人乳头瘤病毒多肽与人免疫球蛋白G融合表达

目的 将人乳头瘤病毒16型(Human papillomavirus type 16，HPV-16)的晚期表达蛋白E7上的抗原24肽(从第38位氨基酸到第61位氨基6病毒感染防治酸)与人免疫球蛋白G的重链恒定区融合表达，并以此融合蛋白作为抗原，可能为HPV-1提供免疫治疗方法。方法 利用PCR方法分别扩增HPV-16 E7(38-61)24肽的DNA片段和人免疫球蛋白G的重链恒定区DNA片段，并构建到pEV21a表达载体上，转化入E．coli中表达，利用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western-blotting)的方法对表达结果进行鉴定。结果 构建的表达载体HPV16E7e／hIgGHCCR-pET21a经酶切鉴定和测序显示序列正确；通过SDS-PAGE和Western-blotting的鉴定，重组融合蛋白Mr约40000，表达量可占菌体蛋白的20％左右。结论 成功构建HPV16-E7的抗原多肽片段和人免疫球蛋白G的重链恒定区的融合蛋白，并可在E．coli中高效表达。     

Growth factor pre-treatment differentially regulates phosphoinositide turnover downstream of lysophospholipid receptor and metabotropic glutamate receptors in cultured rat cerebrocortical astrocytes

Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.     

IL-2对垂体瘤细胞系RC-4B/C细胞分泌ACTH的影响

目的 :探讨白细胞介素 2 (IL 2 )对垂体瘤细胞系RC 4B/C细胞ACTH分泌的调控作用及其影响因素 .方法 :以放射免疫方法测定培养的垂体瘤细胞系RC 4B/C细胞的培养液中的ACTH浓度 .结果 :IL 2 (1× 10 4～ 5× 10 5U·L-1)促进RC 4B/C细胞分泌ACTH ;蛋白激酶A的抑制剂H 9(1μmol·L-1)和酪氨酸蛋白激酶的抑制剂tyrphostin2 3 (1μmol·L-1)均可显著性抑制IL 2的促ACTH分泌作用 .结论 :IL 2可促进RC 4B/C细胞分泌ACTH ,该作用与蛋白激酶A和酪氨酸蛋白激酶信号转导途径紧密相关     

大豆蛋白影响肾损害模型钙摄入和钙排出的实验研究

[目的]研究大豆蛋白对肾损害大鼠钙平衡的影响。[方法]选择3月龄断乳雄性SD大鼠40只．按体质量从小到大排序，采用完全随机化原则分为4组。每组10只。标准饲料对照组：喂食含有14％酪蛋白饲料；大豆蛋白饲料组：喂食含有14％大豆分离蛋白饲料；混合饲料1组：喂食含有7％酪蛋白加7％大豆分离蛋白饲料；混合饲料2组：喂食含有7％酪蛋白加14％大豆分离蛋白饲料。实验期用腺嘌呤灌胃共21d，建立肾损害大鼠模型，各组大鼠喂养相应饲料6周，测饲料消耗量、24h尿蛋白、尿钙、钙表观吸收率、储留钙量。[结果]4组实验大鼠饲料摄入量1d～30d无统计学差异，大豆蛋白饲料组和混合饲料1组实验结束时与另外两组相比，尿钙排泄量低，钙表观吸收率和储留钙量高，差异有统计学意义（P〈0．05）。[结论]蛋白质含量水平在14％条件下，含大豆蛋白的两种饲料对肾损害大鼠钙吸收、钙排泄的影响无统计学差异，有利于促进钙吸收，减少钙排泄。     

