High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

Factors associated with elevated ALT in an international HIV/HBV co-infected cohort on long-term HAART

Background

Previous studies have demonstrated that hepatitis B virus (HBV) infection increases the risk for ALT elevations in HIV-HBV co-infected patients during the first year of HAART; however, there is limited data on the prevalence of ALT elevations with prolonged HAART in this patient group.

Methods/Principal findings

To identify factors associated with ALT elevations in an HIV-HBV co-infected cohort receiving prolonged HAART, data from 143 co-infected patients on HAART enrolled in an international HIV-HBV co-infected cohort where ALT measurements were obtained every 6 months was analysed. A person-visit analysis was used to determine frequency of ALT elevation (≥2.5×ULN) at each visit. Factors associated with ALT elevation were determined using multivariate logistic regression with generalized estimating equations to account for correlated data. The median time on HAART at the end of follow-up was 5.6 years (range 0.4–13.3) years. During follow-up, median ALT was 36 U/L with 10.6% of person-visits classified as having ALT elevation. Most ALT elevations were grade 2 (86.5%), with only 13.5% of all ALT elevations grade 3 or higher. Univariate associations with ALT elevation (p<0.05) included history of AIDS, HBV DNA ≥2,000 IU/ml, HBeAg positive, study visit CD4 <200 cells/ml and nadir CD4 <200 cells/ml. In the multivariate analysis, only study visit CD4 <200 cells/ml (OR 2.07, 95%CI 1.04–4.11, p = 0.04) and HBeAg positive status (OR 2.22, 95%CI 1.03–4.79, p = 0.04) were independently associated with ALT elevation.

Conclusions

In this HIV-HBV co-infected cohort, elevated ALT after >1 year of HAART was uncommon, and severe ALT elevations were rare. HIV-HBV co-infected patients on long-term HAART who are either HBeAg positive or have a CD4 count of <200 cells/ml are at increased risk for ALT elevations.     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Evolution of extraordinary female-biased sex ratios: the optimal schedule of sex ratio in local mate competition

Female-biased sex ratio in local mate competition has been well studied both theoretically and experimentally. However, some experimental data show more female-biased sex ratios than the theoretical predictions by Hamilton [1967. Science 156, 477-488] and its descendants. Here we consider the following two effects: (1) lethal male-male combat and (2) time-dependent control (or schedule) of sex ratio. The former is denoted by a male mortality being an increasing function of the number of males. The optimal schedule is analytically obtained as an evolutionarily stable strategy (ESS) by using Pontrjagin's maximum principle. As a result, an ESS is a schedule where only males are produced first, then the proportion of females are gradually increased, and finally only females are produced. Total sex ratio (sex ratio averaged over the whole reproduction period) is more female-biased than the Hamilton's result if and only if the two effects work together. The bias is stronger when lethal male combat is severer or a reproduction period is longer. When male-male combat is very severe, the sex ratio can be extraordinary female-biased (less than 5%). The model assumptions and the results generally agree with experimental data on Melittobia wasps in which extraordinary female-biased sex ratio is observed. Our study might provide a new basis for the evolution of female-biased sex ratios in local mate competition.     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Flagellin redundancy in Caulobacter crescentus and its implications for flagellar filament assembly

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.