Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.Bacteria of the Bacillus, Clostridium, and some related genera form dormant endospores which are resistant to a variety of physical and chemical treatments. A number of characteristics of the spore core or protoplast affect the degree of heat resistance. These include the water content (5, 29), the identity of the cations in the spore associated with dipicolinic acid (DPA) (reviewed in reference 12), and the presence of ␣/␤-type, small, acid-soluble proteins (SASP) associated with the chromosomal DNA (28). We have become particularly interested in the correlation of heat resistance with the water content of the spore core, a correlation which has been demonstrated across a variety of species, with spores prepared at different temperatures, and with spores containing different cations (5, 29), as well as in the mechanism by which the water content of the spore core is reduced.In addition to a correlation with heat resistance, spore core dehydration appears to be required for processing of a spore germination protease, GPR, from its zymogen form to an active form late in sporulation (17, 36). The reduced level of spore core water is theorized to be the factor which prevents degradation of SASP by GPR during late sporulation, since this degradation normally takes place only during spore germination.The integrity and amount of the spore cortex, a thick peptidoglycan structure which surrounds the spore core, have b...

MicroRNAs (miRNAs) play an essential role in the development and progression of acute leukemia (AL). miR-24 promotes the survival of hematopoietic cells. However, little is known concerning the function of miR-24 in human AL. The aim of the present study was to investigate the clinical significance of miR-24 expression in AL. miR-24 expression in 147 patients with AL and 100 healthy individuals was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The results showed that compared with the healthy individuals, the expression of miR-24 in AL patients was significantly higher (p<0.001). In addition, miR-24 was expressed at significantly higher levels in acute myeloid leukemia (AML) patients and at significantly lower levels in acute lymphoblastic leukemia (ALL) (p<0.001). More importantly, Kaplan-Meier analysis showed that AL patients with high miR-24 expression tended to have shorter overall survival (p<0.05). In the multivariate analysis stratified for known prognostic variables, miR-24 was identified as an independent prognostic marker. Our data indicated that miR-24 upregulation was associated with poor prognosis in AL. miR-24 was identified for the first time as an independent marker for predicting the clinical outcome of AL patients.

No abstract

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR delta), the putative pro sequence removed in conversion of the GPR zymogen (termed P46) to the active enzyme (termed P41) was deleted. GPR delta was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPRS) was synthesized as P46S in both B. subtilis and E. coli, but P46S was processed to P41S earlier in B. subtilis sporulation than was wild-type P46. The second variant (GPREI) was made as P46EI but underwent extremely rapid processing to P41EI in both E. coli and B. subtilis. Expression of elevated (> 100-fold) levels of GPR delta or GPREI blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPRS or low levels (< 20% of the wild-type level) of GPR delta or GPREI did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P41 delta, P41EI, or wild-type P41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P39. The sequence in the P(41)-->P(39) cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P(46) to P(41) is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P(41) on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.

The sequence-specific protease (termed GPR) that degrades small, acid-soluble proteins (SASP) during germination of spores of Bacillus species is synthesized during sporulation as an inactive precursor termed P46. Approximately 2 h later in sporulation, P46 is converted proteolytically to a smaller form, termed P41, which is active in vitro, but which does not act significantly on SASP in vivo until spore germination is initiated. While it appears likely that P46-->P41 conversion is an autoprocessing event, the mechanisms regulating P46-->P41 conversion in vivo are not clear. In this work we found that P46-->P41 conversion in vitro was stimulated tremendously in an allosteric manner by pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus Ca2+ but not by Ca2+ in combination with a variety of DPA analogs. The processing reaction stimulated by Ca(2+)-DPA was seen at pH 5.1 but not at pH 6.2 or 7, and under these conditions P46-->P41 conversion exhibited a linear time course and was a first-order reaction, indicative of an intramolecular autoprocessing reaction. At pH 5.1, P46-->P41 conversion was stimulated markedly by very high ionic strength. At pHs from 5.1 to 6.6, P46-->P41 conversion also occurred when P46 was dehydrated to approximately 54% relative humidity. This processing was stimulated markedly when dehydration was carried out in the presence of DPA and NaCl but was greatly decreased when dehydration was to 10, 33, or 75% relative humidity. Since previous work has shown that P(46)-->P(41) processing in vivo takes place (i) after a fall in forespore pH to 6.3 to 6.9 and approximately in parallel with (ii) DPA accumulation by the forespore and (iii) dehydration of the forespore, out new finings in vitro suggest that these three changes may synergistically trigger P(46)-->P(41) autoprocessing in the developing forespore. Presumably the conditions in vivo during this authoprocessing preclude significant attack of the P(41) generated on its SASP substrates.

