大鼠心冠状动脉系统神经肽Y年龄变化的免疫组织化学研究

运用免疫组化ＡＢＣ法对不同月龄ＳＤ大鼠心脏冠状动脉及其分支含神经肽Ｙ神经纤维的分布及其衰老变化作了详细观察．结果表明：左、右冠状动脉及其各级分支具有丰富的神经肽Ｙ能神经分布．在冠状动脉主干及其较大分支上，神经纤维较稠密，主要呈环状或网状分布，在血管围周形成较多束、丛、网，并伸入血管壁内形成壁内的神经网络分布．部分神经纤维的游离末梢有穿过血管内膜伸向腔面的迹象．在细小的冠状动脉分支上，神经纤维密度减低，主要沿血管长袖是纵向分布．两侧冠状动脉系统神经肽Ｙ能纤维分布形式及密度没有差别．老龄组动物，左、右冠状动脉及其分支的神经肽Ｙ能纤维密度趋于下降，纤维变细，呈断续状，膨体数量减少，免疫染色变浅、此外，在心内发现了神经肽Ｙ能神经节．     

Activation of the lama2 gene in muscle regeneration: abortive regeneration in laminin alpha2-deficiency

Mutations in laminin alpha2, a subunit of the basement membrane protein laminin-2/merosin, cause merosin-deficient congenital muscular dystrophy. To gain insight into the molecular mechanism of disease, we generated and used a mutant mouse, dyW, in which the lacZ gene was inserted into the lama2 gene so that beta-galactosidase would be expressed in place of laminin alpha2. Heterozygous and homozygous mutant mice are normal at birth, but homozygous mice develop muscular dystrophy at 2 to 3 weeks of age. The lama2/lacZ gene was highly expressed in muscle in the early stages of embryonic myogenesis, but was down-regulated at later stages in both heterozygous and homozygous mice. No beta-galactosidase activity was detected in skeletal muscle after birth in adult heterozygous mice. In contrast, high beta-galactosidase activity was detected in postnatal homozygous mice. Induction of injury in heterozygous mice resulted in intense reexpression of beta-galactosidase in the injured muscle early in regeneration, with a decline in enzyme activity as repair of the tissue progressed. Although the initial response to injury was similar in heterozygous and homozygous mice with abundant beta-galactosidase-positive, mononucleated cells in the injured area, repair was rarely completed in the homozygous mice, evidently caused by excessive death of cells associated with immature myofibers. The defect in muscle repair was very efficiently corrected in homozygous dyW mice expressing a human LAMA2 transgene in skeletal muscle. The data show the importance of laminin alpha2 in muscle regeneration and suggest that a major contributor to disease in muscular dystrophy is abortive regeneration.     

致癌物分子结构识别中的一种特征提取方法

本文论述基于拓扑参数的致癌物分子结构识别中的一种特征参数－－特征片段的提取方法；对一组活性已知的分子训练集，将其分子结构用ＫＬＮ编码法进行编码并输入计算机，在三个约束条件下用深度第一法对各分子进行结构分解，生成片段文件，并汇总成片段总库，然后用统计检验和聚类分析提取和优化特征片段，从而为识别模型提供合理有效的特征参数。     

Agouti-related peptide-expressing neurons are mandatory for feeding

Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. Here we demonstrate that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis.     

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-gamma, IL-12, TNF, IL-10 and TGF-beta production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-gamma, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-beta. T cell depletion abolished IFN-gamma and decreased IL-12 production, but did not affect IL-10 and TGF-beta production. Conversely, neither IL-10 nor TGF-beta was produced in cultures of B cell-depleted MLN. In addition, CD4(+) T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-gamma when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-beta, but not IFN-gamma, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-gamma-producing CD4(+) T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.     

Requirements for the development of IL-4-producing T cells during intestinal nematode infections: what it takes to make a Th2 cell in vivo

Summary: Components of the type 2 immune response may mediate host protection against both helminthic parasites and harmful allergic responses. A central player in this response is the T‐helper 2 (Th2) effector cell, which produces interleukin (IL)‐4, IL‐5, IL‐13, and other Th2 cytokines during the primary and memory response. Specific aspects of the parasite that trigger Th2‐cell differentiation are not yet defined. Furthermore, the cell types and cell surface and secreted molecules that provide the immune milieu required for the development of Th2 effector cells and also Th2 memory cells are not well understood. They will probably vary with the particular helminth or other antigen inducing the Th2 response. We have used third stage larvae of intestinal nematode parasites as adjuvants to promote naïve nonparasite antigen‐specific T cells to differentiate into Th2 cells. This model system avoids possible parasite antigen‐specific T‐cell clones or cross‐reactive memory T cells that may preferentially differentiate into Th2 effector cells during the course of infection and confound the stereotypical components of parasite‐induced Th2 cell differentiation. We have found that these parasites have a potent adjuvant effect and have used our model system to begin to investigate the events that lead to the development of polarized Th2 cells in vivo.     

