Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli

Among enteric bacteria, the ability to grow on N-acetyl-galactosamine (GalNAc or Aga) and on D-galactosamine (GalN or Gam) differs. Thus, strains B, C and EC3132 of Escherichia coli are Aga+ Gam+ whereas E. coli K-12 is Aga- Gam-, similarly to Klebsiella pneumoniae KAY2026, Klebsiella oxytoca M5a1 and Salmonella typhimurium LT2. The former strains carry a complete aga/kba gene cluster at 70.5 min of their gene map. These genes encode an Aga-specific phosphotransferase system (PTS) or IIAga (agaVWE) and a GalN-specific PTS or IIGam (agaBCD). Both PTSs belong to the mannose-sorbose family, i.e. the IIB, IIC and IID domains are encoded by different genes, and they share a IIA domain (agaF). Furthermore, the genes encode an Aga6P-deacetylase (agaA), a GalN6P deaminase (agaI), a tagatose-bisphosphate aldolase comprising two different peptides (kbaYZ) and a putative isomerase (agaS), i.e. complete pathways for the transport and degradation of both amino sugars. The genes are organized in two adjacent operons (kbaZagaVWEFA and agaS kbaYagaBCDI) and controlled by a repressor AgaR. Its gene agaR is located upstream of kbaZ, and AgaR responds to GalNAc and GalN in the medium. All Aga- Gam- strains, however, carry a deletion covering genes agaW' EF 'A; consequently they lack active IIAga and IIGam PTSs, thus explaining their inability to grow on the two amino sugars. Remnants of a putative recombination site flank the deleted DNA in the various Aga- Gam- enteric bacteria. Derivatives with an Aga+ Gam- phenotype can be isolated from E. coli K-12. These retain the DeltaagaW' EF 'A deletion and carry suppressor mutations in the gat and nag genes for galactitol and N-acetyl-glucosamine metabolism, respectively, that allow growth on Aga but not on GalN.     

Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target

In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19–1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a “catalytic” latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.     

Sequencing and analysis of an Irish human genome

Background

Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence.

Results

Using sequence data from a branch of the European ancestral tree as yet unsequenced, we identify variants that may be specific to this population. Through comparisons with HapMap and previous genetic association studies, we identified novel disease-associated variants, including a novel nonsense variant putatively associated with inflammatory bowel disease. We describe a novel method for improving SNP calling accuracy at low genome coverage using haplotype information. This analysis has implications for future re-sequencing studies and validates the imputation of Irish haplotypes using data from the current Human Genome Diversity Cell Line Panel (HGDP-CEPH). Finally, we identify gene duplication events as constituting significant targets of recent positive selection in the human lineage.

Conclusions

Our findings show that there remains utility in generating whole genome sequences to illustrate both general principles and reveal specific instances of human biology. With increasing access to low cost sequencing we would predict that even armed with the resources of a small research group a number of similar initiatives geared towards answering specific biological questions will emerge.     

Is the Association Between Helicobacter pylori Infection and Anemia Age Dependent?

Background: The relationship between H. pylori infection and anemia in childhood is still unclear. The aim of the study was to examine the association between H. pylori infection and anemia or iron deficiency in school‐age children and in infants. Materials and Methods: Six‐ to 9‐ year‐old Israeli Arab children (N = 202) and infants (N = 197) were examined for hemoglobin and ferritin levels. ELISA was used to detect H. pylori antigens in stool specimens collected from the participants. Household characteristics were obtained through personal interviews with the mothers. Results: The prevalence of anemia was 15.5 versus 5.5% in H. pylori‐positive and ‐negative school‐age children, respectively and 34.5 versus 29.8% in H. pylori‐positive and ‐negative infants, respectively. The Mantel–Haenszel age‐adjusted prevalence ratio (PR) and 95% confidence intervals (CIs) were 1.6 (95%CI 1.0, 2.6). In multivariate analysis controlling for socioeconomic variables, H. pylori infection was associated with 2.8 higher prevalence of anemia only in school‐age children: adjusted PR 2.8 (95% CI 0.9, 9.3). The adjusted mean difference in hemoglobin levels between H. pylori infected school‐age children and uninfected ones was ?0.372 gr/dL (95% CI ?0.704, ?0.039) (p = .04). The respective mean ferritin difference was ?6.74 μg/L (95% CI ?13.38, ?.011) (p = .04). Such differences were not found in infants. Conclusions: H. pylori infection is associated with higher prevalence of anemia in school‐age children independently of socioeconomic variables. Such association was not observed in infants. These findings are of clinical and public health importance.     

