Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement.     

Cytoskeletal structure in cells harboring two mutations: R133C in NOTCH3 and 5650G>A in mitochondrial DNA

We have previously described a patient with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) caused by R133C mutation in NOTCH3 and with a concomitant myopathy caused by a G to A point mutation at base pair 5650 (5650G>A) in the gene encoding tRNA(Ala) in mitochondrial DNA (mtDNA). In the present study, we have examined the morphology of the cytoskeletal components in fibroblasts and myoblasts of this patient. Immunolabeling revealed that tubulin network was sparse and formed asters in these cells, whereas no changes were found in actin and vimentin networks in comparison to the control cell lines. Furthermore, mitochondria were less abundant and the branches of the mitochondrial network were reduced in number. Muscle histochemical analysis showed ragged red fibres (RRFs) and cytochrome c oxidase (COX)-negative fibres. The mean proportion of mtDNA with 5650G>A was lower in histologically normal muscle fibres than in the COX-negative fibres and in the RRFs. These findings suggest that 5650G>A is a pathogenic mtDNA mutation. However, the changes in tubulin network and mitochondrial distribution in patient fibroblasts and myoblasts cannot solely be explained by this mutation.     

Body mass, DRD4, physical activity, sedentary behavior, and family socioeconomic status: the add health study

Objective: To investigate the joint role of the 48‐base pair repeat polymorphism of the dopamine receptor 4 gene (DRD4) and environmental factors in body mass variation among an ethnically diverse sample of U.S. adolescents and young adults. Research Methods and Procedures: Approximately 2600 adolescent and young adults in the National Longitudinal Study of Adolescent Health (Add Health) who provided DNA measures and measures of height and weight were included in the analysis. Mixed regression modeling was used to investigate the effects of the 7R/7R and any5R variants in the DRD4 gene simultaneously with the effects of physical activity (PA), sedentary behavior (SB), and family socioeconomic status (SES) on body mass variation. European Americans, African Americans, and Hispanic Americans were modeled separately. Results and Discussion: Both the 7R/7R and any5R genotypes of the DRD4 gene were associated with age‐ and sex‐specific BMI percentile score (BMI‐P) based on the Centers for Disease Control and Prevention/National Center for Health Statistics 2000 reference curves among African Americans and only among African Americans (N = 413) 20 years old or younger. Neither genetic variants are associated with the BMI measure among white (N = 1386) and Hispanic‐American (N = 331) adolescents. The presence of the 7R/7R genotype was associated with a reduction of 15.1 in BMI percentile (p = 0.005), and the presence of any5R was associated with an increase of 15.5 in BMI percentile (p = 0.003), after adjusting for PA, SB, and family SES. Neither PA nor SB as measured in Add Health is importantly associated with BMI‐P, suggesting a complex relationship between body mass and PA/SB among adolescents and young adults. Family SES is negatively related to BMI‐P in the European‐American sample.     

Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A

Presence of appetite-regulating peptides orexin-A and orexin-B in mucosal endocrine cells suggests a role in physiological control of the intestine. Our aim was to characterize orexin-induced stimulation of duodenal bicarbonate secretion and modulation of secretory responses and mucosal orexin receptors by overnight food deprivation. Lewis x Dark Agouti rats were anesthetized and proximal duodenum cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Orexin-A was administered intra-arterially close to the duodenum, intraluminally, or into the brain ventricles. Total RNA was extracted from mucosal specimens, reverse transcribed to cDNA and expression of orexin receptors 1 and 2 (OX1 and OX2) measured by quantitative real-time PCR. OX1 protein was measured by Western blot. Intra-arterial orexin-A (60-600 nmol.h(-1).kg(-1)) increased (P < 0.01) the duodenal secretion in fed but not in fasted animals. The OX1 receptor antagonist SB-334867, which was also found to have a partial agonist action, abolished the orexin-induced secretory response but did not affect secretion induced by the muscarinic agonist bethanechol. Atropine, in contrast, inhibited bethanechol but not orexin-induced secretion. Orexin-A infused into the brain ventricles (2-20 nmol.kg(-1).h(-1)) or added to luminal perfusate (1.0-100 nM) did not affect secretion, indicating that orexin-A acts peripherally and at basolateral receptors. Overnight fasting decreased mucosal OX1 and OX2 mRNA expression (P < 0.01) as well as OX1 protein expression (P < 0.05). We conclude that stimulation of secretion by orexin-A may involve both receptor types and is independent of cholinergic pathways. Intestinal OX receptors and secretory responses are markedly related to food intake.     

Electrophysiological characterization of responses from gustatory receptor neurons of sensilla chaetica in the moth Heliothis virescens

Discrimination of edible and noxious food is crucial for survival in all organisms. We have studied the physiology of the gustatory receptor neurons (GRNs) in contact chemosensilla (insect gustatory organs) located on the antennae of the moth Heliothis virescens, emphasizing putative phagostimulants and deterrents. Sucrose and the 2 bitter substances quinine and sinigrin elicited responses in a larger proportion of GRNs than inositol, KCl, NaCl, and ethanol, and the firing thresholds were lowest for sucrose and quinine. Variations in GRN composition in individual sensilla occurred without any specific patterns to indicate specific sensillum types. Separate neurons showed excitatory responses to sucrose and the 2 bitter substances quinine and sinigrin, implying that the moth might be able to discriminate bitter substances in addition to separating phagostimulants and deterrents. Besides being detected by separate receptors on the moth antennae, the bitter tastants were shown to have an inhibitory effect on phagostimulatory GRNs. Sucrose was highly appetitive in behavioral studies of proboscis extension, whereas quinine had a nonappetitive effect in the moths.     

Sporadic colorectal cancer and individual susceptibility: a review of the association studies investigating the role of DNA repair genetic polymorphisms

Mutations in one of the DNA repair genes are one of the most common reasons for cancer, and it may be assumed that the individual genetic background modulating the DNA repair capacity may affect the susceptibility to cancer. Numerous polymorphisms (mainly SNPs) have been identified for DNA repair genes, although their functional outcome and phenotypic effect is often unknown. The aim of the present review is to evaluate the studies investigating a possible influence of DNA repair polymorphisms in the risk of sporadic colorectal cancer and/or adenoma. Overall, no relevant common findings emerge among the studies, except for some statistically significant associations between polymorphisms in the XRCC1 and XPD genes, mainly for colorectal adenoma risk. Other individual associations remain to be confirmed. This inconclusive data may suggest that the modulation of cancer risk depends not only on a single gene/SNP, but also on a joint effect of multiple polymorphisms (or haplotypes) within different genes or pathways, in close interaction with environmental factors. The relevance of many low-penetrance genes in cancer susceptibility is supposed to be very subtle. Several reviewed association studies revealed weaknesses in their design. However, there has been a progressive improvement over the years in aspects such as simultaneous genotyping and combined analyses of different polymorphisms in larger numbers of patients and controls, as well as stratification of results by ethnicity, gender, and tumor localization. This gained experience shows that only carefully designed studies of a sufficient statistical power may resolve the relationships between polymorphisms and colorectal cancer risk.     

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α₂β₂ tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1^−/−) leads to embryonic lethality in mouse, whereas P4ha1^+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2^−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1^+/−;P4ha2^−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the T_m of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1^+/−;P4ha2^−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2^−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.