hydrolyzed more readily in an aqueous medium at pH 7.8. A positive relationship was seen between the hydrolysis rates and the concentration of lipase at this pH. There was a negative relationship between the chain length of the acyl moiety and the corresponding hydrolysis rates. The short chain esters were hydrolyzed at rates many times more rapid than the long chain esters. The intermediate chain-lenght ester, p-acetamidophenyl decanoate, p-acetamidophenyl laurate, and p-acetamidophenyl myristate, were hydrolyzed at intermedediate time periods extending over 12 hr, approaching completion at 97.5, 87.5, and 80.5%, respectively, when 18 Wilson units of lipase was used in each milliliter of hydrolysis mixture. The longer chain esters, p-acetamidophenyl palmitate and p-acetamidophenyl stearate, were hydrolyzed to the extent of 16 and 8%, respectively, over 12 hr under the same in vitro conditions.


Kinetics of sulfite-induced anaerobic degradation of epinephrine.

The rate constant-pH profile of the rate of sulfite-induced disappearance of epinephrine from an aqueous solution under anaerobic conditions was determined at 81 degrees in the 3.63-5.00 pH range at the ionic strength of 0.2. The profile shows a linear relationship with a positive slope. The anaerobic rate shows buffer catalysis above pH 4.4 Metabisulfite was found to be more catalytic than either bisulfite or acetone bisulfite. The concentration of epinephrine used was 5.5 times 10- minus 5 mole/liter.


Systems approach to vaginal delivery of drugs I: development of in situ vaginal drug absorption procedure.

In the framework of the development of drug delivery systems for locally administered contraceptive drugs, a reliable method that can afford quantitative evaluation of drug absorption behavior was explored using the rabit doe. A system was constructed based upon perfusing the drug solution in the vaginal tract. For this purpose, a "rib-cage" type cell was constructed and surgically implanted in the rabbit prior to an experiment. The primary purpose of the present paper is to evaluate the method, including the surgical operation and the perfusion system. The absorption experiments were carried out using n-butanol-1minus 14C as the model solute to survey the reproducibility of the absorption behavior. Experiments were conducted with a number of rabbits on several successive days to determine the day-to-day and animal-to-animal variations. The drug disappearance in the reservoir followed first-order kinetics from which the apparent permeability coefficient was calculated. The results indicated that a set of experiments may be carried out on a single animal and that the method generally affords rather high precision.


Vasoconstriction of the isolated rabbit ear artery caused by nicotinic agonists acting on adrenergic neurons.

In the perfused isolated central ear artery of the rabbit, nicotine, acetylcholine (ACh), tetramethylammonium (TMA) and carbachol (CCh) at high concentrations (greater than 1 mug/ml) produced transient vasoconstriction. The order of potency was: nicotine greater than ACh greater than TMA greater than CCh. Over the concentration range used, all of the agonists except ACh gave bell-shaped log concentration-response curves. Methacholine did not cause vasoconstriction. The response to ACh, TMA or CCh, but not to nicotine, was potentiated by atropine (0.1-1.0 mug/ml). Tetraethylammonium, hexamethonium, mecamylamine and d-tubocurarine blocked the response to the nicotinic agonists and to nerve stimulation. Ear arteries from reserpine-treated rabbits gave no, or very little, constrictor response to the nicotinic agonists or to nerve stimulation. These findings support the conclusion that the vasoconstriction produced by the nicotinic agonists is mediated by norepinephrine released from adrenergic nerve terminals as a result of the action of the agents on neuronal nicotinic receptors located at or near the terminals. The potentiation by atropine of the constrictor response to ACh, TMA and CCh is attributed to the blockade by atropine of neuronal muscarinic receptors on which these agonists act to inhibit the release of norepinephrine.


Effects of beta adrenergic blocking agents on erythropoietin production in rabbits exposed to hypoxia.