Intrathecal synthesis of anti-myelin basic protein IgG in HIV-1+ patients

Human immunodeficiency virus type 1 (HIV-1)-infected individuals frequently develop a broad spectrum of neurological syndromes, classified as HIV-1-associated cognitive/motor complex. Diffuse demyelination of hemispheric white matter is a commonly observed in HIV-1 infected brain, but the events leading to myelin destruction are still obscure. Since oligodendrocyte infection by HIV-1 is not proven as yet, myelin damage in HIV-1 infection may result from indirect mechanisms such as the excessive release of myelinotoxic substances or the triggering of autoimmune responses directed to myelin constituents. To verify the latter hypothesis, we searched for elevated anti-myelin basic protein (MBP) IgG levels in the cerebrospinal fluid (CSF) and serum of 25 patients with HIV-1 infection, 12 with multiple sclerosis (MS), and 9 with non-inflammatory neurological diseases (NIND). CSF, but not serum, anti-MBP IgG levels were more frequently elevated in HIV-1⁺ (16/25, 64%) than in MS (3/12, 25%) or NIND (0/9) patients. By using the anti-MBP IgG index, the anti-MBP IgG antibody specificity index (ASI), and the search for anti-MBP oligoclonal IgG, we ascertained that anti-MBP IgG were produced within the CNS in 13 of 25 (52%) HIV-1⁺, in 6 of 12 (50%) MS, and in none of NIND patients. The incidence of increased CSF anti-MBP IgG levels was higher among HIV-1⁺ patients at stage II–III (4/4, 100%) or at stage IV B (7/9, 78%) than among those at stage IV C–IV D (5/12, 42%). Although our data indicate that intrathecal anti-MBP IgG may occur early during HIV-1 infection and that they are more common in patients with HIV-1-associated cognitive/motor complex, the possible demyelinating role of these antibodies remains to be demonstrated.     

Astrocyte RNA in relation to neuronal RNA depletion in Alzheimer's disease

Summary A new double-staining procedure, in which the techniques of immunocytochemistry of glial fibrillary acidic protein (GFAP) and quantitative microdensitometry of azure B-RNA were combined, was used to study nucleic acid alterations in fibrous astrocytes in Alzheimer's disease (AD). RNA contents of GFAP-positive cells of the hippocampal endplate (Rose's H₃-H₅ fields) and the dentate gyrus molecular layer were determined in ten autopsy-proven AD patients (ages 51–88) and ten age-matched, non-demented control. In addition, RNA contents of pyramidal neurons of the endplate were examined. While there were no differences in RNA contents of astrocytes of either region between AD patients and controls, neuronal RNA was markedly depleted. These data suggest that astrocytes maintain protein synthetic capabilities in AD and that RNA loss is limited to the neuronal compartment.Supported by Grants 1P01-AG05119 and 1P50-AG05144 from the National Institutes of Health and by a Small Research Project Award from the University of Kentucky Medical Center     

Nerve biopsy findings in Niemann-Pick type II (NPC)

The severe infantile form of Niemann-Pick disease type II was diagnosed in a 4-year-old girl and confirmed by demonstrating in cultured skin fibroblasts a deficiency of low-density lipoprotein-stimulated cholesterol ester synthesis of < 5% of normal. Electrodiagnostic studies revealed changes of a predominantly demyelinating motor and sensory polyneuropathy. Light microscope and ultrastructural examination of a peroneal nerve biopsy showed unique changes. Compacted myelin sheaths were disproportionately thin with marked globular irregularities in single teased nerve fibres and evidence of chronic demyelination. The majority of axons were preserved but axonal spheroids and cytoskeletal abnormalities akin to neuroaxonal dystrophy were noted. Membrane-bound multilobulated lysosomal inclusions of floccular and electron-dense material were present in Schwann cells (SC), endoneurial fibroblasts, macrophages, pericytes and endothelial cells. SC of myelinated fibres were stuffed with whorls of concentric osmiophilic membranous profiles and electron-lucent material. The findings are diagnostic and differ from those of classical Niemann-Pick disease.     

红细胞同工酶ADA－EAP一AK_1同步电泳分型方法的建立

综合、改良了分别分型的方法，建立了红细胞同工酶ＡＤＡ一ＥＡＰ一ＡＫ１同步电泳分型方法，为ＡＤＡ，ＥＡＰ和ＡＫ１３种红细胞同工酶在法医学鉴定中更广泛的应用，提供了一种可靠、经济、实用的方法。     

Evidence for negative control in protein kinase C substrate specificity

The peptide Leu-Asp-Asp-Ser-Lys-Arg-Val-Ala-Lys-Arg-Lys-Leu-Ile-Glu, which corresponds to sequence 124 to 137 of c-erb-A protein, was synthesized and tested as substrate for protein kinase C (PKC). Although a typical recognition sequence for PKC, consisting of a cluster of basic residues, is found on the C-terminus side of serine, its phosphorylation was totally prevented by the presence of the two acidic residues on the amino-terminus side. Three analogs in which aspartyl residues were successively replaced with alanine were studied and the influence of the acidic side chain in modulating phosphorylation by PKC was thus possible to determine. The results show that the presence of a single aspartyl residue located in positions i-1 or i-2 with respect to the phosphorylable residue can almost totally abolish the positive effect of a highly favorable cluster of basic residues. These observations highlight the role of negative substrate specificity determinants in settling the protein substrate profile of protein kinase C.