Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

Influence of food restriction during gestation on craniofacial growth of the weanling rat

Holtzman rats were subjected to food restriction during gestation or lactation, or both periods (overall stress). At weaning, male pup skulls were measured and female brains and cranial masticatory muscles were weighed and a neuromuscular index was calculated. It was found that gestational protein-calorie malnutrition (PCM) without suckling restoration accounted for about 30% of the growth delay observed under overall stress. That effect disappeared after a normal suckling restoration. Under the same conditions of maternal food restriction in both periods, growth delay in the offspring was greater during lactation than gestation. As in lactation, craniofacial changes during gestational restriction were due to an adjustive response of bone growth to PCM. This response seemed to accrue from an altered relationship between the growth of the brain-less sensitive and highly restorable-and the growth of the masticatory muscles-more sensitive and less restorable. Some degree of delay in orthocephalization would be the skeletal outcome of such adjustive neuromuscular response.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Effect of dietary level of protein on the metabolism of mouse liver nuclear proteins.

The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Effect of gonadal activity on the cranial dimorphism of the rat

Weanling rats of both sexes were submitted to either castration, or castration together with periodic injections of testicular extract to males or of ovarian extract to females. Control and experimental animals were sampled at 63 days of age. Cranial differentiation between sexes was estimated by Mahalanobis D2 distances. The controls showed a significant sexual cranial difference. Orchidectomy decreased cranial differences and this effect was compensated by injections of testicular extract. On the other hand, oophorectomy increased cranial differences, which were diminished by injections of ovarian extract. Sexual cranial dimorphism in the normal rat seems to be the result of a counteracting effect between testicular and ovarian hormonal secretions.     

Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4–5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases.     

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence

Bacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular-growth-attenuator-A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC-YojN-RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC-YojN-RcsB system. IgaA-mediated repression of the RcsB-YojN-RcsC system occurred at the post-translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC-YojN-RcsB system, monitored with the product of a tagged gmd-3xFLAG gene (positively regulated by RcsC-YojN-RcsB), was totally abolished by wild-type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC-YojN-RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC-YojN-RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10(-6). Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non-functional RcsC-YojN-RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.