Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.     

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.     

Effects of nanomolar concentration dihydroouabain on calcium current and intracellular calcium in guinea pig ventricular myocytes

The effects of nanomolar concentration of dihydroouabain (DHO) on L-type calcium current (ICa-L), TTX-sensitive calcium current (ICa(TTX)), and intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes. The whole-cell patch-clamp technique was used to record ICa-L and ICa(TTX); [Ca2+]i was detected and recorded with the confocal microscopy. The nanomolar concentration of DHO increased the ICa-L, ICa(TTX), and [Ca2+]i, which could be partially inhibited by nisoldipine or TTX, but still appeared in the absence of extracellular K+ and Na+. These data suggest that DHO could increase [Ca2+]i in non-beating myocytes via stimulating the ICa-L and ICa(TTX), or perhaps triggering directly a release of intracellular calcium.     

Lack of increased genetic damage in 1,3-butadiene-exposed Chinese workers studied in relation to EPHX1 and GST genotypes

1,3-Butadiene (BD) is an important industrial chemical and pollutant. Its ability to induce genetic damage and cause hematological malignancies in humans is controversial. We have examined chromosome damage by fluorescence in situ hybridization (FISH) and mutations in the HPRT gene in the blood of Chinese workers exposed to BD. Peripheral blood samples were collected and cultured from 39 workers exposed to BD (median level 2 ppm, 6 h time-weighted average) and 38 matched controls in Yanshan, China. No difference in the level of aneuploidy or structural changes in chromosomes 1, 7, 8, and 12 was detected in metaphase cells from exposed subjects in comparison with matched controls, nor was there an increase in the frequency of HPRT mutations in the BD-exposed workers. Because genetic polymorphisms in glutathione S-transferase (GST) enzymes and microsomal epoxide hydrolase (EPHX1) may affect the genotoxic effects of BD and its metabolites, we also related chromosome alterations and gene mutations to GSTT1, GSTM1 and EPHX1 genotypes. Overall, there was no effect of variants in these genotypes on numerical or structural changes in chromosomes 1, 7, 8 and 12 or on HPRT mutant frequency in relation to BD exposure, but the GST genotypes did influence background levels of both hyperdiploidy and HPRT mutant frequency. In conclusion, our data show no increase in chromosomal aberrations or HPRT mutations among workers exposed to BD, even in potentially susceptible genetic subgroups. The study is, however, quite small and the levels of BD exposure are not extremely high, but our findings in China do support those from a similar study conducted in the Czech Republic. Together, these studies suggest that low levels of occupational BD exposure do not pose a significant risk of genetic damage.     

Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus.

A cDNA clone was isolated after difference screening from cotyledons of two-week cold-treated Ammopiptanthus mongolicus. The full-length cDNA sequence [designated as AmCIP (A. mongolicus cold-induced protein) gene] was 806 bp long and contained a 465 bp open reading frame (ORF) encoding a 16.6 kD protein of 154 amino acids. Bioinformatic analyses indicated that CIP belongs to dehydrin family with the features of high hydrophilicity, a helix K-segment, a long Gly-rich region and a phosphorylatable tract of Ser as well as deficiency in Cys and Trp. The expression of CIP gene increased after two weeks of cold treatment and more expression was detected in radicle than in cotyledon. And PCR amplification of the AmCIP gene from genome of A. mongolicus revealed this gene has no intron. Function prediction suggested this protein seems to protect the stabilization of membrane structure and prevent macromolecular coagulation or sequestrate calcium ions by association or disassociation with membrane under low temperature conditions.     

鱼腥藻PCC7120藻蓝蛋白α亚基体内重组研究

为了研究藻蓝蛋白α亚基的生物合成途径,通过构建相容的3种重组质粒pETDuet-cpcA、pCOLADuet-cpcE-cpcF和pACYCDuet-ho1-pcyA,将裂合酶基因cpcE和cpcF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻蓝蛋白α亚基基因cpcA共同转入大肠杆菌BL21(DE3)。通过色素蛋白锌电泳和光谱检测表明产生了生物活性的CpcA-PCB。成功实现了大肠杆菌内藻蓝蛋白α亚基84位半胱氨酸残基与PCB的连接。而在裂合酶基因cpcE和cpcF不转入大肠杆菌的情况下,大肠杆菌内只有0.2%的CpcA-PCB产生。以上研究为进一步在大肠杆菌内合成天然的藻蓝蛋白奠定了基础。     

美洲黑杨次生维管组织发育的内在节律性与细胞内含物的动态变化

美洲黑杨(Populus deltoids)是一种重要的商业用材树种, 在我国多用作胶合板和纸浆原料, 其速生性的特点也很早为人们所认识。众所周知, 树木次生维管组织的发育具有内在的节律性, 与此同时维管组织的细胞内含物也会发生动态变化。利用电镜技术及细胞化学方法, 研究了美洲黑杨次生维管组织的发育以及细胞内含物分布的变化, 发现次生维管组织的发育具有内在节律性, 在年周期中表现为分化期和休眠期交替出现; 次生韧皮部与次生木质部交替分化形成。在维管组织发育过程中, 早期蛋白质和脂类物质首先减少, 然后淀粉粒解体消失, 发育期内维管组织的蛋白质、脂类和淀粉的积累最少, 休眠期内维管组织细胞内最早出现淀粉的积累, 然后蛋白质和脂类物质积累大大增加。杨树维管组织发育过程的内在节律性与细胞内含物的动态变化密切相关。     

高纯度大豆种子线粒体的分离

利用不连续Percoll梯度分离得到高纯度的大豆(Glycine max)种子线粒体。线粒体制剂中没有检测到胞液、过氧化物体和叶绿素的污染。线粒体的完整性达到97%以上, 在0~4 ℃下至少稳定2小时。