Impacts of the invasive plant <Emphasis Type="Italic">Fallopia japonica</Emphasis> (Houtt.) on plant communities and ecosystem processes

Fallopia japonica (Japanese knotweed) invades riparian areas and roadsides in New England. This large clonal species drastically alters the appearance of habitats by forming highly productive near-monocultures. To understand how these invasions affect ecosystem processes in New England, we quantified the impacts of F. japonica on species diversity, primary productivity, and nitrogen cycling at five locations in central Massachusetts, USA. In stands of F. japonica and in adjacent uninvaded areas, we recorded the cover of each plant species and measured the aboveground biomass and nitrogen (N) concentrations in plants, along with N retranslocation from F. japonica leaves and several soil characteristics. In addition, we severed rhizomes of peripheral F. japonica shoots to determine if clonal integration contributes to the species’ rapid spread and dominance. Stands of F. japonica had lower species diversity, but greater aboveground biomass and standing N than uninvaded areas. Nitrogen and carbon concentrations in biomass and N mineralization rates in soil did not differ between stands and adjacent areas. Rhizome severing temporarily reduced growth of F. japonica, suggesting that retranslocation of photoassimilates and/or nutrients between shoots via rhizomatal connections may maximize stand level growth rates and facilitate dominance by F. japonica.     

Replacement of damaged arteries and veins with vein grafts when replanting crushed, amputated fingers

Two cases are presented in which multiple vein grafts were used to revascularize several digits in hands injured by severe crushes. One case had partial amputations of 4 digits, and the other had 4 complete amputations. After debriding the damaged parts of the vessels, we interposed vein grafts in both the arterial and venous circulations to the digits. Seven of 8 revascularized digits survived. In crushing injuries, it is essential to debride the vessels proximally and distally to where the intima has not been injured. To bridge the gaps, vein grafts can be used successfully where other methods are undesirable or impossible.     

Effect of acid-base status on plasma phosphorus response to lactate

The mechanism(s) underlying the hyperphosphatemia of lactic acidosis is uncertain. We assessed the interacting influence of the acid anion and acid-base status on plasma phosphorus concentration by administering lactic acid alone, lactic acid plus sodium bicarbonate, sodium bicarbonate alone, and sodium lactate alone to four different groups of dogs. The findings of (1) no increase in plasma phosphorus concentration with lactic acid plus sodium bicarbonate versus a marked increment with lactic acid alone, and (2) no difference in the plasma phosphorus response to sodium lactate versus sodium bicarbonate indicate that acidemia is necessary for the expression of lactate-induced hyperphosphatemia. The apparent greater propensity for marked hyperphosphatemia in lactic acidosis than in other types of metabolic acidosis remains unexplained, but conceivably might relate to differences in intracellular pH and in the rate of glycolysis.     

Pathogenesis of hyperphosphatemia in lactic acidosis: disparate effects of racemic (DL-) and levo (L-) lactic acid on plasma phosphorus concentration

The mechanism(s) for the hyperphosphatemia associated with lactic acidosis is unknown. Experimental lactate-induced hyperphosphatemia appears to require acidemia because we have shown that prevention of acidemia with NaHCO3 obviates increases in plasma phosphorus concentration ([P]). Since the rate of lactate metabolism (by utilizing NAD or other mechanisms) might modulate transcellular movement of phosphorus, we assessed the plasma [P] response to 3-h infusions of DL-lactic acid versus L-lactic acid. The dog metabolizes primarily the L-moiety of DL-lactic acid (thereby consuming H+), so more L-lactic acid is needed to produce the degree of acidemia attained with DL-lactic acid. Group 1 (n = 6) mongrel dogs received 12 mequiv./kg DL-lactic acid; group 2 (n = 6) 12 mequiv./kg L-lactic acid, and group 3 (n = 7) 16-19 mequiv./kg L-lactic acid. Prior to acid loading, the plasma [P] and acid-base status of the three groups were similar. After 3 h, blood pH and [HCO3] and change from base line in plasma [P], in both milligrams per decilitre and percent, were as follows: group 1: 7.05 +/- 0.02, 9 +/- 2 mM, 1.9 +/- 0.4 mg/dL, 38 +/- 10%; group 2: 7.28 +/- 0.02, 18 +/- 1, 0.9 +/- 0.3, 17 +/- 6; group 3: 7.06 +/- 0.04, 12 +/- 1, 1.1 +/- 0.3, 26 +/- 10, respectively. Thus, there was a tendency for both infusion rates of L-lactic acid to increase [P] less than DL-lactic acid, suggesting the importance of other factors in addition to pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of alpha1-acid-glycoprotein in human liver parenchymal cells.

alpha1-Acid glycoprotein, a major human serum glycoprotein was detected and localized in human liver parenchymal cells of a biopsy specimen. A heavy metal salt containing fixative was required to retain sufficient antigen determinants of alpha1-acid glycoprotein in order to visualize this protein by the peroxidase-anti-peroxidase unlabeled antibody enzyme method.     

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.