ASH2L Regulates Ubiquitylation Signaling to MLL: trans-Regulation of H3 K4 Methylation in Higher Eukaryotes

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Highlights? ASH2L WH motif is important for trans-regulation of H3 K4 methylation by H2Bub ? Ub stimulates MLL activity in trans, in contrast to DOT1L regulation by H2Bub ? H2Bub enhances MLL, but not MLL3, methyltransferase activity ? H2Aub directly represses MLL methyltransferase activity     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

Crystal structure of cytochrome c peroxidase compound I

We have compared the 2.5-A crystal structure of yeast cytochrome c peroxidase (CCP) with that of its semistable two-equivalent oxidized intermediate, compound I, by difference Fourier and least-squares refinement methods. Both structures were observed at -15 degrees C. The difference Fourier map reveals that formation of compound I causes only small positional adjustments of a few tenths of an angstrom. The map's most pronounced feature is a pair of positive and negative peaks bracketing the heme iron position. Least-squares refinement shows that the iron atom moves about 0.2 A toward the distal side of the heme. No significant difference density is evident near the side chains of Trp-51 or Met-172, each of which has been proposed to be the site of the electron paramagnetic resonance (EPR) active radical in compound I. However, the second most prominent feature of difference density is a negative peak near the side chain of Thr-180, which, according to the results of least-squares refinement, moves by 0.15 A in the direction of Met-230. These observations, together with the results of mutagenesis experiments [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360; Goodin, D. B., Mauk, A. G., & Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299] in which Trp-51 and Met-172 have been replaced without loss of the EPR radical signal in compound I, lead us to consider the possibility that the radical site lies within a cluster composed of the side chains of Met-230, Met-231, and Trp-191.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroxyethylamine-based inhibitors of BACE1: P1–P3 macrocyclization can improve potency,selectivity, and cell activity

We describe a systematic study of how macrocyclization in the P₁–P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.     

Omega 3 Fatty Acids Promote Macrophage Reverse Cholesterol Transport in Hamster Fed High Fat Diet

The aim of this study was to investigate macrophage reverse cholesterol transport (RCT) in hamster, a CETP-expressing species, fed omega 3 fatty acids (ω3PUFA) supplemented high fat diet (HFD). Three groups of hamsters (n = 6/group) were studied for 20 weeks: 1) control diet: Control, 2) HFD group: HF and 3) HFD group supplemented with ω3PUFA (EPA and DHA): HFω3. In vivo macrophage-to-feces RCT was assessed after an intraperitoneal injection of ³H-cholesterol-labelled hamster primary macrophages.Compared to Control, HF presented significant (p<0.05) increase in body weight, plasma TG (p<0.01) and cholesterol (p<0.001) with an increase in VLDL TG and in VLDL and LDL cholesterol (p<0.001).Compared to HF, HFω3 presented significant decrease in body weight. HFω3 showed less plasma TG (p<0.001) and cholesterol (p<0.001) related to a decrease in VLDL TG and HDL cholesterol respectively and higher LCAT activity (p<0.05) compared to HF. HFω3 showed a higher fecal bile acid excretion (p<0.05) compared to Control and HF groups and higher fecal cholesterol excretion (p<0.05) compared to HF. This increase was related to higher gene expression of ABCG5, ABCA1 and SR-B1 in HFω3 compared to Control and HF groups (<0.05) and in ABCG1 and CYP7A1 compared to HF group (p<0.05). A higher plasma efflux capacity was also measured in HFω3 using ³H- cholesterol labeled Fu5AH cells.In conclusion, EPA and DHA supplementation improved macrophage to feces reverse cholesterol transport in hamster fed HFD. This change was related to the higher cholesterol and fecal bile acids excretion and to the activation of major genes involved in RCT.     

Feasibility of Tomotherapy-Based Image-Guided Radiotherapy to Reduce Aspiration Risk in Patients with Non-Laryngeal and Non-Pharyngeal Head and Neck Cancer

Purpose

The study aims to assess the feasibility of Tomotherapy-based image-guided radiotherapy (IGRT) to reduce the aspiration risk in patients with non-laryngeal and non-hypopharyngeal cancer. A retrospective review of 48 patients undergoing radiation for non-laryngeal and non-hypopharyngeal head and neck cancers was conducted. All patients had a modified barium swallow (MBS) prior to treatment, which was repeated one month following radiotherapy. Mean middle and inferior pharyngeal dose was recorded and correlated with the MBS results to determine aspiration risk.

Results

Mean pharyngeal dose was 23.2 Gy for the whole group. Two patients (4.2%) developed trace aspiration following radiotherapy which resolved with swallowing therapy. At a median follow-up of 19 months (1–48 months), all patients were able to resume normal oral feeding without aspiration.

Conclusion and Clinical Relevance

IGRT may reduce the aspiration risk by decreasing the mean pharyngeal dose in the presence of large cervical lymph nodes. Further prospective studies with IGRT should be performed in patients with non-laryngeal and non-hypopharyngeal head and neck cancers to verify this hypothesis.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Studies on the primary sequence requirements for PKC-alpha, -beta 1 and -gamma peptide substrates.

The substrate specificity of purified PKC-alpha, -beta and -gamma has been investigated. A series of synthetic peptides based upon the sequence surrounding serine-7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC-alpha and -beta are indistinguishable in their phosphorylation of these peptides, PKC-gamma shows a distinct specificity profile for these synthetic substrates.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.