The effects of dl-propranolol, d-propranolol, butoxamine or practolol on erythropoietin (ESF) production in rabbits exposed to hypoxia (0.42 atmosphere) for 18 hours were investigated. Rabbits treated with dl-propranolol (4 or 8 mg/kg i.p.) or butoxamine (15 or 30 mg/kg i.p.) produced significantly less ESF in response to hypoxia than did saline-treated control animals. ESF production in rabbits given d-propranolol or practolol during hypoxia was not significantly different from that of control animals exposed to hypoxia. Based on the proposed selectivity of butoxamine for beta2 adrenergic receptors and of practolol for beta1 adrenergic receptors, it is suggested that ESF production in rabbits exposed to hypoxia may involve the activation of beta2 adrenergic receptors.


Oxilorphan (l-N-cyclopropylmethyl-3,14-dihydroxymorphinan): a new synthetic narcotic antagonist.

Oxilorphan is a fully synthetic morphinan derivative containing the 14-hydroxy group characteristics of naloxone and naltrexone. As a narcotic antagonist, oxilorphan was 4 times more potent than dl-cyclazocine, equipotent to naloxone and about one-fourth as potent as naltrexone parenterally. Duration studies in rodents were inconclusive, but in antagonism of morphine analgesia and miosis in the dog, oxilorphan was longer acting than naloxone and equivalent to dl-cyclazocine. Oxilorphan was inactive in the conventional animal thermal analgesic tests. However, oxilone did exhibit relatively weak analgesic activity in preventing phenylquinone-induced abdominal contraction at doses about 700 times higher than those required for antagonist activity. The analgesic potency of oxilorphan was only 120 and 1/300 the potency of morphine and dl-cyclazocine, respectively, but was significantly greater than naltrexone and naloxone. Mice chronically treated with increasing doses of oxilorphan failed to exhibit withdrawal jumping after naloxone challenge. General central nervous system activity studies showed oxilorphan to be relatively free from central side effects at doses at which dl-cyclazocine produced pronounced effects.


The pharmacology of batrachotoxin. VII. Structure-activity relationships and the effects of pH.

The effects of the depolarizing agent, batrachotoxin (BTX), and of various analogs were studied on rat phrenic nerve-diaphragm muscle preparations at 37 degrees C. The structural modifications of BTX included: 1) replacement of the 20alpha-pyrrole-3-carboxylate moiety; 2) alterations of substituents on the pyrrole moiety; 3) clevage of the 3alpha, 9alpha-hemiketal linkage; and 4) quaternization of the tertiary nitrogen of BTX. All of the compounds except batrachotoxinin A (BTX-A), which lacks the 20alpha-substituent, depolarized the postsynaptic membrane, transiently increased the frequency of spontaneous transmitter release to 400 to 600 sec- minus 1 and finally produced blockade of the directly and indirectly elicited muscle twitches. Of the compounds tested, only BTX-A potentiated the muscle twitches. The concentration which elicits a 50% depolarization of the muscle membrane in 1 hour was determined for all the compounds except for BTX-A and for dihydrobatrachotoxin which lacks the 3alpha, 9alpha-hemiketal linkage; these two analogs never depolarized the postsynaptic membrane by more than 10 to 15%. BTX, the 20alpha-2, 4, 5-trimethylpyrrole-3-carboxylate of BTX-A and the 20alpha-ester of BTX-A with 2-ethyl-4-methylpyrrole-3-carboxylic acid (homobatrachotoxin) were the three most potent toxins with doses of 4.5, 12 and 18 times 10- minus 9 M eliciting a 50% membrane depolarization in 1 hour. The quaternary derivative of BTX, the 20alpha-4, 5-dimethylpyrrole-3-carboxylate of BTX-A and 20alpha-2,4-dimethyl-5-acetylpyrrole-3-carboxylate of BTX-A were 24-, 65- and 110-fold less potent than BTX as depolarizing agents, whereas the 20alpha-p-bromobenzoate of BTX-A was 220-fold less potent. Each of these derivatives had the ability to increase sodium permeability since the increase in spontaneous miniature end-plate potential frequency and membrane depolarization were reversed by tetrodotoxin or by reducing the external sodium concentration. BTX was found to be more effective at alkaline pH (pH 9.0), at which it exists almost entirely in the un-ionized form, than at physiological or acidic pH(6.0). The results indicate that the analogs of BTX act by a mechanism similar to that of the parent compound, but that their potency differs and certain compounds may have a more selective action on either the pre- or postsynaptic membrane. For maximal depolarizing activity, a substituted pyrrole moiety is necessary at the 20alpha-position of BTX-A and 3alpha, 9alpha-hemiketal linkage must remain intact providing rigidity for the pentacyclic steroid nucleus.


