Identification of residues involved in binding of IL5 to betacom using betaIL3 and betacom chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, betacom and betaIL3. betacom is used by the IL3, IL5 and GM-CSF receptors whereas betaIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta1L3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the betaIL3 B'-C' loop were replaced with betacom sequence a form of betaIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to betaIL3 in the absence of alpha chain. It is therefore an important structural motif of betacom and betaIL3 responsible for both ligand interaction and specificity.     

Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells

The effect of ATP-induced Ca2+ oscillations on ciliary activity was examined in airway epithelial cells by simultaneously measuring the ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) near the base of the cilia. Exposure to extracellular ATP (ATPo) induces a rapid and large increase in both [Ca2+]i and CBF, followed by oscillations in [Ca2+]i and a sustained elevation in CBF. After each Ca2+ oscillation, the [Ca2+]i returned to near basal values. By contrast, the CBF remained elevated during these Ca2+ oscillations, although each Ca2+ oscillation induced small variations in CBF. During Ca2+ oscillations, increases in CBF closely followed the rising phase of increases in [Ca2+]i, but declines in CBF lagged behind declines in [Ca2+]i. Higher frequency Ca2+ oscillations reduced variations in CBF, producing a stable and sustained elevation in CBF. The maximal CBF was induced by Ca2+ oscillations and was 15% greater than the CBF induced by the substantially larger initial [Ca2+]i increase. These data demonstrate that the rate of CBF is not directly dependent on the absolute [Ca2+]i, but is dependent on the differential changes in [Ca2+]i and suggest that CBF in airway epithelial cells is regulated by frequency-modulated Ca2+ signaling.     

Cutting edge: CD40-induced expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T cells in the periphery

It has been speculated that autoimmune diseases are caused by failure of central tolerance. However, this remains controversial. We have suggested that CD40 expression identifies autoaggressive T cells in the periphery of autoimmune prone mice. In this study, we report that CD40 was cloned from autoaggressive T cells and that engagement induces expression and nuclear translocation of the recombinases, recombination activating gene (RAG) 1 and RAG2 in the autoaggressive, but not in the nonautoaggressive, peripheral T cell population. Furthermore, we demonstrate that CD40 engagement induces altered TCR Valpha, but not Vbeta, expression in these cells. Therefore, CD40-regulated expression of RAG1 and RAG2 in peripheral T cells may constitute a novel pathway for the generation of autoaggressive T cells.     

Molecular data from 27 proteins do not support a Precambrian origin of land plants

Heckman et al. (Science 293: 1129-1133) used sequences obtained from GenBank to infer divergence times in fungi and green plants. They estimated that the crown group of land plants originated in the Precambrian, at 703 ± 45 mya, a date much older than dates implied by the fossils, which are no older than about 450 mya. This paper presents an analysis of an entirely different set of sequence data from 27 plastid protein-coding genes in 10 land plants and a green algal outgroup. It uses a calibration point closer to the origin of land plants and inference methods that do not assume a molecular clock. This leads to estimates ranging from 425 to 490 mya, which brackets the age suggested by the fossil record. Possible explanations for the differing conclusions in the two studies include differences in calibration points and use of single-copy plastid genes rather than nuclear gene families.     

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.     

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.     

Assessment of the accuracy of matrix representation with parsimony analysis supertree construction

Despite the growing popularity of supertree construction for combining phylogenetic information to produce more inclusive phylogenies, large-scale performance testing of this method has not been done. Through simulation, we tested the accuracy of the most widely used supertree method, matrix representation with parsimony analysis (MRP), with respect to a (maximum parsimony) total evidence solution and a known model tree. When source trees overlap completely, MRP provided a reasonable approximation of the total evidence tree; agreement was usually > 85%. Performance improved slightly when using smaller, more numerous, or more congruent source trees, and especially when elements were weighted in proportion to the bootstrap frequencies of the nodes they represented on each source tree ("weighted MRP"). Although total evidence always estimated the model tree slightly better than nonweighted MRP methods, weighted MRP in turn usually out-performed total evidence slightly. When source studies were even moderately nonoverlapping (i.e., sharing only three-quarters of the taxa), the high proportion of missing data caused a loss in resolution that severely degraded the performance for all methods, including total evidence. In such cases, even combining more trees, which had positive effects elsewhere, did not improve accuracy. Instead, "seeding" the supertree or total evidence analyses with a single largely complete study improved performance substantially. This finding could be an important strategy for any studies that seek to combine phylogenetic information. Overall, our results suggest that MRP supertree construction provides a reasonable approximation of a total evidence solution and that weighted MRP should be used whenever possible.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.