Structure of the AML1-ETO NHR3-PKA(RIIα) Complex and Its Contribution to AML1-ETO Activity

AML1-ETO is the chimeric protein product of t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the nervy homology region (NHR) 3 domain, which shares homology with A-kinase anchoring proteins and interacts with the regulatory subunit of type II cAMP-dependent protein kinase A (PKA(RIIα)). We determined the solution structure of a complex between the AML1-ETO NHR3 domain and PKA(RIIα). Based on this structure, a key residue in AML1-ETO for PKA(RIIα) association was mutated. This mutation did not disrupt AML1-ETO's ability to enhance the clonogenic capacity of primary mouse bone marrow cells or its ability to repress proliferation or granulocyte differentiation. Introduction of the mutation into AML1-ETO had minimal impact on in vivo leukemogenesis. Therefore, the NHR3-PKA(RIIα) protein interaction does not appear to significantly contribute to AML1-ETO's ability to induce leukemia.     

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3(+) regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1- and VAP-1-rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.     

Increased myocardial stiffness with maintenance of length-dependent calcium activation by female sex hormones in diabetic rats

A decrease in peak early diastolic filling velocity in postmenopausal women implies a sex hormone-related diastolic dysfunction. The regulatory effect of female sex hormones on cardiac distensibility therefore was evaluated in ovariectomized rats by determining the sarcomere length-passive tension relationship of ventricular skinned fiber preparations. Diabetes also was induced in the rat to assess the protective significance of female sex hormones on diastolic function. While ovariectomy had no effect on myocardial stiffness, collagen content, or titin ratio, a significant increase in myocardial stiffness was observed in diabetic rat only when female sex hormones were intact. The increased stiffness in diabetic-sham rats was accompanied by an elevated collagen content resulting from increases in the levels of procollagen and Smad2. Surprisingly, the increased myocardial stiffness in diabetic-sham rats was accompanied by a shift toward a more compliant N2BA of cardiac titin isoforms. The pCa-active tension relationship was analyzed at fixed sarcomere lengths of 2.0 and 2.3 μm to determine the magnitude of changes in myofilament Ca(2+) sensitivity between the two sarcomere lengths. Interestingly, high expression of N2BA titin was associated with a suppressed magnitude of changes in myofilament Ca(2+) sensitivity only in the diabetic-ovariectomized condition. Estrogen supplementation in diabetic-ovariectomized rats partially increased myocardial stiffness but completely reversed the change in myofilament Ca(2+) sensitivity. These results indicate a restrictive adaptation of myocardium governed by female sex hormones to maintain myofilament activity in compensation to the pathophysiological induction of cardiac dilatation by the diabetic condition.     

Influenza Vaccination Guidelines and Vaccine Sales in Southeast Asia: 2008–2011

Background

Southeast Asia is a region with great potential for the emergence of a pandemic influenza virus. Global efforts to improve influenza surveillance in this region have documented the burden and seasonality of influenza viruses and have informed influenza prevention strategies, but little information exists about influenza vaccination guidelines and vaccine sales.

Methods

To ascertain the existence of influenza vaccine guidelines and define the scope of vaccine sales, we sent a standard three-page questionnaire to the ten member nations of the Association of Southeast Asian Nations. We also surveyed three multinational manufacturers who supply influenza vaccines in the region.

Results

Vaccine sales in the private sector were <1000 per 100,000 population in the 10 countries. Five countries reported purchasing vaccine for use in the public sector. In 2011, Thailand had the highest combined reported rate of vaccine sales (10,333 per 100,000). In the 10 countries combined, the rate of private sector sales during 2010–2011 (after the A(H1N1)2009pdm pandemic) exceeded 2008 pre-pandemic levels. Five countries (Indonesia, Malaysia, Singapore, Thailand and Vietnam) had guidelines for influenza vaccination but only two were consistent with global guidelines. Four recommended vaccination for health care workers, four for elderly persons, three for young children, three for persons with underlying disease, and two for pregnant women.

Conclusions

The rate of vaccine sales in Southeast Asia remains low, but there was a positive impact in sales after the A(H1N1)2009pdm pandemic. Low adherence to global vaccine guidelines suggests that more work is needed in the policy arena.     

Crystal structure and properties of CYP231A2 from the thermoacidophilic archaeon Picrophilus torridus

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.