The human B cell response to IL-13 is dependent on cellular phenotype as well as mode of activation.

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Ralpha1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Ralpha1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Ralpha1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.     

Secondary structure model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis.

A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.     

SPRIT: Identifying horizontal gene transfer in rooted phylogenetic trees

Background  

Phylogenetic trees based on sequences from a set of taxa can be incongruent due to horizontal gene transfer (HGT). By identifying the HGT events, we can reconcile the gene trees and derive a taxon tree that adequately represents the species' evolutionary history. One HGT can be represented by a rooted Subtree Prune and Regraft (RSPR) operation and the number of RSPRs separating two trees corresponds to the minimum number of HGT events. Identifying the minimum number of RSPRs separating two trees is NP-hard, but the problem can be reduced to fixed parameter tractable. A number of heuristic and two exact approaches to identifying the minimum number of RSPRs have been proposed. This is the first implementation delivering an exact solution as well as the intermediate trees connecting the input trees.     

Detailed Assessment of the Kinetics of Hg-Cell Association,Hg Methylation,and Methylmercury Degradation in Several Desulfovibrio Species

The kinetics of inorganic Hg [Hg(II)_i] association, methylation, and methylmercury (MeHg) demethylation were examined for a group of Desulfovibrio species with and without MeHg production capability. We employed a detailed method for assessing MeHg production in cultures, including careful control of medium chemistry, cell density, and growth phase, plus mass balance of Hg(II)_i and MeHg during the assays. We tested the hypothesis that differences in Hg(II)_i sorption and/or uptake rates drive observed differences in methylation rates among Desulfovibrio species. Hg(II)_i associated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ∼3 h. All MeHg produced was rapidly exported. We also tested the idea that all Desulfovibrio species are capable of Hg(II)_i methylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in the Desulfovibrio is not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylating Desulfovibrio species even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive cross-species differences in Hg methylation rates. As part of this study, we identified four new Hg methylators (Desulfovibrio aespoeensis, D. alkalitolerans, D. psychrotolerans, and D. sulfodismutans) and four nonmethylating species (Desulfovibrio alcoholivorans, D. tunisiensis, D. carbinoliphilus, and D. piger) in our ongoing effort to generate a library of strains for Hg methylation genomics.     

The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension

Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca(2+) contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca(2+)-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca(2+) and a decrease in SR Ca(2+) release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca(2+)](i) possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes.     

miR390, Arabidopsis TAS3 tasiRNAs,and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating Lateral Root Growth

Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.     

Evidence for monomeric and dimeric forms of CD45 associated with a 30-kDa phosphorylated protein.

Glycoprotein CD45, a transmembrane protein tyrosine phosphatase of leukocytes, is topographically similar to the epidermal growth factor receptor, a transmembrane tyrosine kinase. Since the latter is thought to be allosterically regulated through conversion between monomeric and dimeric forms, we sought to determine whether CD45 undergoes similar oligomerization. Our analysis, employing a thiol-cleavable and homobifunctional chemical cross-linker, dithiobis succinimidyl propionate, revealed that CD45 indeed formed homodimers. In addition, a protein of molecular mass 30,000 daltons (30 kDa) was found to be associated with both the CD45 monomer and dimer. The 30-kDa protein was phosphorylated and was not labeled by cell surface radioiodination. Distinct differences in protein tyrosine phosphatase activity were detected among the various populations of CD45 separated by sucrose gradient ultracentrifugation. However, the differences observed could not be explained simply by dimerization and instead suggest the presence of other factor(s) involved in the regulation of CD45 enzyme activity.     

Estimation of the irreversible inhibition reaction rate constants and of the concentration of cholinesterase active sites under commensurable concentrations of enzyme and inhibitors]

Kinetic analysis of the interaction of butyrylcholinesterase and the phosphoorganic inhibitor GT-161 [(C2H2O)2P(O)SC2H4+N(CH3)2C6H5-1-] is carried out. Short time incubations of an enzyme and an inhibitor (1-3 sec), even under commensurable concentrations, were shown to enable the rate constants of irreversible enzyme inhibition to be calcualted by the formula for pseudomonomoleuclar reactions. A simple analytical method for the estimation of the enzyme active sites concentrations is proposed.