Hydrolysis ofγ-glutamyl linkages byFusobacterium nucleatum

The cell extracts of two human oral strains (FN2 and FN3) ofFusobacterium nucleatum displayed exceptionally high-glutamylpeptidase activity as determined withN--l-glutamyl-2-naphthylamine as substrate. This activity was so dominant that the hydrolysis of otherN-aminoacyl-2-naphthylamines progressed at a rate <10% of the former. Two major enzymes (I and II) were partially purified from FN2. I had a molecular weight of 115,000 and did not hydrolyze-glutamylcysteinylglycine (glutathione). II had a molecular weight of 70,000 and rapidly liberated only glutamic acid from glutathione. Strain FN3 contained several enzymes hydrolyzing-glu-2NA. Direct anion exchange chromatography of FN3 cell extracts separated one enzyme that liberated both glutamic acid and glycine from glutathione, one that was inactive against glutathione (but hydrolyzed-glu-2NA), and one that liberated only glutamic acid. Although-glu-2NA was a good synthetic substrate, glutathione was hydrolyzed at least 500 times faster by an enzyme present in both strains. These results indicate that the presence of-glutamylpeptidase activity is very characteristic of theseF. nucleatum strains.     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.     

Correlation of tumor specific delayed type hypersensitivity reaction and tumor protection to SV40-induced mKSA fibrosarcoma

Summary Mice immunized by excision of a primary, subcutaneously growing SV₄₀-induced mKSA solid tumor which resisted challenge of homologous tumor cells administered at a contralateral site, were found to develop a specific DTH response to SV₄₀ tumor associated transplantation antigens (TATA).In a two-way criss-cross experiment, this DTH response (assessed by direct challenge) was found to be one-way SV₄₀ specific in that chemically induced, non SV₄₀, MCA tumor failed to elicit a DTH response in mice primed by excision of mKSA tumor.These mice also showed a corresponding one-way specific protection against challenge with live homologous mKSA sarcoma cells. In contrast immunization and challenge of MCA-excised mice with either MCA or mKSA tumor cells, exhibited cross-reactivity in both DTH response and protection against either tumor.Unlike this cross-immunity by the direct challenge method, transfer of immune spleen cells from mKSA or MCA excision-primed mice demonstrated a specific DTH response and protection to the original immunizing, homologous but not heterologous tumor. Tumor resistant, DTH-primed mice remained DTH reactive to the primary tumor cells over a period of 4 weeks. Characterization of the splenic T-DTH cells in mice primed by excision of mKSA tumor, indicated a Lyt 1⁺2⁺ phenotype of cells conferring both the DTH response and the immune protection against mKSA sarcoma in a local (Winn) adoptive transfer assay, thus reinforcing the correlation between the DTH response and the antitumor protection.     

Interaction of influenza virus proteins with planar bilayer lipid membranes II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (ΔU), transversal elasticity module (E₁) and membrane conductivity (G₀) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 · 10⁵ M^?1 and 1.3 · 10⁴ M^?1 for rimantadine and amantadine, respectively. The changes in G₀ took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 μg/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Oxidation of 2,3,4,6-tetra-O-methyl-d-glucose with alkaline hydrogen peroxide

Treatment of 2,3,4,6-tetra-O-methyl-d-glucose with 10 molar equivalents ofn 30% aqueous hydrogen peroxide and 2 molar equivalents of potassium hydroxide afforded, after chromatographic separation, 2,3,4,6-tetra-O-methyl-d-gluconolactone. 1-O-formyl-2,3,5-tri-O-methyl-d-arabinose methyl hemiacetal (7), 2,3,5-tri-O-methyl-d-arabinonolactone, methyl 2,3,5-tri-O-methyl-d-arabinoside, O-(2,4-di-O-methyl-d-erythrose)-(1'→3)-2,4-di-O-methyl-d-erythronic acid, and O-(2,4-di-O-methyl-d-erythrose)-(1′→2)-3-O-methyl-d-glyceraldehyde. The proportions of the products depended on the reaction conditions, especially the time, temperature, and the presence or absence of magnesium hydroxide. Formation of the products is explained by a series of reactions beginning with the addition of hydrogen peroxide to the carbonyl form of the methylated sugar. The adduct, with the help of superoxide radical and a molecule of hydrogen peroxide, breaks up in two ways, giving 2,3,4,6-tetra-O-methyl-d-gluconic acid and 7. The formic ester, on hydrolysis, gives 2,3,5-tri-O-methyl-d-arabinose, which undergoes a similar series of reactions, affording 2,3,5-tri-O-methyl-d-arabinonic acid, and presumably, 1-O-formyl-2,4-di-O-methyl-d-erythrose methyl hemiacetal. Apparently, the latter compound, on hydrolysis, forms a dimer, which, with alkaline hydrogen peroxide, undergoes a similar series of reactions, ultimately affording O-(2,4-di-O-methyl-d-erythrose)-(1→3)-2,4-di-O-methyl-d-erythronic acid and O-(2,4-di-O-methyl-d-erythrose)-(1→2)-3-o-methyl-d-glyceraldehyde. With a larger amount of alkali, under more-severe conditions, oxidation of 2,3,4,6-tetra-O-methyl-d-glucose proceeds further, with production of up to 3 moles of formic acid per mole of methylated sugar.     

