Comparative in vivo and in vitro analyses of putative virulence factors of Burkholderia pseudomallei using lipopolysaccharide, capsule and flagellin mutants

Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of melioidosis. We evaluated host–pathogen interaction at different levels using three separate B. pseudomallei mutants generated by insertional inactivation. One of these mutants is defective in the production of the polysaccharide side chains associated with lipopolysaccharide; one does not produce the capsular polysaccharide with the structure -3)-2- O -acetyl-6-deoxy-β- d - manno -heptopyranose-(1-; and the third mutant does not produce flagellin. We compared the in vivo virulence in BALB/c mice, the in vitro fate of intracellular survival inside human polymorphonuclear cells (PMNs) and macrophages (Mφs) and the susceptibility to killing by 30% normal human serum, reactive nitrogen and oxygen intermediates and antimicrobial peptides with that of their wild-type counterpart. The lipopolysaccharide and capsule mutants demonstrated a marked reduction in virulence for BALB/c mice, but the flagellin mutant was only slightly less virulent than the parent strain. The results from the BALB/c mice experiments correlated with survival in Mφs. The lipopolysaccharide and capsule mutants were also more susceptible to killing by antimicrobial agents. All bacteria were equally susceptible to killing by PMNs. Altogether, the data suggest that lipopolysaccharide and capsule and, to a much lesser extent, flagella, are most likely associated with the virulence of this bacterium and highlight the importance of intracellular killing by PMNs and Mφs in disease pathogenesis.     

Comparison of two plethysmography systems in assessment of forearm blood flow.

Venous occlusion plethysmography is widely used to assess forearm blood flow (FBF). We compared the established Hokanson system (HEC4) with a newly developed Filtrass 2001 system (F2001). The HEC4 uses mercury-in-Silastic strain gauges, whereas F2001 detects volume changes with a nonmercury linear displacement device. The aim of this study was to evaluate the new F2001 against the HEC4 in terms of repeatability and systematic bias. Ten subjects were studied on 4 separate days in random order using either the HEC4 on both arms, the F2001 on both arms, the HEC4 on the right arm with the F2001 on the left, or the F2001 on the right arm and the HEC4 on the left. Stroop's colored word conflict test and postocclusive hyperemia were used to increase FBF, and lower body negative pressure was used to lower FBF. Stroop's colored word conflict test and lower body negative pressure increased (24.6 +/- 1.5%, n = 240, P < 0.0001) and decreased (18.7 +/- 0.8%, n = 240, P < 0.0001) FBF, respectively. Postocclusive hyperemia after occlusion times of 5, 8, and 13 min substantially increased FBF by 390 +/- 86, 756 +/- 217, and 851 +/- 132%, respectively. Repeatability was not different between the devices (0.10 +/- 2.37 vs. -0.47 +/- 1.92 l/min, n = 125, P > 0.05), and there was no systematic bias. The F2001 is a newly developed plethysmography system that does not utilize mercury and is suitable for assessing changes of FBF in physiological studies.     

On the origins of esterases

Comparisons among the primary sequences of five cloned eukaryotic esterases reveal two distinct lineages, neither bearing any significant overall sequence similarity to the functionally related serine protease multigene family. We have not eliminated the possibility that the esterases may have residual conformational similarities to the serine proteases. However, our profile analysis and analyses of the predicted conformations of the esterases reveal little similarity to the serine proteases. Four of the esterase proteins share 27%-53% overall sequence similarity and evidence of a catalytic mechanism involving the same Arg- Asp-Ser or His-Asp-Ser charge relay. We propose that these four esterases, three of them cholinesterases, form part of a multigene family essentially separate from the serine proteases.      

Microbicidal effect of the lactoferrin peptides Lactoferricin17–30, Lactoferrampin265–284, and Lactoferrin chimera on the parasite Entamoeba histolytica

Entamoeba histolytica is a parasitic protozoan that produces amoebiasis, an intestinal disease characterized by ulcerative colitis and dysentery. In some cases, trophozoites can travel to the liver leading to hepatic abscesses and death. Recently, lactoferrin and lactoferricin B have been shown to be amoebicidal in axenic cultures. The aim of this work was to determine whether the lactoferrin-peptides lactoferricin amino acids 17–30, lactoferrampin amino acids 265–284, and lactoferrin chimera which is a fusion product of the two peptides, are capable of producing a microbicidal effect to trophozoites of E. histolytica. We evaluated the killing effect of these peptides in growth kinetics carried out in axenic culture medium to which different concentrations of peptides were added. At 50 μM of peptide concentration, lactoferricin and lactoferrampin had a moderate amoebicidal effect, since a 45–50% of trophozoites remained viable at 24 h culture. However, at 50 μM of the lactoferrin chimera 75% amoeba were killed whereas at 100 μM all cells died. These data indicate that of lactoferrin-peptides mainly the chimera have amoebicidal activity in a time- and concentration-dependent manner. The lactoferrin-peptides might be useful as therapeutic agents against amoebiasis and thereby diminish the use of metronidazole, which is extremely toxic for the host.     

Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

Background  

Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation.