Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

The potent colon carcinogen, 1,2-dimethylhydrazine induces mutations primarily in the colon

1,2-Dimethylhydrazine (DMH) is a potent colon carcinogen that is commonly used as an initiator in studies of the effects of diet on colon cancer. Previous studies have shown that although this compound produces multiple tumors in the colons in most individuals of every species tested, it is, at best, marginally mutagenic in the bone marrow (micronuclei) and small intestine (Dlb-1 mutations). Here we report its mutagenicity in the primary target tissue, the colonic epithelium, by means of the Mutatrade markMouse cII assay, an assay for intragenic mutations in a lambda shuttle vector that is integrated into the genome of these mice. Animals were treated with 0, 10, 20, or 30 mg/ml of DMH, either as a single injection or as multiple weekly injections, and mutations were measured in both the small intestine and colon. In the small intestine, there was an increase in mutant frequency following a single injection of DMH, but this was significant only at 30 mg/kg [induced mutant frequency (MF) = 18 x 10(-5) mutants/plaque]. In the colon, following a single treatment of DMH, there was a significant increase in mutant frequency at doses of 20 and 30 mg/kg (induced MF = 17 x 10(-5) and 23 x 10(-5) mutants/plaque, respectively). Following ten injections of 20 mg/kg of DMH, there was a greater than ten-fold increase in mutations in the colon (MF = 275 x 10(-5) mutants/plaque) than the small intestine (MF = 25 x 10(-5) mutants/plaque). These results show that DMH, under the conditions typically used for dietary studies, induces large numbers of mutations in the tissue in which it induces most cancers.     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Children who acquire HIV infection perinatally are at higher risk of early death than those acquiring infection through breastmilk: a meta-analysis

BACKGROUND: Assumptions about survival of HIV-infected children in Africa without antiretroviral therapy need to be updated to inform ongoing UNAIDS modelling of paediatric HIV epidemics among children. Improved estimates of infant survival by timing of HIV-infection (perinatally or postnatally) are thus needed. METHODOLOGY/PRINCIPAL FINDINGS: A pooled analysis was conducted of individual data of all available intervention cohorts and randomized trials on prevention of HIV mother-to-child transmission in Africa. Studies were right-censored at the time of infant antiretroviral initiation. Overall mortality rate per 1000 child-years of follow-up was calculated by selected maternal and infant characteristics. The Kaplan-Meier method was used to estimate survival curves by child's HIV infection status and timing of HIV infection. Individual data from 12 studies were pooled, with 12,112 children of HIV-infected women. Mortality rates per 1,000 child-years follow-up were 39.3 and 381.6 for HIV-uninfected and infected children respectively. One year after acquisition of HIV infection, an estimated 26% postnatally and 52% perinatally infected children would have died; and 4% uninfected children by age 1 year. Mortality was independently associated with maternal death (adjusted hazard ratio 2.2, 95%CI 1.6-3.0), maternal CD4<350 cells/ml (1.4, 1.1-1.7), postnatal (3.1, 2.1-4.1) or peri-partum HIV-infection (12.4, 10.1-15.3). CONCLUSIONS/RESULTS: These results update previous work and inform future UNAIDS modelling by providing survival estimates for HIV-infected untreated African children by timing of infection. We highlight the urgent need for the prevention of peri-partum and postnatal transmission and timely assessment of HIV infection in infants to initiate antiretroviral care and support for HIV-infected children.     

Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA-protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells

Abstract In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His→Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.     

Strategies and Decision-Support Tools for Optimizing Long-Term Groundwater Monitoring Plans—MAROS 2.0

Existing long-term groundwater monitoring programs can be optimized to increase their effectiveness/efficiency with the potential to generate considerable cost savings. The optimization can be achieved through an overall evaluation of conditions of the contaminant plume and the monitoring network, focused spatial and temporal sampling analyses, and automated and efficient management of data, analyses, and reporting. Version 2.0 of the Monitoring and Remediation Optimization System (MAROS) software, by integrating long-term monitoring analysis strategies and innovative optimization methods with a data management, processing, and reporting system, allows site managers to quickly and readily develop cost-effective long-term groundwater monitoring plans. The MAROS optimization strategy consists of a hierarchical combination of analysis methods essential to the decision-making process. Analyses are performed in three phases: 1) evaluating site information and historical monitoring data to obtain local concentration trends and an overview of the plume status; 2) developing optimal sampling plans for future monitoring at the site with innovative optimization methods; and 3) assessing the statistical sufficiency of the sampling plans to provide insights into the future performance of the monitoring program. Two case studies are presented to demonstrate the usefulness of the developed techniques and the rigor of the software.