Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Isolation and Characterisation of Lytic Bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca

Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.     

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

Raman and FTIR microscopic imaging of colon tissue: a comparative study

Colon tissue constitutes a valid model for the comparative analysis of soft tissue by Raman and Fourier transform infrared (FTIR) imaging because it contains four major tissue types such as muscle tissue, connective tissue, epithelium and nerve cells. Raman microscopic images were recorded in the mapping mode using 785 nm laser excitation and a step size of 10 μm from three regions within a thin section that encompassed mucus, mucosa, submucosa, and longitudinal and circular muscle layers. FTIR microscopic images that were composed of 4, 8 and 9 individual images of 4096 spectra each were recorded from the same regions using a FTIR spectrometer coupled to a microscope with a focal plane array detector. Furthermore, Raman microscopic images were recorded at a step size of 2.5 μm from three ganglia that belong to the myenteric plexus. The results are discussed with respect to lateral resolution, spectral resolution, acquisition time and sensitivity of both modalities. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nature,Source and Function of Pigments in Tardigrades: In Vivo Raman Imaging of Carotenoids in Echiniscus blumi

Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.     

Diversity of Bacillus anthracis strains in Georgia and of vaccine strains from the former Soviet Union

Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.     

Hepatitis C Virus Co-Infection Increases the Risk of Anti-Tuberculosis Drug-Induced Hepatotoxicity among Patients with Pulmonary Tuberculosis

Background

The country of Georgia has a high prevalence of tuberculosis (TB) and hepatitis C virus (HCV) infection.

Purpose

To determine whether HCV co-infection increases the risk of incident drug-induced hepatitis among patients on first-line anti-TB drug therapy.

Methods

Prospective cohort study; HCV serology was obtained on all study subjects at the time of TB diagnosis; hepatic enzyme tests (serum alanine aminotransferase [ALT] activity) were obtained at baseline and monthly during treatment.

Results

Among 326 study patients with culture-confirmed TB, 68 (21%) were HCV co-infected, 14 (4.3%) had chronic hepatitis B virus (HBV) infection (hepatitis B virus surface antigen positive [HBsAg+]), and 6 (1.8%) were HIV co-infected. Overall, 19% of TB patients developed mild to moderate incident hepatotoxicity. In multi-variable analysis, HCV co-infection (adjusted Hazards Ratio [aHR]=3.2, 95% CI=1.6-6.5) was found to be an independent risk factor for incident anti-TB drug-induced hepatotoxicity. Survival analysis showed that HCV co-infected patients developed hepatitis more quickly compared to HCV seronegative patients with TB.

Conclusion

A high prevalence of HCV co-infection was found among patients with TB in Georgia. Drug-induced hepatotoxicity was significantly associated with HCV co-infection but severe drug-induced hepatotoxicity (WHO grade III or IV) was rare.     

Diversity of Bacillus anthracis Strains in Georgia and of Vaccine Strains from the Former Soviet Union

Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.