Mouse ephrinB3 augments T-cell signaling and responses to T-cell receptor ligation

Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-gamma but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solid-phase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function.     

Interacting genetic loci on chromosomes 20 and 10 influence extreme human obesity

Obesity is a multigenic trait that has a substantial genetic component. Animal models confirm a role for gene-gene interactions, and human studies suggest that as much as one-third of the heritable variance may be due to nonadditive gene effects. To evaluate potential epistatic interactions among five regions, on chromosomes 7, 10, and 20, that have previously been linked to obesity phenotypes, we conducted pairwise correlation analyses based on alleles shared identical by descent (IBD) for independent obese affected sibling pairs (ASPs), and we determined family-specific nonparametric linkage (NPL) scores in 244 families. The correlation analyses were also conducted separately, by race, through use of race-specific allele frequencies. Conditional analyses for a qualitative trait (body mass index [BMI] >/=27) and hierarchical models for quantitative traits were used to further refine evidence of gene interaction. Both the ASP-specific IBD-sharing probability and the family-specific NPL score revealed that there were strong positive correlations between 10q (88-97 cM) and 20q (65-83 cM), through single-point and multipoint analyses with three obesity thresholds (BMI >/=27, >/=30, and >/=35) across African American and European American samples. Conditional analyses for BMI >/=27 found that the LOD score at 20q rises from 1.53 in the baseline analysis to 2.80 (empirical P=.012) when families were weighted by evidence for linkage at 10q (D10S1646) through use of zero-one weights (weight(0-1)) and to 3.32 (empirical P<.001) when proportional weights (weight(prop)) were used. For percentage fat mass, variance-component analysis based on a two-locus epistatic model yielded significant evidence for interaction between 20q (75 cM) and the chromosome 10 centromere (LOD = 1.74; P=.024), compared with a two-locus additive model (LOD = 0.90). The results from multiple methods and correlated phenotypes are consistent in suggesting that epistatic interactions between loci in these regions play a role in extreme human obesity.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.     

Effect of circular motion exercise on bone modeling and bone mass in young rats: an animal model of isometric exercise

The aims of the study are to develop a non-invasive animal model of circular motion exercise and to evaluate the effect of this type of exercise on bone turnover in young rats. The circular motion exercise simulates isometric exercise using an orbital shaker that oscillates at a frequency of 50 Hz and is capable of speeds from 0-400 rpm. A cage is fixed on top of the shaker and the animals are placed inside. When the shaker is turned on, the oscillatory movement should encourage the animals to hold on to the cage and use various muscle forces to stabilize themselves. Rats at 8 weeks of age were trained on the shaker for 6 weeks and static and dynamic histomorphometric analyses were performed for the proximal tibial metaphysis and the tibial shaft. The exercise resulted in no significant effect on animal body weight, gastrocnemius muscle weight and femoral weight. Although the bone formation rate of cancellous and cortical periosteum was increased by the exercise, trabecular bone volume was decreased. The exercise increased periosteal and marrow perimeters and the cross-sectional diameter of cortical bone from medial to lateral without a significant increase in the cortical bone area. These results suggest that circular motion exercise under force without movement or additional weight loading will cause bone-modeling drift with an increase in bone turnover to reconstruct bone shape in adaptation to the demand in strength. Since there is no additional weight loading during circular motion exercise, the net mass of bone is not increased. The bone mass lost in trabecular bone could possibly be due to a re-distribution of mineral to the cortical bone.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

兔左室壁三层心肌细胞的分离及动作电位、钙和钾电流分布的异质性

探讨兔左室壁三层心肌单个细胞的分离方法以及电生理特征,实验以胶原酶按二步消化法分离兔心肌细胞,其中用剃须刀分离左室游离壁内,中,外三层心肌,采用全细胞膜片钳记录AP和离子电流,结果显示：（1）中层细胞上的动作电位时程明显长于内膜下心肌和外膜下心肌,且存在显著的1相切迹和2相驼峰;（2）中层细胞的Ica,L和Lto较内,外膜下的大,IK,s相反,可见三层心肌细胞上多种电流存在显著差异。     

SNPkit: an efficient approach to systematic evaluation of candidate single nucleotide polymorphisms in public databases

There is widespread interest in devising genotyping methods for SNPs that are robust, inexpensive, and simple to perform. Although several high-throughput SNP genotyping technologies have been developed, including the oligonucleotide ligation assay, real-time PCR, and mass spectrometry, the issues of simplicity and cost-effectiveness have not been adequately addressed. Here we describe the application of a novel computer software package, SNPkit, which designs SNP genotyping assays based on a classical approach for discriminating alleles, restriction enzyme digestion. SNPkit can be used in genotyping assays for almost any SNPs including those that do not alter "natural" restriction sites. Using this method, 164 SNPs have been evaluated in DNA samples from 48 immortalized cell lines of randomly selected Chinese subjects. Sixty-two (37.8%) of the SNPs appeared to be common (frequencies of the minor alleles are > or = 5%) and were subsequently applied to a larger population-based sample. Overall, by using SNPkit, we have been able to validate and genotype accurately a large fraction of publicly available SNPs without sophisticated instrumentation.     

Identification of essential residues in the type II Hsp40 Sis1 that function in polypeptide binding

Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins.