Substrate diversity of macrophomate synthase catalyzing an unusual multistep transformation from 2-pyrones to benzoates

Macrophomate synthase, which we have recently purified, catalyzes an unusual multistep transformation from 5-acetyl-4-methoxy-6-methyl-2-pyrone to 4-acetyl-3-methoxy-5-methyl-benzoic acid (macrophomic acid). To investigate the substrate diversity of the enzyme, 40 analogs of 2-pyrone were prepared and their relative efficiency was examined in the enzymatic conversions. The experimental results reveal the structural requirements of the substrates and the rough size of the enzyme active site, and eliminate the ambiguity caused by contamination by other enzymes in the whole-cell experiments.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Cystathionine β-Synthase Inhibition Is a Potential Therapeutic Approach to Treatment of Ischemic Injury

Hydrogen sulfide (H₂S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H₂S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H₂S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H₂S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H₂S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.     

Novel recombination system using Cre recombinase alpha complementation

A major limitation for the use of Cre recombinase is its toxicity and a lack of temporal control over its activity. We have developed a new recombination system using Cre recombinase α-complementation. Cre recombinase was divided and one fragment (β) was introduced into cells between two loxP sites with a CMV promoter in the upstream. The gene of interest (EGFP) was positioned just downstream of this construct. Cre recombinase activity was recovered by adding the other part of the molecule (α) to cells as a protein fragment, as evidenced by the expression of EGFP under the control of the CMV promoter. The activity of fragmented cre reached 68% of that of the wild type enzyme at 1 μM α-protein.     

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies. Financial support and conflicts of interest: This study was supported by grants from Medinet (Japan), and Suncorp Metway and Gallipoli Research Foundation (Australia). No financial or commercial interests arise from this study. Informed consent: This study was approved by Human Research Ethics Committees of the University of Queensland and Greenslopes Private Hospital and informed consent was obtained from all subjects.     

Mycophenolic acid as a latent agonist of PPARgamma

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor gamma (PPARgamma).     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

Neonatal mortality levels for 193 countries in 2009 with trends since 1990: a systematic analysis of progress, projections, and priorities

Background

Historically, the main focus of studies of childhood mortality has been the infant and under-five mortality rates. Neonatal mortality (deaths <28 days of age) has received limited attention, although such deaths account for about 41% of all child deaths. To better assess progress, we developed annual estimates for neonatal mortality rates (NMRs) and neonatal deaths for 193 countries for the period 1990–2009 with forecasts into the future.

Methods and Findings

We compiled a database of mortality in neonates and children (<5 years) comprising 3,551 country-years of information. Reliable civil registration data from 1990 to 2009 were available for 38 countries. A statistical model was developed to estimate NMRs for the remaining 155 countries, 17 of which had no national data. Country consultation was undertaken to identify data inputs and review estimates. In 2009, an estimated 3.3 million babies died in the first month of life—compared with 4.6 million neonatal deaths in 1990—and more than half of all neonatal deaths occurred in five countries of the world (44% of global livebirths): India 27.8% (19.6% of global livebirths), Nigeria 7.2% (4.5%), Pakistan 6.9% (4.0%), China 6.4% (13.4%), and Democratic Republic of the Congo 4.6% (2.1%). Between 1990 and 2009, the global NMR declined by 28% from 33.2 deaths per 1,000 livebirths to 23.9. The proportion of child deaths that are in the neonatal period increased in all regions of the world, and globally is now 41%. While NMRs were halved in some regions of the world, Africa''s NMR only dropped 17.6% (43.6 to 35.9).

Conclusions

Neonatal mortality has declined in all world regions. Progress has been slowest in the regions with high NMRs. Global health programs need to address neonatal deaths more effectively if Millennium Development Goal 4 (two-thirds reduction in child mortality) is to be achieved. Please see later in the article for the Editors'' Summary     

Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.