Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.     

Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20-200muM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.     

Anaerocolumna sedimenticola sp. nov., isolated from fresh water sediment

Strain CBA3638^T was isolated from the Geum River sediment, Republic of Korea. The cells of strain CBA3638^T were Gram-stain-positive, strictly anaerobic, rod-shaped, and 0.5–1.0 μm wide, and 4.0–4.5 μm long. Optimal growth occurred at 37 °C, pH 7.0, and 1.0% (w/v) NaCl. Based on the 16S rRNA gene sequence, the phylogenetic analysis showed that strain CBA3638^T belongs to the genus Anaerocolumna in the family Lachnospiraceae, and is most closely related to Anaerocolumna cellulosilytica (94.6–95.0%). The DDH value with A. cellulosilytica SN021^T showed 15.0% relatedness. The genome of strain CBA3638^T consisted of one circular chromosome that is 5,500,435 bp long with a 36.7 mol% G?+?C content. The genome contained seven 16S-5S-23S rRNA operons and one antibiotic resistance-related transporter gene (mefA). Quinones were not detected. The predominant cellular fatty acids were C_16:0 and C_14:0 and the polar lipids were diphosphatidylglycerol, phosphatidylcholine, and uncharacterised polar lipids. Based on the polyphasic taxonomic analysis, we propose strain CBA3638^T as a novel species in the genus Anaerocolumna, with the name Anaerocolumna sedimenticola sp. nov. The type strain is CBA3638^T (=?KACC 21652^T?=?DSM 110663^T).

     

Functional and Molecular Surveillance of Helicobacter pylori Antibiotic Resistance in Kuala Lumpur

Background

Helicobacter pylori is the etiological agent for diseases ranging from chronic gastritis and peptic ulcer disease to gastric adenocarcinoma and primary gastric B-cell lymphoma. Emergence of resistance to antibiotics possesses a challenge to the effort to eradicate H. pylori using conventional antibiotic-based therapies. The molecular mechanisms that contribute to the resistance of these strains have yet to be identified and are important for understanding the evolutional pattern and selective pressure imposed by the environment.

Methods and Findings

H. pylori was isolated from 102 patients diagnosed with gastrointestinal diseases, who underwent endoscopy at University Malaya Medical Centre (UMMC). The isolates were tested for their susceptibility on eleven antibiotics using Etest. Based on susceptibility test, 32.3% of the isolates were found to have primary metronidazole resistance; followed by clarithromycin (6.8%) and fluoroquinolones (6.8%). To further investigate the resistant strains, mutational patterns of gene rdxA, frxA, gyrA, gyrB, and 23S rRNA were studied. Consistent with the previous reports, metronidazole resistance was prevalent in the local population. However, clarithromycin, fluoroquinolone and multi-drug resistance were shown to be emerging. Molecular patterns correlated well with phenotypic data. Interestingly, multi-drug resistant (MDR) strains were found to be associated with higher minimum inhibitory concentration (MIC) than their single-drug resistant (SDR) counterparts. Most importantly, clarithromycin-resistant strains were suggested to have a higher incidence for developing multi-drug resistance.

Conclusion

Data from this study highlighted the urgency to monitor closely the prevalence of antibiotic resistance in the Malaysian population; especially that of clarithromycin and multi-drug resistance. Further study is needed to understand the molecular association between clarithromycin resistance and multi-drug resistance in H. pylori. The report serves a reminder that a strict antibiotic usage policy is needed in Malaysia and other developing countries (especially those where H. pylori prevalence remained high).     

Predominance of Th2 response in human abdominal aortic aneurysm: mistaken identity for IL-4-producing NK and NKT cells?

Abdominal aortic aneurysm (AAA) is a complex remodeling process that involves both synthesis and degradation of extracellular matrix proteins in the aortic wall, leading to decreased tensile strength, progressive dilation and eventual rupture. Chronic inflammation, increased local production of elastin-degrading proteases by inflammatory cells and destruction of medial elastic lamellae play important roles in aneurysm progression. Neovascularization in all layers of the arterial wall is prominent and angiogenesis can facilitate chronic inflammation. It is still unclear what initiates aneurysmal dilation and what determines its progression. The complex nature of the process has defied elucidation. Apart from macrophages, the predominant immune cell infiltrates reported so far are CD3(+)T cells that express CD4 and CD8. Infiltrates of type 2 Th cells and their production of IL-4 and IL-5 have been implicated in AAA development. However, NKT and NK cells have a Th0 cytokine profile and can also produce type 2 as well as type 1 (IL-2 and IFNgamma) cytokines. We have demonstrated the presence of NK and NKT cells in AAA tissue. With their growing importance in autoimmunity and transplantation, they may play a role in AAA development. Therefore, there is a need to use a combination of T and NK markers to fully characterize both innate and adaptive lymphoid cell subsets in local inflammatory infiltrates in order to elucidate their roles in AAA progression.