Experimental study of a potential anti-asthmatic agent: SCH 15280.

A forced oscillations technique for measuring total respiratory system resistance was used to quantitate the bronchomotor activity of Sch 15280: (5[4-(N-methyl)-piperidylidine]5H-[1]-benzopyrano[2,3,b]-pyridine maleate) and to compare its potency to that of standard bronchodilator agents. By the intravenous route, Sch 15280 was 48 times more potent than aminophylline and 1/78 as potent as atropine in inhibiting methacholine-induced bronchoconstriction in rabbits. In cats Sch 15280 was 1/12 as potent as ephedrine in inhibiting histamine-induced bronchoconstriction and had a greater protective activity against histamine-induced bronchoconstriction and had a longer duration of action than 0.5% solution of isoproterenol. Statistical tests of parallelism revealed a significant difference between the log dose-response curves of Sch 15280 and ephedrine but not between those of Sch 15280, atropine and aminophylline. The results suggest that Sch 15280 can act via, a nonadrenergic mechanism to block both histaminergically and cholinergically mediated responses in the tracheobronchial tree. This pharmacologie profile may have important therapeutic application in the type I immediate hypersensitivity reactions of man.


Effects of 6-hydroxydopamine and reserpine on amphetamine-induced release of norepinephrine in rat cerebral cortex.

Amphetamine released 3-H-norepinephrine from rat cerebral cortex tissue which had previously accumulated the 3-H-amine. Destruction of noradrenergic nerve endings by pretreatment of the rats with 6-hydroxydopamine inhibited the accumulation of 3-H-norepinephrine by the tissue and reduced the proportion of the 3-H-amine which was released by amphetamine. Inhibition of storage of 3-H-norepinephrine within nerve endings by pretreatment of the animals with reserpine also reduced accumulation of 3-H-norepinephrine but did not reduce the proportion of the accumulated 3-H-amine which was released by amphetamine. The addition of desipramine (an inhibitor of neuronal uptake) further reduced the accumulation of 3-H-norepinephrine in animals pretreated with reserpine but had no further effect in animals pretreated with 6-hydroxydopamine. A greater proportion of the 3-H-norepinephrine was converted to 3-H-deaminated metabolites in tissues of reserpine-treated animals than in the tissues of control or 6-hydroxydopamine-treated rats. Amphetamine-induced release of 3-H-norepinephrine was partially calcium dependent in tissues from control animals. After reserpine treatment, amphetamine-induced release of norepinephrine was independent of calcium, whereas potassium-mediated release was still markedly calcium dependent. These experiments indicate that amphetamine releases 3-H-norepinephrine primarily from storage sites within central adrenergic nerve endings. An analysis of the time course of release from tissues of rats treated with reserpine suggests that amphetamine is equally capable of releasing 3-H-norepinephrine from granular sites which are susceptible to reserpine and from reserpine-insensitive sites.


Effect of alpha-methyldopa on dopamine synthesis and release in rat striatum in vitro.

