Regulation of apoptotic effects by erythrocarpine E, a cytotoxic limonoid from Chisocheton erythrocarpus in HSC-4 human oral cancer cells

The aim of this study was to determine the cytotoxic and apoptotic effects of erythrocarpine E (CEB4), a limonoid extracted from Chisocheton erythrocarpus on human oral squamous cell carcinoma. Based on preliminary dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, CEB4 treated HSC-4 cells demonstrated a cytotoxic effect and inhibited cell proliferation in a time and dose dependent manner with an IC(50) value of 4.0±1.9 μM within 24 h of treatment. CEB4 was also found to have minimal cytotoxic effects on the normal cell line, NHBE with cell viability levels maintained above 80% upon treatment. Annexin V-fluorescein isothiocyanate (FITC), poly-ADP ribose polymerase (PARP) cleavage and DNA fragmentation assay results showed that CEB4 induces apoptosis mediated cell death. Western blotting results demonstrated that the induction of apoptosis by CEB4 appeared to be mediated through regulation of the p53 signalling pathway as there was an increase in p53 phosphorylation levels. CEB4 was also found to up-regulate the pro-apoptotic protein, Bax, while down-regulating the anti-apoptotic protein, Bcl-2, suggesting the involvement of the intrinsic mitochondrial pathway. Reduced levels of initiator procaspase-9 and executioner caspase-3 zymogen were also observed following CEB4 exposure, hence indicating the involvement of cytochrome c mediated apoptosis. These results demonstrate the cytotoxic and apoptotic ability of erythrocarpine E, and suggest its potential development as a cancer chemopreventive agent.     

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H⁺/g, 1.4 mmol H⁺/g, and 17 mmol H⁺/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ?7.2 mg bovine serum albumin/g, ?7.4 hen egg‐white lysozyme (HEWL) mg/g, and ?108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

IL-1beta and TNF-alpha induce increased expression of CCL28 by airway epithelial cells via an NFkappaB-dependent pathway

CCL28 is a mucosal chemokine that attracts eosinophils and T cells via the receptors CCR3 and CCR10. Consequently, it is a candidate mediator of the pathology associated with asthma. This study examined constitutive and induced expression of CCL28 by A549 human airway epithelial-like cells. Real-time RT-PCR and ELISA of cultured cells and supernatants revealed constitutive levels of CCL28 expression to be low, whereas IL-1beta and TNF-alpha, induced significantly increased expression. Observations from induced sputum and human airway biopsies supported this. Signal transduction studies revealed that IL-1beta and TNF-alpha stimulation induced NFkappaB phosphorylation in A549 cells, but antagonist inhibition of NFkappaB p50-p65 phosphorylation correlated with marked reduction of IL-1beta or TNF-alpha induced CCL28 expression. Together these studies imply a role for CCL28 in the orchestration of airway inflammation, and suggest that CCL28 is one link between microbial insult and the exacerbation of pathologies such as asthma, through an NFkappaB-dependent mechanism.     

Patterns of evolution of genes disrupted in expression in Drosophila species hybrids

Divergence between species in regulatory pathways may contribute to hybrid incompatibilities such as sterility. Consistent with this idea, genes involved in male fertility often evolve faster than most other genes both in amino acid sequence and in expression. Previously, we identified a panel of male-specific genes under-expressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to pure species, and we showed that this under-expression is associated with infertility. In a preliminary effort to assess the generalities in the patterns of evolution of these genes, I examined patterns of mRNA expression in three of these genes in sterile F 1 hybrid males of D. pseudoobscura and D. persimilis . F 1 hybrid males bearing D. persimilis X chromosomes under-expressed all these genes relative to the parental species, while hybrids bearing D. pseudoobscura X chromosomes under-expressed two of these three genes. Interestingly, the third gene, CG5762 , has undergone extensive amino acid evolution within the D. pseudoobscura species group, possibly driven by positive natural selection. We conclude that some of the same genes exhibit disruptions in expression within each of the two species groups, which could suggest commonalities in the regulatory architecture of sterility in these groups. Alternative explanations are also considered.     