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.     

Cloning and sequencing of putative acetylcholinesterase cDNAs from the American dog tick, Dermacentor variabilis, and the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae)

Two putative cDNAs of acetylcholinesterase (AChE), one from Dermacentor variabilis, and the other from Rhipicephalus sanguineus, were amplified and sequenced. The deduced amino acid sequences have high amino acid identities (between 70 and 94%) to known tick AChE sequences deposited in GenBank. Furthermore, these two AChEs also possess common features in their primary AChE structure such as catalytic active sites. A 2,220-bp contiguous sequence, containing a 1,791-bp open reading frame encoding an AChE precursor with 596 amino acid residues, was obtained from D. variabilis. The deduced proteins of R. sanguineus are different in size by 6 amino acids because of alternative splicing at the 5' end. A gene tree deduced from phylogenetic analysis indicates that there are at least three lineages of AChE in arthropods.     

Detection of expansion regions in Portuguese bipolar families

We have studied 24 families with multiple affected members with bipolar disorder to test the hypothesis that in those families clinically showing genetic anticipation [Macedo et al., 1999] we would find large repeat expansions. The families meeting inclusion criteria had a minimum of two affected members over two generations and showed marked anticipation both in terms of age of onset and disease severity. We used the repeat expansion detection (RED) method to test patients (n = 24) and controls from these families and unrelated controls (n = 53). We also genotyped patients and family members from two families with large expansions at the known expansion loci on chromosomes 13, 17, and 18. The RED method revealed a higher number of large expansions in patients compared with controls (t-test; P < 0.0055: Mann-Whitney U; P = 0.02). The patients with the largest expansions were typed at the specific loci on chromosomes 13, 17, and 18 and the chromosome 18 expansion locus segregated with disease in one family, and a second family showed segregation with the expansion located at the SCA8 locus on chromosome 13. Genetic anticipation had been analyzed in this cohort of families, with correction for potential ascertainment bias, possible proband effects, cohort effects, regression to the mean, gender effects, and maternal vs. paternal transmission. None of these potential confounds appeared to account for the observed anticipation. We also identified that the presence of large expansions in affected family members derives primarily from two families from the genetically isolated Azores population. One family shows segregation with the chromosome 18 locus, whereas the other family segregates with expansions at the SCA8 locus. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:854-857, 2000.     

Increased inducible apoptosis in CD4+ T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

Recent studies suggest that increased lymphocyte apoptosis (Ao) detected in peripheral blood T cells from burn patients appears to contribute to decreased lymphocyte immunoresponsiveness. However, while it is known that sepsis induces a marked depression in the splenocyte immune response (i.e. decreased interleukin-2, interferon-gamma production and proliferation) in response to the T-cell mitogen concanavalin A (Con A), it is unknown whether this depression is associated with an increase in inducible Ao and if so, which mediators control this process. To assess this, splenocytes were harvested from mice at 24 hr (a period associated with decreased Con A response) after the onset of polymicrobial sepsis [caecal ligation and puncture (CLP)] or sham-CLP (Sham) and then stimulated with 2.5 microg Con A/ml (24 hr). Septic mouse splenocytes stimulated with Con A, while not showing a change in their phenotypic make-up, did exhibit a marked increase in the percentage of splenocyte that were Ao+ which was associated with altered cytokine release. This appears to be due to an increase in the percentage of Ao+ cells in the CD4+ CD8- population and was associated with enhanced Fas antigen expression as well as an increase in mRNA for the Fas-FasL gene family. To determine if the changes in Ao are due to either endotoxin (a product of Gram-negative bacteria seen in CLP mice) or the expression of Fas ligand (FasL; a mediator of activation-induced lymphocyte Ao), a second set of studies examining Con A-inducible Ao was performed with splenocytes harvested from septic endotoxin-tolerant C3H/HeJ and the FasL-deficient C3H/HeJ-Fasl gld mice. The results show that increased splenocyte Ao detected following CLP is due to a FasL-mediated process and not to endotoxin. Thus the inadvertent up-regulation of FasL-mediated splenocyte Ao may contribute to the depression of splenocyte immune responses seen during polymicrobial sepsis.