SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain

Although extracellular matrix (ECM) glycoproteins play important roles in neural development, their levels are generally believed to decrease in the adult brain. Immunohistochemical analysis indicates that the anti-adhesive ECM glycoprotein SPARC/osteonectin, which contains a follistatin ‘module’, is expressed in the adult rabbit nervous system. In the cerebellum, SPARC is present in Bergmann glia, with a strong signal along their radial fibres. SPARC, while enriched in membrane fractions, is not a transmembrane protein. In the hippocampus, colocalization of SPARC is observed in cells which express the astrocytic marker GFAP. The expression of SPARC by a subset of astrocytes, particularly in synaptic enriched areas, suggests a continuing role for the ECM in the adult brain.     

Retardation of pain development: a case of recovery from congenital insensitivity to pain

Congenital analgesia is a rare genetic disorder. We report here that a 12-year-old boy was able to recover from congenital insensitivity to pain. Neurological examinations revealed that there was a 'stocking' distribution of pain decrement on the lower extremities under the patient's knee joints. Magnetic Resonance Imaging (MRI) of his brain showed gyrus thinning with sulcus widening at both sides of the parietal lobe. Southern blot hybridization probed with cDNAs of various opioid receptors did not detect any significant abnormality. Our results suggest that this rare case may not be genetically determined.     

赖型钩端螺旋体重组DNApCX7的特异性鉴定和限制性内切酶谱分析

本实验将赖型钩端螺旋体（简称钩体）ＤＮＡ基因库的克隆ｐＣＸ７制备成￣（３２）Ｐ－重组ＤＮＡ探针，对８个不同血清群的１７株问号状钩体、双曲钩体ＰａｔｏｃⅠ株以及细螺旋体３０５５株ＤＮＡ进行打点杂交；同时用１５种ＤＮＡ片断进行限制性内切酶谱分析。结果表明，该重组ＤＮＡ具有问号状钩体种（Ｓｐｅｃｉｅｓ）特异性，但与不同问号状钩体之间的同源性程度有差别；限制性内切酶谱分析发现ｐＣＸ７重组ＤＮＡ片段长约１．７ｋｂ，具有１个Ｂｇ１Ⅱ识别位点和３个ＢｓｔＢ１识别位点。     

Detection of IgA protease from Haemophilus influenzae by immunoblotting

IgA protease produced by various strains of Haemophilus infuenzae can digest serum IgA and yield its fragments which can react with anti-IgA serum. We assayed IgA protease activity by detecting the digests of IgA by SDS-PAGE and immunoblotting. The digests were separated with SDS-PAGE, transferrend to nitrocellulose membranes and detected with anti- ( chain of human IgA, its Fab and its Fc) immunoglobulin conjugated peroxidases.Using this method, we can determine which type of IgA protease is produced by various of H. infuenzae strains. All the 20 strains isolated from respiratory tracts produced IgA protease.     

Recognition of an extensive range of IgE-reactive proteins in cod extract

Allergy to fish is one of the most common food allergies. Gad c 1 is the only fish allergen which has been purified and characterized. Other allergens have been detected by Western blot in cod extracts. We have now improved the Western-blot procedure in order to characterize fish IgE-reactive proteins from extracts prepared under different conditions: pre-rigor mortis and postrigor mortis. EDTA addition or not. and DEAE ion-exchange chromatography. Several IgE-reactive protein bands have been identified over a wide molecular-weight range. In particular, the 104- and 130-kDa IgEreactive protein bands were detected. These new bands may correspond to aggregates, as EDTA increased the relative amount of the 60-, 67-, 104-, and 130-kDa IgE-reactive protein bands in Western blot. All these bands were also detected by an antiparvalbumin monoclonal antibody, specific to the first calcium-binding site. The longer period of storage increased the relative amounts of the 41-, 80-, 104-. and 130-kDa IgE-reactive protein bands. The 18-kDa band was detected only in fish stored for several days. In conclusion, we have described IgE-reactive protein bands over a wide molecular-weight range (12–130 kDa) in Western blot of cod extract, and shown that EDTA and storage conditions may influence the relative distribution of IgE-reactive protein bands.     

Tumor cells induced by the v-src oncogene are heterogeneous for expression of markers of mesenchyme differentiation

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.     