Changing Emergence of Shigella Sero-Groups in Bangladesh: Observation from Four Different Diarrheal Disease Hospitals

Background

Shigellosis continues to be a public health challenge for developing countries, including Bangladesh. The aim of the study is to demonstrate recent changes in Shigella sero-groups and their geographical diversity.

Methods

Data were extracted from data archive of four diarrheal disease surveillance systems. A 2% sub sample from urban Dhaka Hospital (2008–2011; n = 10,650), and 10% from urban Mirpur Treatment Centre (2009–2011; n = 3,585), were enrolled systematically; whereas, all patients coming from the Health and Demographic Surveillance System area in rural Matlab (2008–2011; n = 6,399) and rural Mirzapur (2010–2011; n = 2,812) were included irrespective of age, sex, and disease severity. A fresh stool specimen was collected for identification of Shigella spp. Of them, 315 (3%) were positive for Shigella in Dhaka, 490 (8%) from Matlab, 109 (3%) from Mirpur and 369 (13%) from Mirzapur and considered as analyzable sample size.

Results

Among all Shigella isolates regardless of age, significant decreases in percentage of S. flexneri over time was observed in Mirpur (55→29%; p value of χ²-for trend = 0.019) and Mirzapur (59→47%; p = 0.025). A non-significant decrease was also seen in Dhaka (58→48%), while in Matlab there was a non-significant increase (73→81%). Similar patterns were observed among under-5 children at all sites. Emergence of S. sonnei was found in Dhaka (8→25%; p<0.001) and Mirpur (10→33%; p = 0.015), whereas it decreased in Mirzapur (32→23%; p = 0.056). The emergence of S. boydii was seen in all ages in Mirzapur [(3→28%; p<0.001); (3→27%; p<0.001)]. On the other hand, we saw non-significant percent reductions in S. boydii in Dhaka [overall (25→16%); under-5 (16→9%)]. Decreasing rates of Shigella dysenteriae were observed in Matlab, Mirpur and Mirzapur; whereas, in Dhaka it remained unchanged.

Conclusion and Significance

Emergence of S. sonnei and S. boydii as important infectious diarrhea etiologies and variations in geographical diversity underscore the need for monitoring, with possible implications for vaccine development.     

Streptomyces muensis sp. nov.

A strain of Streptomyces, MBRL 179^T, isolated from a sample from a Limestone quarry located at Hundung, Manipur, India, was characterized by polyphasic taxonomy. The strain formed a monophyletic clade with Streptomyces spinoverrucosus NBRC 14228^T (16S rRNA gene sequence similarity of 99.3 %) in the Neighbour-joining tree. DNA–DNA hybridization experiment gave a DNA–DNA relatedness value of 34.7 % between MBRL 179^T and S. spinoverrucosus NBRC 14228^T. Strain MBRL 179^T contained LL-diaminopimelic acid, xylose, glucose, and mannose in the whole cell-wall hydrolysates along with small amount of ribose. The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, with other unknown phospholipids and aminophospholipid. MK-9(H₆), MK-9(H₈) and MK-9(H₄) were the predominant menaquinones detected. The major fatty acids were anteiso-C_16:0 (28.1 %), iso-C_16:0 (20.3 %), C_16:0 (9.4 %) and anteiso-C_17:0 (8.3 %). The G+C content of the genomic DNA was 71.1 %. Based on the polyphasic experiment results, the strain MBRL 179^T merits recognition as a representative of a novel species of the genus Streptomyces for which the name Streptomyces muensis sp. nov. is proposed; the type strain is MBRL 179^T (=JCM 17576^T = KCTC 29124^T).     

Clinical Characteristics,Etiology and Antimicrobial Susceptibility among Overweight and Obese Individuals with Diarrhea: Observed at a Large Diarrheal Disease Hospital,Bangladesh

Background

The present study aimed to determine the clinical characteristics and etiology of overweight and obese (OO) individuals with diarrhea attending an urban Dhaka Hospital, International Centre for Diarrheal Disease Research (icddr,b), Bangladesh.

Methods

Total of 508 under-5 children, 96 individuals of 5–19 years and 1331 of >19 years were identified as OO from the Diarrheal Disease Surveillance System (DDSS) between 1993–2011. Two comparison groups such as well-nourished and malnourished individuals from respective age stratums were selected.