Abnormal expression and promoter methylation of microRNAs (miRNAs) are common events during cervical carcinogenesis. Worldwide, infection by types 18 and 16 of human papillomaviruses (HPVs) is considered the major risk factor for cervical cancer development. It has been reported that expression of the miRNAs can be deregulated by specific HPV genotypes. In this study we analyzed the promoter methylation of 22 miRNAs and the expression of three miRNAs in 10 non-squamous intraepithelial lesions (Non-SIL) without HPV16 infection, and 7 Non-SIL, 16 low-grade SIL (LSIL) and 16 cervical cancer samples, all with HPV16 infection. The methylation status was determined using Human Cancer miRNA EpiTect Methyl II Signature PCR Array® and the expression of miR-124, miR-218 and miR-193b was determined by qRT-PCR using individual TaqMan assays. Comparisons of groups defined were performed using the Fisher exact test for categorical variables and Mann-Whitney test for continuous variables. A p-value of <0.05 was considered statistically significant. The methylation levels of miR-124-2, miR-218-1, miR-218-2 and miR-34b/c promoters were significantly higher in cervical cancer than in LSIL samples. The methylation levels of miR-193b promoter were significantly lower in cervical cancer than in LSIL samples. The expression of miR-124 and miR-218 was significantly lower in cervical cancer than in LSIL samples. The expression of miR-193b was significantly higher in cervical cancer than in LSIL and Non-SIL samples. Our results suggest that the abnormal promoter methylation and expression of miR-124, miR-218 and miR-193b are common events during cervical carcinogenesis.

BackgroundThe association between proinflammatory cytokine gene polymorphisms and gastric diseases related to Helicobacter pylori varies by population and geographic area.Our objective was to determine if the IL-1B -511 T>C and -31 C>T polymorphisms and H. pylori vacA genotypes are associated with risk of chronic gastritis and gastric ulcer in a Mexican population.MethodsWe conducted endoscopic studies in 128 patients with symptoms of dyspepsia. We took two biopsies from the body, antrum, or ulcer edge from each patient, and classified our histopathological findings according to the Sydney System. H. pylori infection and vacA genotyping were accomplished via PCR from total DNA of the gastric biopsies. We confirmed the presence of anti-H. pylori serum IgG and IgM in 102 control subjects. In both case subjects and control subjects, the IL-1B -511 T>C polymorphism was genotyped by PCR-RFLPs and the IL-1B -31 C>T polymorphism was genotyped by pyrosequencing.ResultsSixty-two point seven (62.7%) of the 102 control subjects were H. pylori-seropositive. Among the case subjects, 100 were diagnosed with chronic gastritis and 28 with gastric ulcer. We found that 77% of the patients with chronic gastritis and 85.7% of the patients with gastric ulcer were H. pylori-positive. The predominant H. pylori genotype was vacA s1m1 (58.4%) and the most frequent subtype was vacA s1. The -511 TC, (rs16944 -511 T>C) genotype and the -511C allele were associated with chronic gastritis (OR = 3.1, 95% CI = 1.4-6.8 and OR = 3.0, 95% CI = 1.4-6.0, respectively). The subjects carrying -31T (rs1143627 -31 C>T) were found to be at a higher risk of having chronic gastritis (OR = 2.8, 95% CI = 1.3-5.8). The IL-1B -511C/-31T haplotype was associated with chronic gastritis (OR = 2.1, 95% CI = 1.2-3.8) but not with gastric ulcer.ConclusionsThe H. pylori vacA genotypes identified herein were similar to those reported for other regions of Mexico. The vacA s1m1 genotype was not associated with gastric ulcer. In the southern Mexican population, the IL-1B -511C and -31T alleles and the -511C/-31T and -511T/-31T haplotypes are associated with increased risk of chronic gastritis and gastric ulcer.

BackgroundHelicobacter pylori chronic infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. Cytotoxin-associated gene A (cagA)-positive H. pylori strains increase the risk of gastric pathology. The carcinogenic potential of CagA is linked to its polymorphic EPIYA motif variants. The goals of this study were to investigate the frequency of cagA-positive Helicobacter pylori in Mexican patients with gastric pathologies and to assess the association of cagA EPIYA motif patterns with peptic ulcer and gastric cancer.MethodsA total of 499 patients were studied; of these, 402 had chronic gastritis, 77 had peptic ulcer, and 20 had gastric cancer. H. pylori DNA, cagA, and the EPIYA motifs were detected in total DNA from gastric biopsies by PCR. The type and number of EPIYA segments were determined by the electrophoretic patterns. To confirm the PCR results, 20 amplicons of the cagA 3′ variable region were sequenced, and analyzed in silico, and the amino acid sequence was predicted with MEGA software, version 5. The odds ratio (OR) was calculated to determine the associations between the EPIYA motif type and gastric pathology and between the number of EPIYA-C segments and peptic ulcers and gastric cancer.ResultsH. pylori DNA was found in 287 (57.5%) of the 499 patients, and 214 (74%) of these patients were cagA-positive. The frequency of cagA-positive H. pylori was 74.6% (164/220) in chronic gastritis patients, 73.6% (39/53) in peptic ulcer patients, and 78.6% (11/14) in gastric cancer patients. The EPIYA-ABC pattern was more frequently observed in chronic gastritis patients (79.3%, 130/164), while the EPIYA-ABCC sequence was more frequently observed in peptic ulcer (64.1%, 25/39) and gastric cancer patients (54.5%, 6/11). However, the risks of peptic ulcer (OR = 7.0, 95% CI = 3.3–15.1; p < 0.001) and gastric cancer (OR = 5.9, 95% CI = 1.5–22.1) were significantly increased in individuals who harbored the EPIYA-ABCC cagA gene pattern.ConclusionscagA-positive H. pylori is highly prevalent in southern Mexico, and all CagA variants were of the western type. The cagA alleles that code for EPIYA-ABCC motif patterns are associated with peptic ulcers and gastric cancer.