The effect of alpha-methyldopa and alpha-methyldopamine (alpha-MDA) on the rate of hydroxylation of radioactive tyrosine was studied in striatal slices from rat brain. This was done by measuring the formation of 3-H-H2O as well as the accumulation of 3-H-dopamine (3-H-DA) from L-3, 5-3-H-tyrosine. alpha-Methyldopa, at tissue concentrations similar to those found in vivo after systemic administration, produced a decrease in both 3-H-H2O and 3-H-DA. The marked decrease (91thyldopa injection, also inhibited 3-H-H2O formation. The inhibitory effect of alpha-methyldopa on 3-H-H2O formation was not reduced by the addition of brocresine, which prevents the formation of alpha-MDA. The effects of alpha-methyldopa and alpha-MDA on the release of 3-H-DA that had been taken up into brain slices, was also studied. Although alpha-methyldopa, 1000 muM, did not increase the release of 3H-DA from tissue, alpha-MDA did. However, the latter was more potent in inhibiting 3-H-H2O formation from 3-H-tyrosine than in releasing 3-H-DA. These results, as well as the close similarity between the percent reduction of 3-H-H2O formation and tissue 3-H-DA levels, suggest that alpha-methyldopa decreases tissue levels of dopamine by inhibiting tyrosine hydroxylase activity in DA neurons.


Induction of tyrosine hydroxylase elicited by beta adrenergic receptor agonists in normal and decentralized sympathetic ganglia: role of cyclic 3',5' - adenosine monophosphate.

A subcutaneous injection of an oil suspension of l-epinephrine (270 mumol/kg), dopamine (270 mumol/kg) or l-norepinephrine (270 mumol/kg), when administered with phenoxybenzamine (32 mumol/kg i.p.) to blocl alpha adrenergic effects, increases the cyclic 3', 5'-adenosine monophosphate (cAMP) content in superior cervical ganglia (SCG) of rats. The increase is highest after l-epinephrine and dopamine and is barely detectable after l-norepinephrine; it lasts longer than 2 hours after l-epinephrine, about 30 minutes after dopamine and is fleeting after l-norepinephrine. The duration of the increase in cAMP elicited by l-epinephrine in SCG of rats is dose-related. Furthermore, when the cAMP increase lasts longer than 90 minutes, 48 hours later the tyrosine hydroxylase (TH) activity in SCG is increased. l0Epinephrine (150 mol/kg s.c.) induces TH in decentralized ganglia. One injection of l-isoproterenol (77 mol/kg i.p.) increases cAMP concentrations in intact and decentralized SCG. This increase lasts only 30 minutes and fails to induce TH 48 hours later. However, if the increase of cAMP concentration is prolonged by four successive injections of l-isoproterenol (15 30-minute intervals) the TH activity of intact and decentralized SCG is increased 48 hours later.l-Isoproterenol (four injections of 77 mumol/kg, each) and l-epinephrine (270 mumol/kg) fail to induce TH in the adrenal medulla. dl-Propranolol (125 mumol/kg i.p.) injected 30 minutes before l-isoproterenol blocks the increase of cAMP content and the delayed induction of TH activity in SCG. The elevation of TH activity elicited in SCG by beta adrenergic receptor agonists is always preceded by an increase of cAMP concentration lasting 90 minutes or longer. However, the induction of TH elicited by cold exposure or by reserpine administration can occur without an apparent increase in ganglionic cAMP concentration.


The distribution and movements of carbon dioxide, carbonic acid and bicarbonate between blood and milk in the goat.

1. A-V differences and milk concentrations of respiratory gases, pH, HCO3 and H2CO3 have been measured in lactating goats and cows. 2. The pH and [HCO3 minus] of milk were significantly lower than those of plasma while milk PCO2 was virtually identical to that of mammary venous blood. [H2CO3+ dissolved CO2] was similar in milk and blood. 3. 14-C (from injected [14-C]HCO3 minus was found to cross the mammary epithelium in both directions. 14-C also passed across the duct epithelium and since this epithelium has previously been shown to be impermeable to ions it is argued that 14-C crossed in an unionized form, i.e. as CO2 and/or H2CO3. 4. Hourly milking with the aid of oxytocin raised milk pH, [HCO3 minus], [H2CO3], [Na] and E1Cl], and lowered [K], [lactose] and [phosphate]. These effects are discussed in relation to the hypothesis proposed previously for the action of oxytocin on milk composition. 5. A scheme for the distribution and movements of CO2, H2CO3 and HCO3 minus between extracellular fluid and milk is suggested, and discussed in relation to Cl minus transport.


