The Bw4/Bw6 difference between HLA-B*0802 and HLA-B*0801 changes the peptides endogenously bound and the stimulation of alloreactive T cells

 HLA-B^*0801 is unique among HLA-B allotypes in having dominant amino acid anchors at positions 3 and 5 of the peptide-binding motif. HLA-B^*0802 is a variant of HLA-B^*0801 in which the Bw6 sequence motif is replaced by a Bw4 sequence motif. This change, involving substitutions at positions 77, 80, 81, 82, and 83 of the B^*08 heavy chain, is probably the result of a single evolutionary event of interallelic conversion. Moreover, the difference between B^*0802 and B^*0801 is sufficient to stimulate a cytotoxic T-cell response. To assess further the functional impact of the Bw4 motif on a B8 background, we compared the peptide-binding specificity of the B^*0801 and B^*0802 allotypes by sequencing the mixture of peptides endogenously bound to B^*0802 and 12 individual peptides purified from that mixture. The HLA-B^*0802 allotype, while able to bind some peptides bound by B^*0801, has a broader repertoire of endogenously bound peptides than B^*0801: the peptides bound by B^*0802 are more variable in length and exhibit greater diversity in the carboxyl-terminal amino acid which interacts with the F pocket. Received: 29 October 1997     

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 mol/L aminoguanidine (I), 100 mol/L aminoguanidine and 100 mol/L N,N-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 mol/L aminoguanidine and 200 mol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity.The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (160%) and spermine (145%) and decreased the spermidine content (75%) without any modification of the total amount of polyamine.The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.     

Low activity of amine-oxidases and accumulation of conjugated polyamines in disfavour of organogenic programs in Chrysanthemum leaf disc explants

Foliar discs (8 mm diameter) from expanding leaves of the middle part of vegetative shoots of Chrysanthemum morifolium Ramat raised in vitro were induced to form directly on specific media in vitro either roots or vegetative buds, or callus. The budding programme, on its specific medium, was deviated to callus formation by the addition of 2 mM β-OH-E (β-OH-ethyldrazine, an inhibitor of diamine oxidase). Conversely vegetative buds instead of callus were formed on the callus medium in the presence of 2 mM DFMO (difluoromethylornithine, an inhibitor of ornithine decarboxylase). Callus formation was characterized by high accumulation of free and particularly conjugated polyamines (PA), very low or undetectable activities of diamine- and polyamine oxidases, and transglutaminase. DFMO-deviation of callus initiation in favour of bud formation lowered the accumulation of PA and increased the activity of amine-oxidases. The high catabolism of PA in the organogenic (rooting, budding) programs was questioned as to its role in developmental processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe₃O₄@nSiO₂ Core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂). The Fe₃O₄@nSiO₂@mSiO₂ microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high‐surface areas of the microspheres were demonstrated to have good size‐exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re‐used Fe₃O₄@nSiO₂@mSiO₂ microspheres was also studied. Large‐scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano‐LC‐ESI‐MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel Fe₃O₄@nSiO₂@mSiO₂ microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.     

Chromatin reorganization and endogenous auxin/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay. Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole totipotency process of dedifferentiation and redifferentiation. We identified two phases of chromatin decondensation during the dedifferentiation and redifferentiation. At the same time, sharp increase in the ratio of indoleacetic acid (IAA), isopentenyladenosine group (iPAs) at the same stage of cell dedifferentiation and redifferentiation process serve as distinct biochemical maker of dedifferentiation and SE initiation with the unique feature. Our results suggest the two phases of chromatin reorganization associated with endogenous auxin/cytokinin dynamic activity may underlie dedifferentiation and redifferentiation during the entire SE process in cotton.     

Self-excreted mediator from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 for electron transfer to carbon electrodes

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (−0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

‘砀山酥梨’及其褐皮芽变果皮中多胺类物质含量和相关基因表达分析

为研究多胺类物质在‘砀山酥梨’芽变品系‘锈酥’果皮褐色形成中的作用,以‘砀山酥梨’和‘锈酥’花后不同时期果皮为试材,采用HPLC方法测定果皮中多胺类物质的含量,利用RACE技术克隆多胺合成过程中的关键基因,通过Protparam网站与MEGA5.0软件分析相关基因蛋白的理化性质及其系统进化,用荧光定量qRT-PCR方法分析不同时期果皮中相关基因的相对表达量。结果表明:(1)除花后50和150d外,其它时期‘砀山酥梨’果皮中腐胺含量均显著高于‘锈酥’,花后50d、75d、150d时,‘锈酥’果皮中的亚精胺、精胺含量显著高于‘砀山酥梨’果皮。(2)克隆出多胺合成的基因ADC、SPDS和SPMS(GenBank登录号分别为KM923903、KM923905和KM923906),预测ADC和SPMS均为水溶性蛋白、SPDS为疏水性蛋白,它们与苹果的亲缘关系最近。(3)荧光定量PCR结果显示,花后50d,多胺合成中ADC、SPDS和SPMS在‘锈酥’果皮中表达量均显著高于‘砀山酥梨’。研究推测,‘锈酥’果皮中多胺代谢及相关基因的上调表达可能与‘锈酥’的果皮褐色形成有关。