The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.     

Effects of length of forest fallow on fertility dynamics in a Mexican ultisol

Changes with time in the fertility of a Mexican ultisol were evaluated from analyses of soil samples from fields representing 50 years of pineapple slash-and-burn agriculture. Sixteen fields with similar soil profile charactersitics representing eight ages of forest fallow were sampled. The fields, within an area of 5 km² of nearly level tropical forest, had been cleared by hand, initially intercropped with corn and manioc, followed with 3 to 4 y of pineapple, and then abandoned. The surface soils contained relatively high levels of C and N (5% and 0.3% respectively), were strongly acid (pH 5 to 5.5), and supplied very low levels of available P and K. Soil contents of both C and N declined rapidly after clearing to levels approximately 20% lower at 10y, then increased steadily during the fallow period to values not different from their initial levels. Since little of the N released was utilized by the low-yielding, P-deficient crops, this loss of C, N and accompanying cations is a most serious consequence of this rotation. Both the exchangeable and reserve K levels of these ultisols are low. Thus, K is identified as second to P as a growth limiting factor and the most difficult fertility management problem.     

Effect of manganese and zinc ions on the growth of Rhizobium

Five strains, representing four species of Rhizobium, were grown in specially purified synthetic culture solutions with manganese or zinc ion concentrations ranging from 0 to 10(-5)m. Omission of Mn(2+) restricted growth to 0.4 to 10% of that of controls, depending on the species. Growth was generally maximum with 10(-6)m Mn(2+) and nearly so with 10(-5)m. Omission of Zn(2+) restricted growth to 1 to 20% of that of controls. Growth was generally maximum with 10(-7)m Zn(2+). The 10(-5)m level of Zn(2+) was toxic to a variable degree to all species, suppressing the growth of one strain of R. meliloti to 49% of that of controls, and of R. phaseoli to only 0.4% of that of controls. A Mn(2+) x Zn(2+) interaction was also obtained as evidenced by a higher apparent Zn(2+) requirement with 10(-7)m Mn(2+) than with 10(-6)m Mn(2+).     

Human jejunal brush border folate conjugase. Characteristics and inhibition by salicylazosulfapyridine

Human jejunal brush border folate conjugase (EC 3.4.22.-) was partially purified and characterized. Three drugs known to be associated with clinical folate deficiency were tested for inhibition of the partially purified enzyme. Using jejunal mucosa from obese patients undergoing intestinal bypass surgery, brush border folate conjugase was purified 50-80-fold by centrifugation, Triton X-100 solubilization and DEAE-Sephadex and Sephacryl S-200 chromatography. Using synthetic pteroyldiglutamyl[14C]glutamate as substrate, the enzyme was found to have a pH optimum of 6.5 and an apparent Km of 1.6 micro M. Incubation of the enzyme with synthetic pteroyl[14C]glutamylhexaglutamate resulted in a spectrum of shorter-chain 14C-labeled pteroylglutamates at 60 min. Pteroyl[14C]glutamate was the major product at 120 min, with quantitative recovery of free glutamate in the incubation medium. Salicylazosulfapyridine was a competitive inhibitor of the enzyme (Ki = 0.13 mM), while ethanol, diphenylhydantoin and salicylazosulfapyridine metabolites had no effect. These data suggest that brush border folate conjugase is an exopeptidase which progressively hydrolyzes glutamyl units from pteroylpolyglutamate, leaving pteroylmonoglutamate as the folate form available for intestinal transport. Inhibition of brush border folate conjugase by salicylazosulfapyridine provides a mechanism for folate malabsorption and deficiency in chronic users of this drug.     

Osteogenesis imperfecta: the audiological phenotype lacks correlation with the genotype

Background

Neurofibromatosis 1 (NF1), a common autosomal dominant disorder, was shown in one study to be associated with a 15-year decrease in life expectancy. However, data on mortality in NF1 are limited. Our aim was to evaluate mortality in a large retrospective cohort of NF1 patients seen in France between 1980 and 2006.

Methods

Consecutive NF1 patients referred to the National French Referral Center for Neurofibromatoses were included. The standardized mortality ratio (SMR) with its 95% confidence interval (CI) was calculated as the ratio of observed over expected numbers of deaths. We studied factors associated with death and causes of death.

Results

Between 1980 and 2006, 1895 NF1 patients were seen. Median follow-up was 6.8 years (range, 0.4-20.6). Vital status was available for 1226 (65%) patients, of whom 1159 (94.5%) survived and 67 (5.5%) died. Overall mortality was significantly increased in the NF1 cohort (SMR, 2.02; CI, 1.6-2.6; P < 10^-4). The excess mortality occurred among patients aged 10 to 20 years (SMR, 5.2; CI, 2.6-9.3; P < 10^-4) and 20 to 40 years (SMR, 4.1; 2.8-5.8; P < 10^-4). Significant excess mortality was found in both males and females. In the 10-20 year age group, females had a significant increase in mortality compared to males (SMR, 12.6; CI, 5.7-23.9; and SMR, 1.8; CI, 0.2-6.4; respectively). The cause of death was available for 58 (86.6%) patients; malignant nerve sheath tumor was the main cause of death (60%).

Conclusions

We found significantly increased SMRs indicating excess mortality in NF1 patients compared to the general population. The definitive diagnosis of NF1 in all patients is a strength of our study, and the high rate of death related to malignant transformation is consistent with previous work. The retrospective design and hospital-based recruitment are limitations of our study. Mortality was significantly increased in NF1 patients aged 10 to 40 years and tended to be higher in females than in males.     

Irisin immunohistochemistry in gastrointestinal system cancers

Cancer is the leading cause of morbidity and mortality worldwide. Some studies have shown that high heat kills cancer cells. Irisin is a protein involved in heat production by converting white into brown adipose tissue, but there is no information about how its expression changes in cancerous tissues. We used irisin antibody immunohistochemistry to investigate changes in irisin expression in gastrointestinal cancers compared to normal tissues. Irisin was found in human brain neuroglial cells, esophageal epithelial cells, esophageal epidermoid carcinoma, esophageal adenocarcinoma and neuroendocrine esophageal carcinoma, gastric glands, gastric adenosquamous carcinoma, gastric neuroendocrine carcinoma, gastric signet ring cell carcinoma, neutrophils in vascular tissues, intestinal glands of colon, colon adenocarcinoma, mucinous colon adenocarcinoma, hepatocytes, hepatocellular carcinoma, islets of Langerhans, exocrine pancreas, acinar cells and interlobular and interlobular ducts of normal pancreas, pancreatic ductal adenocarcinoma, and intra- and interlobular ducts of cancerous pancreatic tissue. Histoscores (area × intensity) indicated that irisin was increased significantly in gastrointestinal cancer tissues, except liver cancers. Our findings suggest that the relation of irisin to cancer warrants further investigation.     

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.