Results

Isolation rate of rotavirus was higher among OO under-5 children compared to malnourished group (46% vs. 28%). Rotavirus infection among OO individuals aged 5–19 years (9% vs. 3%) (9% vs. 3%) and >19 years (6% vs. 4%) (6% vs. 3%) was higher compared to well-nourished and malnourished children. Conversely, Vibrio cholerae was lower among all OO age groups compared to well-nourished and malnourished ones. Shigella (4% vs. 6%) (4% vs. 8%), and Campylobacter (3% vs. 5%) (3% vs. 5%) were lower only among OO in >19 years individuals compared to their counterparts of the same age stratum. Salmonella was similarly isolated in all age strata and nutritional groups. In multinomial logistic regression among under-5 children, significant association was observed only with use of antimicrobials at home [OR-1.97] and duration of hospital stay [OR-0.68]. For individuals aged 5–19 years, use of antimicrobials at home (OR-1.83), some or severe dehydration (OR-3.12), having received intravenous saline (OR-0.46) and rotavirus diarrhea (OR-2.96) were found to be associated with OO respectively. Moreover, significant associations were also found for duration of diarrhea before coming to hospital (>24 hours) (OR-1.24), Shigella (OR-0.46), and Campylobacter (OR-0.58) among >19 years OO individuals along with other associated co-variates in 5–19 years group (all p<0.05).

Conclusion and significance

Higher proportion of OO were infected with rotavirus and a greater proportion of them used antimicrobials before coming to the hospital.     

Hydrocarbon Degradation and Biosurfactant Production by an Acenaphthene-degrading Pseudomonas Species

An acenaphthene-degrading bacterium putatively identified as Pseudomonas sp. strain KR3 and isolated from diesel-contaminated soil in Lagos, Nigeria was investigated for its degradative and biosurfactant production potentials on crude oil. Physicochemical analysis of the sampling site indicates gross pollution of the soil with high hydrocarbon content (2100 mg/kg) and detection of various heavy metals. The isolate grew luxuriantly on crude oil, engine oil and acenaphthene. It was resistant to septrin, amoxicillin and augmentin but was susceptible to pefloxacin, streptomycin and gentamycin. It was also resistant to elevated concentration of heavy metals such as 1–15 mM lead, nickel and molybdenum. On acenaphthene, the isolate exhibited specific growth rate and doubling time of 0.098 day^?1 and 3.06 days, respectively. It degraded 62.44% (31.2 mg/l) and 91.78% (45.89 mg/l) of 50 mg/l acenaphthene within 12 and 21 days. On crude oil, the specific growth rate and doubling time were 0.375 day^?1 and 1.85 days with corresponding percentage degradation of 33.01% (903.99 mg/l) and 87.79% (2403.71 mg/l) of crude oil (2738.16 mg/l) within 9 and 18 days. Gas chromatographic analysis of residual crude oil at the end of 18 days incubation showed significant reductions in the aliphatic, alicyclic and aromatic fractions with complete disappearance of benzene, propylbenzene, pristane, phytane, and nC₁₈-octadecane fractions of the crude oil. The isolate produced growth-associated biosurfactant on crude oil with the highest emulsification index (E₂₄) value of 72% ± 0.23 on Day 10 of incubation. The partially purified biosurfactant showed zero tolerance for salinity and had its optimal activity at 27°C and pH 2.0.     

Inducible Nitric Oxide Synthase Promoter Haplotypes and Residential Traffic-Related Air Pollution Jointly Influence Exhaled Nitric Oxide Level in Children

BackgroundExhaled nitric oxide (FeNO), a biomarker of airway inflammation, predicts asthma risk in children. We previously found that the promoter haplotypes in inducible nitric oxide synthase (NOS2) and exposure to residential traffic independently influence FeNO level. Because NOS2 is inducible by environmental exposures such as traffic-related exposure, we tested the hypothesis that common NOS2 promoter haplotypes modulate the relationship between residential traffic-related exposure and FeNO level in children.MethodsIn a cross-sectional population-based study, subjects (N = 2,457; 7–11 year-old) were Hispanic and non-Hispanic white children who participated in the Southern California Children’s Health Study and had FeNO measurements. For residential traffic, lengths of local roads within circular buffers (50m, 100m and 200m radii around homes) around the subjects’ homes were estimated using geographic information system (GIS) methods. We interrogated the two most common NOS2 promoter haplotypes that were found to affect FeNO level.ResultsThe relationship between local road lengths within 100m and 200m circular buffers and FeNO level varied significantly by one of the NOS2 promoter haplotypes (P-values for interaction between road length and NOS2 promoter haplotype = 0.02 and 0.03, respectively). In children who had ≤250m of local road lengths within 100m buffer around their homes, those with two copies of the haplotype had significantly lower FeNO (adjusted geometric mean = 11.74ppb; 95% confidence intervals (CI): 9.99 to 13.80) than those with no copies (adjusted geometric mean = 15.28ppb; 95% CI: 14.04 to 16.63) with statistically significant trend of lower FeNO level with increasing number of haplotype copy (P-value for trend = 0.002). In contrast, among children who had >250m of local road lengths within 100m buffer, FeNO level did not significantly differ by the haplotype copy-number (P-value for trend = 0.34). Similar interactive effects of this haplotype and local road lengths within 200m buffer on FeNO were also observed.ConclusionsHigher exposure from residential traffic nullifies the protective effect of one common NOS2 promoter haplotype on FeNO level. Regulation of traffic-related pollution may protect children’s respiratory health.