T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.     

Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Increased resistance to Salmonella infection of hypoferremic mice fed a low-protein diet

BALB/c and CBA/CA mice fed a protein-deficient diet developed a plasma hypoferremia corresponding to a 30 percent lowering of serum iron concentration. This hypoferremia persisted as long as the diet was maintained. Hypoferremic CBA/CA mice had increased resistance to Salmonella typhimurium C5 infection, as shown by the reduced lethal activity and the decreased growth of the bacteria in the spleen and in the peritoneal exudate of the deficient animals. This induced resistance was abolished after injection of iron or Desferal into the restricted animals. Such resistance was not observed with BALB/c mice fed a protein-deficient diet, in spite of the plasma hypoferremia. The growth of S. typhimurium C5 in the spleen and in the peritoneal exudate of these animals did not differ from the growth observed in control animals fed a protein-sufficient diet. This study suggests that hypoferremia induced by a protein-deficient diet is probably involved in the enhancement of resistance of CBA/CA mice to Salmonella infection, and that the phenomenon is host-strain dependent.     

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera,Tettigoniidae) and protein analysis of their secretions

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two‐part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the “first‐order” glands, a number of large anterior tubules, and the “second‐order” glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second‐order glands. The mesoderm‐derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron‐dense granules and globules or electron‐transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first‐order glands, and corresponds to an important protein component of the spermatophylax. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Phosphoenolpyruvate carboxykinase and glucose-6-phosphate dehydrogenase expression in fetal hepatocyte primary cultures under proliferative conditions

Fetal hepatocytes cultured for 64 h in the presence of glucagon and dexamethasone maintain a quiescent state, showing a low expression of glucose-6-phosphate dehydrogenase (G6PD) and a high induction of phosphoenolpyruvate carboxykinase (PEPCK). Under these culture conditions, the presence of EGF produced hepatocyte proliferation, with a concomitant increase of DNA synthesis, DNA content, and G6PD expression, meanwhile the expression of PEPCK was drastically reduced. The presence of forskolin plus IBMX nearly suppressed the increase in DNA synthesis and G6PD expression induced by EGF, showing a very high expression of PEPCK. Accordingly, it is possible to establish an inverse relation between G6PD, highly expressed in proliferating fetal hepatocytes, and PEPCK expression, highly expressed in quiescent fetal hepatocytes under specific hormonal stimulation.     

Stabilization of the monoolein Pn3m cubic structure on trehalose glasses

Trehalose is known to protect some organisms from various stresses due to drought and high temperature. To explore the molecular mechanism of the protective function, the mesomorphic properties of the monoolein-water system, dried in the presence of trehalose, were studied by X-ray diffraction. While, in pure water, two bicontinuous inverse cubic structures (the Pn3m and Ia3d phases) and a lamellar Lα phase exist as a function of concentration, only the Pn3m cubic phase has been detected in concentrated trehalose solutions or in trehalose glasses, even under extremely dry conditions. Depending on the sugar concentration, or after glass dehydration, the Pn3m cubic unit cell decreases to very low values, much below the smaller one observed in pure water. However, as no phase transitions occur, a simple osmotic mechanism can be excluded. An additional stabilization of the lipid phase, arising from interfacial free energy changes due to trehalose-water-lipid direct interactions, and large enough to affect the energetic balance between the Pn3m and the Ia3d cubic phases, evidently occurs. Moreover, no differences in the Pn3m cubic structure were observed when the sugar platelets convert to the glassy state; no apparent structural modifications that can be related to mechanical pressure exerted on the lipid phase have been detected. Received: 5 October 1998 / Accepted: 13 November 1998     

Postimperative negativity of the contigent negative variations: a critical index of a stress reaction in the normal subject?]

21 subjects were recorded during two experimental sessions: 1st session: 2 CNVs in control conditions; 2nd session: 1) control CNV, 2) CNV and arithmetic calculation, 3) CNV and labyrinthine stimulation, 4) CNV, arithmetic calculation and labyrinthine stimulation, 5) control CNV. The results obtained show a post-imperative extension of the CNV in the situation 2: arithmetic calculation (P less than 0.025) and 4: double interference (P less than 0.05).     

Application of spheroid culture to human hepatocytes and maintenance of their differentiation

Summary— Human hepatocytes cultured with a hormonally defined medium on non-adherent poly-(2-hydroxyethyl methacrylate) coated surface were able to form spheroids. The maintenance of liver-specific functions was assessed by following secretion of albumin, transferrin and α-antitrypsin that were still detectable after 4 months of spheroidal culture. Moreover, cytochrome P-450 IA was induced by methylcholanthrene for up to 2 weeks. This cell system is very promising for long-term in vitro studies of human hepatocyte functions.     

Field performance of temporary immersion bioreactor-derived sugarcane plants

Summary The temporary immersion bioreactor has been found to be an important tool for sugarcane micropropagation, allowing higher shoot formation rates and cost reduction. This research was conducted to demonstrate the agricultural value of temporary immersion bioreactor-derived sugarcane plants. The experiment was carried out for about 2 yr to study the field performance of these plants. Two control treatments were also evaluated representing the conventional forms of micro- and macropropagation. Growth of sugarcane stools, first ratoon and the use of micropropagated plants for macropropagation were recorded. Some botanical and chemical characteristics were evaluated. Differences among propagation systems were only found in the first 6 mo. of field growth, regarding the stem length and diameter. Such differences disappeared with the course of the experiment.