Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion

There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4-7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.     

Rat mature sympathetic neurones derive neurotrophin 3 from peripheral effector tissues

In a previous study we have demonstrated that endogenous neurotrophin 3 (NT3) is required for the survival of most sympathetic neurones in postnatal rats. However, the mechanisms underlying the action of NT3 on sympathetic neurones is not known. Neither is it understood whether NT3 is retrogradely transported from peripheral tissues or acts locally in an autocrine fashion. In the present study, NT3-mRNA was quantified in sympathetic effector tissues and NT3 protein was localized in intact and lesioned sympathetic nerves in rats. NT3-mRNA is expressed and developmentally regulated in many effector tissues including mesenteric arteries, salivary gland, heart and kidney but hardly detectable in the superior cervical ganglia of adult animals. The majority of sympathetic neurones express immunoreactivity for TrkA and TrkC in both neonatal and adult rats. Sympathetic somata are normally immunoreactive for NT3, but the immunoreactivity is abolished by systemic administration of NT3 antibodies or axotomy of postganglionic neurones, suggesting an accumulation of NT3 from extraneuronal sources. Furthermore, the detection of NT3-immunoreactivity in the internal carotid nerve as early as 3 h following a compression and only on the distal side indicates endogenous NT3 is retrogradely transported by sympathetic neurones. These studies provide evidence indicating that NT3, like nerve growth factor, is an effector tissue-derived neurotrophic factor for sympathetic neurones both during development and in the adult animal. Thus, we have provided a clear example that one type of neurone derives, through a retrograde transport mechanism, two neurotrophic factors simultaneously from its peripheral effector tissues.     

低功率激光治疗血管性头痛24例

低功率激光穴位照射治疗血管性头痛24例,总治愈率54.2%,好转率29.2%,无效率16.6%,但头痛部位、治疗经穴的不同,疗效差别很大。     

Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.     

激光角膜矫正术中所用激光波长的选择

用分光光度计测定了12例兔角膜和1例人角膜的吸收光谱,结果表明波长小于300nm的准分子激光和波长在1.88μm～2.64μm,2.36μm～40μm的激光,均可对角膜进行切割并可望用于激光角膜矫正术。     

The BsmI vitamin D receptor restriction fragment length polymorphism (bb) influences the effect of calcium intake on bone mineral density

Previous studies of the vitamin D receptor (VDR) polymorphisms and bone mineral density (BMD) have suggested that there may be differences in calcium absorption among groups of women with different VDR genotypes, and that the association may be stronger in younger women. To investigate the association between the VDR polymorphisms and BMD, this study was undertaken in the Framingham Study Cohort and a group of younger volunteers. Subjects from the Framingham Study (ages 69-90 years) included those who underwent BMD testing and who had genotyping for the VDR alleles (n = 328) using polymerase chain reaction methods and restriction fragment length polymorphisms with BsmI (B absence, b presence of cut site). A group of younger volunteer subjects (ages 18-68) also underwent BMD testing and VDR genotyping (n = 94). In Framingham Cohort subjects with the bb genotype, but not the Bb or BB genotypes, there were significant associations between calcium intake and BMD at five of six skeletal sites, such that BMD was 7-12% higher in those with dietary calcium intakes greater than 800 mg/day compared with those with intakes < 500 mg/day. The data also suggested that BMD was higher in persons with the bb genotype only in the group with calcium intakes above 800 mg/day. No significant differences were found in the Framingham Cohort for age-, sex-, and weight-adjusted BMD at any skeletal site between those with the BB genotype and those with the bb genotype regardless of 25-hydroxyvitamin D levels or country of origin. In the younger volunteers, BMD of the femoral neck was 5.4% higher (p < 0.05) in the bb genotype group compared with the BB group and 11% higher (p < 0.05) in males with the bb genotype compared with the BB group. There were no significant differences at the lumbar spine. In this study, the association between calcium intake and BMD appeared to be dependent upon VDR genotype. The findings of an association between dietary calcium intake and BMD only in the bb genotype group suggests that the VDR genotype may play a role in the absorption of dietary calcium. Studies that do not consider calcium intake may not detect associations between VDR genotype and BMD. In addition, the association between VDR alleles and BMD may become less evident in older subjects.     

Double coupling Edman chemistry for high-sensitivity automated protein sequencing

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.     

Molecular mechanisms of transforming growth factor-beta signaling

The present experiments examined the effects of posttraining intrahippocampal injections of the degradative enzyme-resistant methylcarbamyl analog of the bioactive phospholipid platelet-activating factor (mc-PAF) and the platelet-activating factor (PAF) receptor antagonists BN52021 and BN 50730 on memory in male Long-Evans rats trained in a hidden platform version of the Morris water maze. Following an eight-trial training session, rats received a unilateral intrahippocampal injection of mc-PAF (0.5, 1.0, or 2.0 microgram/0.5 microliter), lyso-PAF (1.0 microgram/0.5 microliter), the cell surface PAF receptor antagonist BN 52021 (0.25, 0.5, or 1.0 micrigram/0.5 microliter/, the intracellular PAF receptor antagonist BN 50730 (2.0, 5.0, or 10.0 microgram/0.5 microliter), or vehicle (50% DMSO in 0.9% saline; 0.5 microliter). On a retention test conducted 24 h after training, the escape latencies of rats administered mc-PAF (1.0 or 2.0 microgram) were significantly lower than those of the vehicle-injected controls, demonstrating a memory-enhancing effect of mc-PAF. Injections of lyso-PAF, a structurally similar metabolite of PAF, had no influence on memory, indicating that the memory-enhancing effect of mc-PAF is not caused by membrane perturbation by the phospholipid. The retention test escape latencies of rats administered BN 52021 (0.5 microgram) and BN 50730 (5.0 or 10 microgram) were significantly higher than those of the controls, indicating a memory impairing effect of both PAF antagonists. When mc-PAF, BN 52021, or BN 50730 was administered 2 h posttraining, no effect on retention was observed, indicating a time-dependent effect of the neuroactive substances on memory storage. The findings suggest a role for endogenous PAF in hippocampal-dependent memory processes.     

不可追踪电子货币的实现

电子货币技术的实现是电子商务领域研究的重要课题之一,而不可追踪的电子货币的实现则是能否使电子商务普及的重要因素之一。即如何解决电子货币的匿名性和其它特性同时实现的矛盾。本文分析了理想的不可追踪的电子货币应具备的特点,运行模式及安全方案。给出了现阶段较完善的电子货币实现方案——基于RSA盲数字签名方案设计的一个在线电子货币方案和Brands系统。     

妊娠相关血浆蛋白A的分离及纯化

采用人胎盘血为原料,利用Sephadex G-200,DEAE-Sephadex A-50层析,Sepharose 4B反相免疫亲和层析技术,获得妊娠相关血浆蛋白A(PAPP-A)纯品,经8.0％聚丙烯酰胺凝胶电泳、免疫电泳、双向免疫扩散证明,所提取的PAPP-A纯品为单一成分。免疫家兔获得抗血清,其效价在1:16以上。