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.     

A Study of Rb Gene in Oral Squamous Cell Carcinoma

Thefirsttumorsuppressorgenemolecularlyisolatedistheretinoblastoma(Rb).ThedeletionofRbplaysanessentialroleinRetinoblastoma[1].TheosteosarcomaisalsoassociatedwiththedoublelossoftheRbgene[2].AbnormalitiesinthestructureandexpressionoftheRbgenehadbeenidentifiedinsmallcellcanceroflung[3]andbreastcancers[4].ThesefindingssuggestthattheRbgeneisapleiotropicsuppressorgene.Oralsquamouscellcarcinomaisthecommonmalignanttumor,whichaccountsfor80%ofthetotalofmalignanttumorsinoralmaxillofacialregion.Theprog…     

Tissue-specific distribution of the Goodpasture antigen demonstrated by 2-D electrophoresis and Western blotting

The target of autoantibodies in Goodpasture's disease, the Goodpastureantigen has recently been characterized as the NC1 domain ofthe 3 chain of type IV collagen. In order to study the Goodpastureantigen in different organs, NC1 domains were isolated frombasement membranes (BM) of human glomeruli (GBM), tubules (TBM),alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparationswere separated by 2-D electrophoresis, and silver stained orimmunoblotted to determine the subunit structure and antigenicityof different basement membranes. All basement membranes containedmonomeric components of MW 26 kDa and 24 kDa, and associateddimers, corresponding to the 2-D location of 1(IV) and cc2(IV)chains respectively. However, GBM, ABM, and to a lesser extentTBM possessed an extra set of monomeric components of MW 28kDa and associated dimers corresponding to the proposed locationof 3(IV) and 4(IV) chains. 2-D-separated polypeptides were Westernblotted with autoantibodies from patients with Goodpasture'sdisease, a monoclonal antibody to the Goodpasture antigen (P1)and a monoclonal antibody to the bovine 3(IV) chain. The predominantbinding of all these reagents was to cationic 28 kDa monomersof GBM, ABM and TBM, corresponding to the 3(IV) chain, althoughautoantibodies and P1 also bound to neutral 28 kDa monomers,corresponding to the 4(IV) chain. Autoantibodies bound weaklyto more neutral components of PBM and VBM, but neither monoclonalantibody bound to these basement membranes. This study suggeststhat there are two separate type IV collagen networks: one containingthe l(IV) and 2(IV) chains appears to be present in all basementmembranes, and the other containing the 3(IV) and 4(IV) chainshas a tissue specific distribution. The latter network bearingthe Goodpasture antigen is expressed in the basement membranesof both kidney and lung, the organs involved in Goodpasture'sdisease.     

Constitutive and cytokine-regulated expression of presenilin-1 and presenilin-2 genes in human neural cell lines

To investigate the role of pleiotropic neuronal and glial cytokines in the regulation of presenilin (PS) gene expression in human neural cells, both presenilin-1 (PS1) and presenilin-2 (PS2) mRNA levels were analysed by Northern blotting in SK-N-SH neuroblastoma, IMR-32 neuroblastoma, NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N) and U-373MG astrocytoma cells following exposure to proinflammatory cytokines (TNF-alpha, IFN-gamma, or IL-1beta), anti-inflammatory cytokines (IL-10 or TGF-beta1), dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate (PMA). The constitutive expression of PS1 (3.0 kb) and PS2 (2.3 kb) mRNA was identified in all these cell lines, in which PS1 mRNA levels were unaltered following treatment with any cytokines and factors examined. By contrast, PS2 mRNA expression was upregulated substantially in SK-N-SH cells by exposure to TNF-alpha and in U-373MG cells by treatment with IFN-gamma, whereas it was downregulated in both NTera2-N and U-373 MG cells following exposure to IL-1beta or PMA. The levels of PS2 mRNA remained unchanged in IMR-32 cells after these treatments. These results indicate that PS1 and PS2 genes are expressed constitutively in a panel of human neural cell lines where PS2 mRNA expression is affected by a distinct set of cytokines via cell type-specific mechanisms that do not alter PS1 mRNA levels, suggesting the existence of separated regulatory systems controlling the expression of PS1 and PS2 genes in human neural cells.