Proteomics approach to identifying ATP-covalently modified proteins

This study aims to investigate functionally similar proteins based on their capacity to remain bound to ATP under stringent resolving conditions. Using two-dimensional gel electrophoresis and capillary liquid chromatography on-line mass spectrometry, we have identified several mammalian and E. coli proteins that appear to covalently bind ATP. To validate this approach, we obtained commercially purified forms of proteins identified from two-dimensional protein maps and tested their capacity to bind alpha 32P phosphate labeled ATP. This proteomics approach provides an initial screening method of identifying functionally similar proteins for further scrutiny by a more traditional analysis.     

Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-gamma secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn's disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4(+) and CD8(+) lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-gamma production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.     

Kinetic nonoptimality and vibrational stability of proteins

Scaling of folding times in Go models of proteins and of decoy structures with the Lennard-Jones potentials in the native contacts reveal power law trends when studied under optimal folding conditions. The power law exponent depends on the type of native geometry. Its value indicates lack of kinetic optimality in the model proteins. In proteins, mechanical and thermodynamic stabilities are correlated.     

Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization,degradation, and mRNA destabilization

Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.     

Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam)

Aedes aegypti, the main vector of dengue viruses in Asia, displays variation in population density over time. The larval habitats of this species being unevenly distributed and transient (depending on cycles of drought and flood), the forces generating temporal variation in gene frequencies in populations are studied. We sampled seven mosquito populations from Ho Chi Minh City (Vietnam) and its suburbs on five occasions between April 1999 and August 2000. We investigated genetic variation by studying isoenzyme and microsatellite polymorphism and susceptibility to a dengue 2 virus strain. Ae. aegypti populations collected during the dry season (January-April) showed genetic differentiation (F(ST) = 0.016, P < 10(-6) for isoenzymes) and showed more differentiated infection rates of the dengue 2 virus. The genetic structure of the population is less marked during the rainy season (F(ST) = 0.081, P < 10(-6)). Thus, environmental factors, such as rainfall and factors related to human activity, such as breeding site density and insecticide treatment, control the genetic structure of Ae. aegypti populations in the short term. The implications of studies of this kind for the design of future control programmes are discussed.     

Recombinant equine cytochrome c in Escherichia coli: high-level expression,characterization, and folding and assembly mutants

To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to the ribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variants tailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform.     

IL-4 Haplotype -590T, -34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background

Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T_H1 and T_H2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases.

Methods

We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese).

Results

The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ² ₃ = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%).

Conclusions

Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.     

The strength of phenotypic selection in natural populations

How strong is phenotypic selection on quantitative traits in the wild? We reviewed the literature from 1984 through 1997 for studies that estimated the strength of linear and quadratic selection in terms of standardized selection gradients or differentials on natural variation in quantitative traits for field populations. We tabulated 63 published studies of 62 species that reported over 2,500 estimates of linear or quadratic selection. More than 80% of the estimates were for morphological traits; there is very little data for behavioral or physiological traits. Most published selection studies were unreplicated and had sample sizes below 135 individuals, resulting in low statistical power to detect selection of the magnitude typically reported for natural populations. The absolute values of linear selection gradients |beta| were exponentially distributed with an overall median of 0.16, suggesting that strong directional selection was uncommon. The values of |beta| for selection on morphological and on life-history/phenological traits were significantly different: on average, selection on morphology was stronger than selection on phenology/life history. Similarly, the values of |beta| for selection via aspects of survival, fecundity, and mating success were significantly different: on average, selection on mating success was stronger than on survival. Comparisons of estimated linear selection gradients and differentials suggest that indirect components of phenotypic selection were usually modest relative to direct components. The absolute values of quadratic selection gradients |gamma| were exponentially distributed with an overall median of only 0.10, suggesting that quadratic selection is typically quite weak. The distribution of gamma values was symmetric about 0, providing no evidence that stabilizing selection is stronger or more common than disruptive selection in nature.