Using comparative genomic data to test for fast-X evolution

Genes may acquire nonsynonymous substitutions more rapidly when X-linked than when autosomal, but evidence for "fast-X evolution" has been elusive. Fast-X evolution could explain the disproportionate contribution of X-linked genes to hybrid sterility and other traits. Here, we use a comparative genomic approach, with sequences of 30-110 genes in four Drosophila species, to test for fast-X evolution. Specifically, the 3L autosome arm in D. melanogaster and D. simulans is homologous to the right arm of the X chromosome in D. pseudoobscura and D. miranda. We executed two paired comparisons to determine how often genes on this chromosome arm exhibit higher rates of nonsynonymous substitution in the D. pseudoobscura species group, as predicted by fast-X evolution. We found a statistically significant pattern consistent with fast-X evolution in one comparison and a similar trend in the other comparison. Variation in functional constraints across genes may have masked the signature of fast-X evolution in some previous studies, and we conclude paired comparisons are more powerful for examining rates of evolution of genes when X-linked over autosomal.     

Variation in recombination rate may bias human genetic disease mapping studies

The availability of the human genome sequence and variability information (as from the International HapMap project) will enhance our ability to map genetic disorders and choose targets for therapeutic intervention. However, several factors, such as regional variation in recombination rate, can bias conclusions from genetic mapping studies. Here, we examine the impact of regional variation in recombination rate across the human genome. Through computer simulations and literature surveys, we conclude that genetic disorders have been mapped to regions of low recombination more often than expected if such diseases were randomly distributed across the genome. This concentration in low recombination regions may be an artifact, and disorders appearing to be caused by a few genes of large effect may be polygenic. Future genetic mapping studies should be conscious of this potential complication by noting the regional recombination rate of regions implicated in diseases.     

Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease

Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the central region of the retina. AMD is clinically heterogeneous, leading to geographic atrophy (GA) and/or choroidal neovascularization (CNV) at advanced stages. Considerable data exists in support of a genetic predisposition for AMD. Recent linkage studies have provided evidence in favor of several AMD susceptibility loci. We have performed a high-resolution (5-cM) genome scan of 412 affected relative pairs that were enriched for late-stage disease (GA and/or CNV). Nonparametric linkage analysis was performed using two different diagnostic criteria and also by dividing the affected individuals according to GA or CNV phenotype. Our results demonstrate evidence of linkage in regions that were suggested in at least one previous study at chromosomes 1q (236-240 cM in the Marshfield genetic map), 5p (40-50 cM), and 9q (111 cM). Multipoint analysis of affected relatives with CNV provided evidence of additional susceptibility loci on chromosomes 2p (10 cM) and 22q (25 cM). A recently identified Gln5345Arg change in HEMICENTIN-1 on chromosome 1q25 was not detected in 274 affected members in the restricted group with AMD, 346 additional patients with AMD, and 237 unaffected controls. Our results consolidate the chromosomal locations of several AMD susceptibility loci and, together with previous reports, should facilitate the search for disease-associated sequence variants.     

Nutritional factors affecting organic solvent-tolerant alkaline protease production by a newBacillus cereus strain 146

An organic solvent-tolerant bacterium designated as 146 capable of producing an organic solvent-stable alkaline protease was isolated from contaminated soil of a wood factory. The strain was a Gram-positive, spore-forming, nitrate-positive, rod-shaped organism capable of hydrolysing gelatine, starch, skim milk and identified asBacillus cereus. Activity of the protease was drastically increased in the presence of 1–decanol, isooctane, n-dodecane and n-tetradecane, but reduced in the presence of ethyl acetate, benzene, toluene, 1-heptanol, ethylbenzene and hexane. The bacterium was shown to require lactose as a carbon source and peptone as a nitrogen source. The optimum fermentation condition for the production of alkaline protease was in the presence of beef and yeast extract. Optimum pH was determined to be at 10.0 at incubation temperature of 37 °C for 48 h. Results from the studies suggest that 146 is a new strain of Bacillus cereus capable of producing organic solvent-tolerant alkaline protease with potential use in industries.     

Biodegradation of hydrocarbon contamination by immobilized bacterial cells

This study examined the capacity of immobilized bacteria to degrade petroleum hydrocarbons. A mixture of hydrocarbon-degrading bacterial strains was immobilized in alginate and incubated in crude oil-contaminated artificial seawater (ASW). Analysis of hydrocarbon residues following a 30-day incubation period demonstrated that the biodegradation capacity of the microorganisms was not compromised by the immobilization. Removal of n-alkanes was similar in immobilized cells and control cells. To test reusability, the immobilized bacteria were incubated for sequential increments of 30 days. No decline in biodegradation capacity of the immobilized consortium of bacterial cells was noted over its repeated use. We conclude that immobilized hydrocarbon-degrading bacteria represent a promising application in the bioremediation of hydrocarbon-contaminated areas.