Lithium ions and the release of transmitter at the frog neuromuscular junction.

1. Transmitter release has been examined at the frog neuromuscular junction when all or part of the Na of the Ringer is replaced by Li ions. 2. No immediate change occurs in either the mean quantal content of the end-plate potential or the miniature end-plate potential frequency on changing to Li Ringer but over the following hour both these quantities increase by more than two orders of magnitude. 3. During thefirst 30-40 min of an exposure to Li-Ringer the m.e.p.p. frequency rises exponentially with a time constant of 10 min, and the mean quantal content of the e.p.p. grows by addition of extra evoked quanta, the increment rising exponentially with a time constant the same as that of the m.e.p.p. frequency. 4. Following this initial period in Li-Ringer the m. e.p.p. frequency accelerates to a peak of several hundred quanta per second and then declines slowly over the next few hours. Just before the m.e.p.p.frequency peak the conduction velocity of the presynaptic action potential declines and shortly afterwards synaptic transmission fails as the action potential no longer conducts into the terminals. 5. The rise in the m.e.p.p. frequency during the first 30-40 min is independent of the [Ca-2+]o. At subsequent times before the peak external Cabecomes progressively more effective in accelerating the m.e.p.p. frequency and in the presence of 1mM-EGTA spontaneous release stabilizes at 60-80 quanta/sec. 6. The [Li-+]o strongly influences the rate of increases in both evoked and spontaneous release but not their extent; replacing only half the Na of the Ringer by Li increases the time constants of the increases to about 30 min. 7. Rises in the m.e.p.p. frequency can be irreversibly accelerated by tetanizing the nerve in a Li-Ringer in which the Ca has been chelated by EGTA. The extent of the increases in the m.e.p.p. frequency is dependent on the number of pulses in the tetanus and is little affected by the frequency of stimulation. Accumulation of Li ions inside the presynaptic terminals probably underlies the changes in spontaneous release. 8. When only 10 percent of the Na of the Ringer is replaced by Li-+ ions the magnitude of post-tetanic potentiation of the e.p.p. and of the post-tetanic rise in the m.e.p.p. frequency is increased. Under these conditions changes in facilitation of the e.p.p. are small. 9. Various mechanisms by which Li could alter transmitter release are discussed and it is suggested that intracellular Ca sequestering mechanisms of the presynaptic terminals are affected when an end-plate is exposed to Li-Ringer.


Chemical stimulatory mechanism in gastric secretion.

1. The serum gastrin level, gastric mucosal blood flow and acid secretion from the canine Heidenhain pouch have been measured in response to the introduction of bovine serum albumin, pepsin-digested albumin, an amino acid mixture, liver extract and mannitol used as control. 2. Distention of the Heidenhain pouch with mannitol or albumnin at pH 5-0 produced a similar pressure-related increase of acid secretion reaching a peak of only 10 percent of the maximal response to histamine. Pepsin-digested albumin was capable of producing larger acid outputs than undigested albumin. The highest acid output, attaining about 80 percent of the maximal response to histamine, was obtained with liver extract both before and after exhaustive dialysis to remove all the amino acids and short peptide fragments. An amino acid mixture containing all essential amino acids was also found to stimulate acid secretion but a lesser degree than liver extract. 3. This concluded that it is not the intact protein but the products of its digestion, the polypeptides and free amino acids, which are potent chemical stimulants of acid secretion from the oxyntic gland area. Since the serum gastrin level was not changed during acid secretion induced by peptic digests bathing the oxyntic gland area, the mechanism of chemical stimulation appears to be gastrin-independent. 4. The response to chemical stimulation by peptic digests can be greatly potentiated by combining this with distention of the oxyntic gland area. Topical application of xylocaine or atropine causes a marked decrease of Heidenhain pouch response to peptic digests, suggesting a possible neural reflex component in the mechanism of chemical stimulation of the oxyntic gland area. 5. When the pH of the liver extract in the Heidenhain pouch was gradually decreased in sequential order from 5-0 to 1-0, this resulted in a pH-related decrease in acid secretion and in the mucosal blood flow falling to the basal level at pH 1-0. Exogenous secretion given in graded doses from 0-5 to 8-0 u./kg. hr caused a small but dose-related inhibition of acid response to liver extract accompanied by a decrease of mucosal blood flow but without any significant change in the serum gastrin level. 6. The results indicate that the chemical stimulation of the oxyntic gland area by peptic digests is capable of inducing acid secretion by a local, gastrin-independent, partially neural reflex mechanism; sensitive to pH, pressure and secretin.


