Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion

Summary DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of thepulC-O operon fromKlebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG,pulH,pulI,pulJ andpulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that thepulC-O operon contains at least 11 pullulanase secretion genes.     

The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of theE. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.     

Cl transport in the frog cornea: An electron-microprobe analysis

Summary The intracellular electrolyte concentrations of the bullfrog corneal epithelium have been determined in thin freezedried cryosections using the technique of electron-microprobe analysis. Under control conditions, transepithelial potential short-circuited and either side of the cornea incubated in Conway's solution, the mean intracellular concentrations (in mmol/kg wet weight) were 8.0 for Na, 18.4 for Cl and 117.3 for K. These values are in good agreement with ion activities previously obtained by Reuss et al. (Am. J. Physiol. 244:C336–C347, 1983) under open-circuit conditions. From a comparison of the chemical concentrations and activities of Na and K a mean intracellular activity coefficient of 0.75 is calculated. For small ions no significant differences between nuclear and cytoplasmic concentration values were detectable. The Cl concentrations in the different epithelial layers were virtually identical and showed parallel changes at varying states of Cl secretion, suggesting that the epithelium represents a functional syncytium. For Na a concentration gradient between theouter and inner epithelial layer was observed, which can be accounted for by two different models of epithelial cooperation. The behavior of the intracellular Na and Cl concentrations after removal of Na, Cl or K from the outer or inner bathing medium provides support for a passive electrodiffusive Cl efflux across the apical membrane and a Na-coupled Cl uptake across the basolateral membrane. The results are inconclusive with regard to the exact mechanism of Cl uptake, indicating either a variable stoichiometry of the symporter or the presence of more than one transport system. Furthermore, a dependence of intracellular Cl on HCO₃ and CO₂ was observed. Extracellular measurements in corneal stroma demonstrated that ion concentrations in this space are in free equilibrium with the inner bath.     

A two-step control of basic and acidic peroxidases and its significance for growth and development

A generalized two-step and interdependent control of basic and acidic peroxidases (EC 1.11.1.7) is observed in plant responses to different physical and chemical stimuli. An interpretative model consisting of a pathway of reactions is presented on the basis of our own data and the literature. Stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation. The degradation of cell membranes is suggested to bring about rapid changes in ionic fluxes (especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio) and in leakage of solutes (among them electron donors such as ascorbic acid and phenolic substances). The increased intracellular relative calcium level results in: 1) activated secretion of basic peroxidases into the free space where, in association with the electron donors and maybe with the circulating indole-3-acetic acid (IAA), they eliminate the peroxides; and 2) facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase (EC 4.31.5) and acidic peroxidases. The resulting lignification and cell wall rigidification determines the growth and/or the developmental response to the initial stress.     

Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats

The neurohumoral pathways mediating intracisternal TRH-induced stimulation of gastric acid secretion were investigated. In urethane-anesthetized rats, with gastric and intrajugular cannulas, TRH or the analog [N-Val2]-TRH (1 microgram) injected intracisternally increased gastric acid output for 90 min. Serum gastrin levels were not elevated significantly. Under these conditions the TRH analog, unlike TRH, was devoid of thyrotropin-releasing activity as measured by serum TSH levels. In pylorus-ligated rats, gastrin values were not modified 2 h after peptide injection whereas gastric acid output was enhanced. TRH (0.1-1 micrograms) stimulated vagal efferent discharge, recorded from a multifiber preparation of the cervical vagus in urethane-anesthetized rats and the response was dose-dependent. The time course of vagal activation was well correlated with the time profile of gastric stimulation measured every 2 min. These results demonstrated that gastric acid secretory stimulation elicited by intracisternal TRH is not related to changes in circulating levels of gastrin or TSH but is mediated by the activation of efferent vagal pathways that stimulated parietal cell secretion.     

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.     

Gastric and intestinal release of vasoactive intestinal polypeptide in the milk-fed lamb

Vasoactive intestinal polypeptide (VIP) has been proposed as the neurotransmitter of the atropine-resistant relaxation of gastric structures in the lamb. To examine this proposal VIP concentrations in plasma from arterial, gastric venous and intestinal venous blood were measured in healthy conscious lambs before, during and after teasing with, and sucking of milk. Basal arterial plasma VIP concentrations were undetectable (less than 3 pmol/l) and remained so during and after feeding. Before feeding VIP was detected in only 2 of 12 gastric venous plasma samples (5 and 13 pmol/l). During teasing with food there were increments in VIP of 19 +/- 4 pmol/l and during feeding of 27 +/- 5 pmol/l. VIP concentration in gastric venous plasma rapidly returned to fasting levels after cessation of sucking. In contrast VIP in the intestinal venous plasma did not rise during teasing or upon commencement of sucking but a peak increment of 34 +/- 6 pmol/l occurred at 5 min after cessation of feeding. The results are consistent with the hypotheses that VIP is released in anticipation of and during sucking from inhibitory neurones involved in relaxation of gastric structures and that intestinal release of VIP is a consequence of entry of digesta into the small intestine.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

Effects of Neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat

Neuropeptide Y (NPY) has been recently localized in several hypothalamic nuclei in the mammalian brain. In order to investigate the possible role of NPY on neuroendocrine function, we have investigated the effects of the peptide on the release of anterior pituitary hormones in the rat. Both intravenous (300 μg) or intraventricular (2 to 15 μg) injection of NPY produced in gonadectomized male rats a significant and long-lasting decrease of plasma LH levels. A short duration stimulating effect on prolactin plasma levels was also observed after the intravenous but not after the intraventricular injection of NPY. Plasma levels of the other pituitary hormones were not significantly modified after NPY injection. When incubated in vitro with anterior pituitary cells in monolayer culture, NPY produced no significant change in release of pituitary hormones. Thus NPY seems to exert a selective effect on LH release. Since this effect can be observed after both intravenous and intraventricular injection, it might be hypothesized that NPY could affect LHRH release in two areas which lack blood-brain barrier: the organum vasculosum of the lamina terminalis (OVLT) which contains LHRH cell bodies and NPY fibers and the median eminence which contains both LHRH and NPY fibers. The effect on prolactin release needs to be carefully evaluated in different experimental conditions.