Estimates of DNA and protein sequence divergence: an examination of some assumptions

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.      

Recovery of poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) from Ralstonia eutropha cultures with non‐halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA‐based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate‐co‐hydroxyhexanoate) (P(HB‐co‐HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non‐halogenated solvents. Several non‐halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel‐like phase. All PHA in solution could be precipitated by addition of threefold volumes of n‐hexane or n‐heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non‐halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.     

Secondary Structural Changes in Globular Protein Induced by a Surfactant: Fourier Self-Deconvolution of FT-IR Spectra

Abstract

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C=O bond shifts from 1639 cm^?1 to 1652 cm^?1. On the other hand, the position of the Amide II band remains fairly unchanged.

Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alters the β sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm^?1 indicates generation of some β turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders.

The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.     

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.     

Oncogenic Pathway Combinations Predict Clinical Prognosis in Gastric Cancer

Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-κB, and Wnt/β-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms.     

Amyloid beta peptide (Abeta42) activates PLC-delta1 promoter through the NF-kappaB binding site

The abnormal deposition of amyloid beta peptide (Abeta) is a hallmark of Alzheimer's disease (AD). Phospholipase C-delta1 (PLC-delta1) is also known to abnormally accumulate in the brains of AD patients, but no report has addressed the relationship between these two events. This study investigated the effect of Abeta42 on the PLC-delta1 expression in human neuroblastoma cell lines. The PLC-delta1 mRNA level was increased by treatment with Abeta42 in a RT-PCR analysis. In the reporter assay, Abeta42 was found to activate the PLC-delta1 promoter activity in a dose-dependent manner. A novel NF-kappaB binding site in the PLC-delta1 promoter appeared to be responsible for the Abeta42 activity. First, the dominant negative forms of the NF-kappaB activating molecules, dominant negative TGF-beta activated kinase 1 (dnTAK1) and dnNIK (dominant negative NF-kappaB-inducing kinase), abolished the Abeta42 activity in the reporter assay. Second, the Abeta42 augmented a factor binding on the NF-kappaB site in the electrophoretic mobility shift assay (EMSA), which was abolished by a molar excess of the unlabeled consensus NF-kappaB oligonucleotide. These results suggest that the PLC-delta1 promoter is under the control of NF-kappaB, which mediates the expression of PLC-delta due to the Abeta42 treatment.     

Requirement for the p75 TNF-alpha receptor 2 in the regulation of airway hyperresponsiveness by gamma delta T cells

In a recent study, we found that TNF-alpha negatively regulates airway responsiveness through the activation of gammadelta T cells. The biological activities of TNF-alpha are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75-deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of gammadelta T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of gammadelta T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-alpha uses the p75 pathway exclusively, the development of AHR is regulated by gammadelta T cells.