Molecular features of organic anion permeablity in ox red blood cell.

1. The penetration of organic anions into bovine red blood cells has been studied under experimental conditions where it could be distinguished from the penetration of undissociated acids which proceeds by diffusion through lipid zones of the membrane. 2. Several lines of evidence suggest that the entry of organic anions cannot be ascribed to simple diffusion across aqueous channels limited by positive charges but needs a specific interaction of the penetrating anion with a component of the membrane. 3. The structural requirements allowing for ionic transfer is a strong polar head for the smallest molecules and in addition an amphiphilic structure for acids with chain length greater than C4. Interaction between substrate and receptor requires at least a three point attachment involving three oxygen atoms in the substrate which react with complementary loci on the receptor to form ionic and hydrogen bonds. Such a three point attachment can be made by a sulphonic group or with carboxylic acid by alpha ketosubstitution, alpha hydroxysubstitution, addition of an amidegroup or addition of a second carboxyl group spatially close to the first. 4. As suggested by the behaviour of the formate anion, in such a transport system any carboxylic acid could interact transiently with the receptor and therefore interfere with the transport of an organic anion even though such ionic interaction with the receptor were insufficient to produce transport of the acid itself.


Effects of lysergic acid diethylamide on autonomic post-ganglionic transmission.

1. Six sites of autonomic post-ganglionic transmission were examined for susceptibility to LSD. Inhibition of transmission by LSD was confined to the three sympathetic junctions. 2. Inhibition of sympathetic transmission was maximal with short trains of pulses and declined considerably as train length was increased. 3. Evidence for a presynaptic mode of action was obtained. This was the predominant effect of LSD in the rat anococcygeus and dog retractor penis because alpha-adrenoceptor-blocking properties were feeble or absent; but in dog splenic strips LSD produced marked post-synaptic alpha-blockade. 4. The presynaptic inhibitory effect of LSD was unrelated to its 5-hydroxytryptamine-blocking property because it was not shared by methysergide. Neither was it mediated by prostaglandin release because it was unaltered by indomethacin, which suppresses prostaglandin synthesis. 5. In the rat anococcygeus and dog retractor penis larger doses of LSD induced slow contractions and, as a result of the concurrent block of motor transmission, revealed relaxation responses on transmural stimulation, caused by the excitation of sacral inhibitory fibres present in these muscles. 6. The LSD contractions were due to stimulation of post-synaptic alpha-adrenoceptors because they were abolished by phentolamine or yohimbine but were present as usual in preparations taken from reserpinized animals. 7. LSD blocked presynaptic alpha-adrenoceptors in the cholinergic motor fibres to the longitudinal muscle of the guinea-pig ileum.


Etonitazene. An improved synthesis.

1-(BETA-Kiethylaminoethyl)-2-(p-ethoxybenzyl)-5-nitrobenzimidazole (u, etonitazene) is a potent analgesic that has value in drug addiction studies. We have developed a simple high-yield synthesis of 1 that is adaptable to alrge scale preparations. The synthesis involves the condensation of 2-(beta-diethylaminoethylamino)-5-mitroaniline and p-ethoxyphenylacetic acid in Thf in the presence of EEDQ.


