Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-D-mannose

Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A-band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF15-4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PA01. With A-band and B-band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel electrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (Spel fragment SpK; Dpnl fragment DpF₂), while genes involved with expression of B-band LPS mapped at 1.9 Mbp (Spel fragments SpC, Spl and SpAl; Dpnl fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A-band synthesis in rd7513, an A^? mutant derived from A⁺ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one-kilobase region capable of complementing the expression of A-band in the A^? strain rd7513. This region was subcloned as a 1.6 kb Kpnl fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted ¹⁴C-labelled-guanosine diphospho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 did not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37kDa protein is involved in this conversion.     

Cardiovascular responses to lower body negative pressure in trained and untrained older men.

To determine whether aerobic conditioning alters the orthostatic responses of older subjects, cardiovascular performance was monitored during graded lower body negative pressure in nine highly trained male senior athletes (A) aged 59-73 yr [maximum O2 uptake (VO2 max) = 52.4 +/- 1.7 ml.kg-1 x min-1] and nine age-matched control subjects (C) (VO2 max = 31.0 +/- 2.9 ml.kg-1 x min-1). Cardiac volumes were determined from gated blood pool scintigrams by use of 99mTc-labeled erythrocytes. During lower body negative pressure (0 to -50 mmHg), left ventricular end-diastolic and end-systolic volume indexes and stroke volume index decreased in both groups while heart rate increased. The decreases in cardiac volumes and mean arterial pressure and the increase in heart rate between 0 and -50 mmHg were significantly less in A than in C. For example, end-diastolic volume index decreased by 32 +/- 4 ml in C vs. 14 +/- 2 ml in A (P < 0.01), mean arterial pressure declined 7 +/- 5 mmHg in C and increased by 5 +/- 3 mmHg in A (P < 0.05), and heart rate increased 13 +/- 3 beats/min in C and 7 +/- 1 beats/min in A (P < 0.05). These data suggest that increased VO2 max among older men is associated with improved orthostatic responses.     

The Effect of Diet and Exercise on Intestinal Integrity and Microbial Diversity in Mice

Background

The gut microbiota is now known to play an important role contributing to inflammatory-based chronic diseases. This study examined intestinal integrity/inflammation and the gut microbial communities in sedentary and exercising mice presented with a normal or high-fat diet.

Methods

Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-weeks and randomly assigned to exercise or sedentary groups. After 12 weeks animals were sacrificed and duodenum/ileum tissues were fixed for immunohistochemistry for occludin, E-cadherin, and cyclooxygenase-2 (COX-2). The bacterial communities were assayed in fecal samples using terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of 16S rRNA gene amplicons.

Results

Lean sedentary (LS) mice presented normal histologic villi while obese sedentary (OS) mice had similar villi height with more than twice the width of the LS animals. Both lean (LX) and obese exercise (OX) mice duodenum and ileum were histologically normal. COX-2 expression was the greatest in the OS group, followed by LS, LX and OX. The TRFLP and pyrosequencing indicated that members of the Clostridiales order were predominant in all diet groups. Specific phylotypes were observed with exercise, including Faecalibacterium prausnitzi, Clostridium spp., and Allobaculum spp.

Conclusion

These data suggest that exercise has a strong influence on gut integrity and host microbiome which points to the necessity for more mechanistic studies of the interactions between specific bacteria in the gut and its host.     

On the Use of Biomineral Oxygen Isotope Data to Identify Human Migrants in the Archaeological Record: Intra-Sample Variation,Statistical Methods and Geographical Considerations

Oxygen isotope analysis of archaeological skeletal remains is an increasingly popular tool to study past human migrations. It is based on the assumption that human body chemistry preserves the δ¹⁸O of precipitation in such a way as to be a useful technique for identifying migrants and, potentially, their homelands. In this study, the first such global survey, we draw on published human tooth enamel and bone bioapatite data to explore the validity of using oxygen isotope analyses to identify migrants in the archaeological record. We use human δ¹⁸O results to show that there are large variations in human oxygen isotope values within a population sample. This may relate to physiological factors influencing the preservation of the primary isotope signal, or due to human activities (such as brewing, boiling, stewing, differential access to water sources and so on) causing variation in ingested water and food isotope values. We compare the number of outliers identified using various statistical methods. We determine that the most appropriate method for identifying migrants is dependent on the data but is likely to be the IQR or median absolute deviation from the median under most archaeological circumstances. Finally, through a spatial assessment of the dataset, we show that the degree of overlap in human isotope values from different locations across Europe is such that identifying individuals’ homelands on the basis of oxygen isotope analysis alone is not possible for the regions analysed to date. Oxygen isotope analysis is a valid method for identifying first-generation migrants from an archaeological site when used appropriately, however it is difficult to identify migrants using statistical methods for a sample size of less than c. 25 individuals. In the absence of local previous analyses, each sample should be treated as an individual dataset and statistical techniques can be used to identify migrants, but in most cases pinpointing a specific homeland should not be attempted.     

Interplant variation in creosotebush foliage characteristics and canopy arthropods

Summary We conducted a field study to test the hypothesis that creosotebush (Larrea tridentata) shrubs growing in naturally nutrient-rich sites had better quality foliage and supported greater populations of foliage arthropods than shrubs growing in nutrient-poor sites. Nutrient-rich sites had significantly higher concentrations of soil nitrogen than nutrient-poor sites. Multivariate analysis of variance revealed significant differences between high nutrient and low nutrient shrubs based on a number of structural and chemical characteristics measured. High nutrient shrubs were larger, had denser foliage, greater foliage production, higher concentrations of foliar nitrogen and water, and lower concentrations of foliar resin than low nutrient shurbs. Numbers of foliage arthropods, particularly herbivores and predators, were significantly higher on high nutrient shrubs. Shrub characteristics and foliage arthropod abundances varied considerably from shrub to shrub. Shrub characteristics representing shrub size, foliage density, foliage growth, and foliar nitrogen and water concentrations were positively correlated with arthropod abundances. Foliar resin concentrations were negatively correlated with foliage arthropod abundances. The positive relationship between creosotebush productivity and foliage arthropods is contradictory to the tenet that physiologically stressed plants provide better quality foliage to insect herbivores.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

A convective mass transfer model for determining intestinal wall permeabilities: laminar flow in a circular tube

A convective mass transfer model as analyzed and developed for use in determining intestinal wall permeabilities from external perfusion experiments. Analysis of the model indicates that the ratio of the exit to inlet concentration $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ is a function of only two dimensionless independent variables, the wall permeability, $\text{P}{}_{\mathrm{w}}{}^1$ and Graetz number, Gz = πDL/2Q. The Graetz number contains the independent variables of interest, length, diflusivity, and flow rate. The radius of the intestine is included implicitly in the flow rate. Since $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ and Gz are the experimental quantities, and the solution to the model system contains P_ω¹ implicitly, a convenient approximate method is developed which allows a direct calculation of P_ω¹. This method is in error by 10–20% in the worst cases. The approach is illustrated by application to the determination of the wall permeabilities for two non polar compounds.