Comparison of male and female physician's associate program applicants.

Using personality measures, the author examined Physician's Associate Program applicants at the Duke University School of Medicine, comparing male and female applicants and the outcome for these applicants in terms of acceptance into paramedical training. The perecentage of women accepted into training was similar to the percentage who applied for training. However, women invited for interview were more aggressive, autonomous, impulsive, playful, and flexible than the males invited to interview, suggesting self-selection by women in applying for paramedical training. Women accepted for training were more "feminine" in the conventional sense, and bias against female applicants less accepting of social mores and the traditional female role was revealed. The implications of these findings are discussed.


Some aspects of proximal tubular sodium chloride reabsorption in Necturus kidney. Renal tubular reabsorption of fluid and sodium was measured by clearance methods in the doubly perfused Necturus kidney in which the bicarbonate concentration was varied between 0 and 60 mEq/liter. The effects of Damox (2.2 times 10-3M), ocubain (10-5M) and ethacrynic acid (10-4M) and of acidosis were also investigated. In addition to clearance experiments, stationary microperfusion experiments were carried out on promimal tubules to measure volume flow and steady-state sodium and chloride concentration differences across the tubular epithelium. In some experiments, the transepithelial electrical potential difference was also measured using an axial electrode system. The following results were obtained: 1) Bicarbonate is not essential to the operation of renal tubular fluid and sodium transport. 2) Total renal and proximal tubular fluid and sodium transport are partially inhibited by Diamox, ouabian and ethacrynic acid. 3) The proximal tubule maintains a significant transepithelial sodium and chloride concentration difference and a significant electrical potential difference (lumen-negative) in the presence of a poorly permeant nonelectrolyte. The direction and magnitude of the electrical polarization fully accounts for the observed chloride concentration difference. The data support the thesis that sodium chloride transport accross the proximal tubular epithelium takes place by active sodium transport and electically coupled passive chloride reabsorption. Important species differences with respect to mammalian transport mechanisms are discussed.


Nature of the acidifying defect after the relief of ureteral obstruction.

Nature of the acidifying defect observed after relief of ureteral obstruction. The acidifying capacity of the normal and experimental kidney was studied in rats three hours after release of complete unilateral ureteral obstruction or after unilateral release of unilateral ureteral obstruction,  mean plasma bicarbonate concentration was 23.5 mEq/liter, and plasma pH, 7.45. Urine pH from the postreleased kidney was 7.47 and 6.01 from the control side. Net acid escretion averaged 0.97 muEq/min in the experimental kidney. Fractional excretion of bicarbonate averaged 0.02% in the control and 3.17% in the experimental kidney. In eight animals with unilateral release of bilateral ureteral obstruction, mean plasma bicarbonate was 16.9 mEq/min and plasma pH was7.35. Mean urine pH in the postreleased Kidney was 6.11, net acid excretion averaged 2.01 plus or minus 0.69 muEq/min and fractional bicarbonate excretion averaged 0.84%.


Renal mitochondrial glutamine metabolism and dietary potassium and protein content. Glutamine distribution, glutamate accumulation, phosphate-dependent glutaminase (PDG) concentrations and intact mitochondrial ammonia production were studied in renal mitochondria from rats fed low, normal and high potassium diets and in mitochondria from rats fed high or low protein diets. The rats given a low potassium diet were potassium-depleted by 10 to 20% but in none of the groups were there any abnormalities of extracellular acid-base status. Glutamine was present in the outer space of mitochondria but could not be depleted in the matrix space in any group. In both the potassium-depleted and the high protein animals, we found increased matrix -14-C-uptake of glutamine (as -14-C-glutamate), increased intact mitochondrial ammonia production and increased concentrations of PDG. In the K+-depleted group there was a decreased matrix -14C-uptake when -14C-gamma-ketoglutarate of -14C-glutamate was present in the medium. Potassium loading produced no change in mitochondrial glutamine metabolism. Protein loading (compared with protein depletion) and potassium depletion induce an increased uptake of glutamine into the renal mitochondrial matrix space which leads to its increased deamidation. This adaption may explain the increased renal ammonia production seen in these situations when compared to their respective controls.


Arterial blood gas tensions and acid-base status of Wistar rats during thiopental and halothane anesthesia.

Arterial blood gas tensions and acid-base status of spontaneously-breathing, unanesthetized Wister rats were compared with values obtained during 4 hr of thiopental and 6 hr of halothane (1%) anesthesia. During thiopental anesthesia, marked respiratory depression occurred (PaCO-2:57.0 plus or minus 10.0 MM Hg, PaO-2:70.4 plus or minus 11.2 MM Hg). Thirty-six percent of the rats died. During inhalation of room air and 1% halothane, PaO-2 decreased also, whereas PaO-2 did not change. Twenty-seven percent of the original number of rats died. Lowered arterial oxygen tension may have caused death; no rats died during inhalation of oxygen and 1% halothane. This technic insured sufficient analgesia for surgical procedures without marked alterations of the acid base status and is recommended for long-term anesthesia of small laboratory animals like rats.


[Loading-stable osteosynthesis of pertrochanteric femoral fractures using a condyle plate].

Also the authors' Department shares SPIER's opinion, according to which the ideal treatment of the proximal femur fractures is the osteosynthesis, with the greatest stability. The aim of surgical treatment of the fracture is the motion-stable and possibly loading-stable osteosynthesis. Since proximal femur fractures occur mostly in aged patients, the survival is considerably augmented by early and efficacious mobilization. If the general state of the patient is suitable, after consultation with the anaesthesiologist and the internist the surgical intervention is carried out. AO-condyle-plate yields further possibility to the loading-stable osteosynthesis of the pertrochanteric fractures.


[Comparative evaluation of various surgical technics in the treatment of dislocated patellar fractures].

From the point of view of the different operative methods 137 patellar fractures operated on in the 3 years' material (1969--1971) of the Central Casualty Rehabilitation Institute have been analysed by the authors. 116 fractures could be re-examined 2--4 years after patients were back at work. If the operation was followed by adequate functional management, satisfactory results were found after all operations. When the patients were back at work, further improvement of the results has been observed. In the time the patients got again to work, the results of partial patellectomy seemed better,--however, after 2--4 years osteosynthesis yielded better final results. Traction loop proved more effective than cerclage. If possible, conservation and exact unionof the patella is of great importance in both juveniles and manual workers. Early functional treatment improves the late results of the operation.


[History of traumatology in Hungary].

The history of the Hungarian Traumatology since the 16th century up to the present days is outlined by the author. The most important works--dealing with the treatment of the fractures and with the activity of other areas of the traumatology--are enumerated. Moreover, the factors promoting the development of the Hungarian traumatology are specified and the important surgeons active in the field of traumatology and the war operative surgery are named, who too have contributed to the development of the Hungarian traumatology. The importance of the Hungarian Institute of Traumatology--founded in 1956--is emphasized,--thus, the traumatology became an independent section of the Hungarian public health; and centrally directed and organized function of the traumatology was assured.


[Bilateral shortening of the m. rectus femoris due to complications after injection].

A case of bilateral shortening of the muscle rectus femoris--developed as complication of the injection--is reported. The mechanisms of the development of the alteration and the examination method are described. The possibility of the prevention of the extension knee constractures--developing as complication of the injection--is similarly discussed.


[Current problems in the treatment of open fractures].

The paper evidently does not discuss several important problems of the management of open fractures, but it is confined to the exposition of some fundamental rules of their traitment. Nowadays, the treatment of open fractures is to be decided individually for all cases. A great number of patients with open fracture owe their recovery to the proper first aid care at scence of accident. This is also in respect of the further treatment in the hospital of great importance. Exagerated activity is usually superfluous in the treatment of open fractures. Less and more proper management is often more useful for the injured patient.