Human bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.     

湖南汉族人群HLA-DP、-DQ位点等位基因多态性与鼻咽癌的相关性

目的 探讨湖南汉族人群HLA—DP、HLA—DQ位点等位基因多态性与鼻咽癌遗传易感性之间的关系。方法 应用聚合酶链反应—特异性寡核苷酸探针基因分型技术对87例湖南汉族鼻咽癌患者与91名健康对照作HLA—DPAl、—DPBl、—DQAl及—DQBl的基因分型，采用x^2检验比较两组各位点等位基因频率，单倍型频率分布的差异。结果 发现鼻咽癌组DPAl*0201、DPBl*1901、DQAl*0201较对照组明显降低，DPBl*0402、DQAl*0101较对照组明显升高；单倍型DPAl*0201—DPBl*1401及DQAl*0201—DQBl*0201较对照明显降低；但P值经Bonferroni校正后，差异均无显著性(Pc＞0．05)。结论 湖南汉族人群HLA—DP和—DQ位点与鼻咽癌无明显相关，与以往用受累同胞对方法在鼻咽癌家系中未能证实鼻咽癌易感基因与HLA—DP和—DQ位点连锁的结果一致。     

Stimulus-dependent activation of c-Fos in neurons and glia in the rat cerebellum

The intent of the present study was to use chemical or electrical stimulation of cerebellar afferents to determine how different stimulation paradigms affect the pattern of activation of different populations of neurons in the cerebellar cortex. Specifically, we analyzed immediate changes in neuronal activity, identified neurons affected by different stimulation paradigms, and determined the time course over which neuronal activity is altered. In the present study, we used either systemic (harmaline) or electrical stimulation of the inferior cerebellar peduncle (10 and 40 Hz) to alter the firing rate of climbing and mossy fiber afferents to the rat cerebellum and an antibody made against the proto-oncogene, c-fos, as a marker to identify activated neurons and glia. In control animals, only a few scattered granule cells express nuclear Fos-like immunoreactivity. Although no other cells show Fos-like immunoreactivity in their nuclei, Purkinje cells express Fos-like immunoreactivity within their somatic and dendritic cytoplasm in control animals. Within 15 min of chemical or electrical stimulation, numerous granule and glial cells express Fos-like immunoreactivity in their nuclei. Cells in the molecular layer express Fos-like immunoreactivity following harmaline stimulation in a time and lobule specific manner; they do not appear to be activated in the electrical stimulation paradigm. Following harmaline injections, there is an initial loss of Fos-like immunoreactivity in the cytoplasm of Purkinje cells; 90 min later, nuclear staining is observed in a few scattered Purkinje cells. Following electrical stimulation, the cytoplasmic staining in Purkinje cells is enhanced; it is never present in the nucleus. Data derived from this study reveal cell-specific temporal and spatial patterns of c-Fos activation that is unique to each paradigm. Further, it reveals the presence of an activity dependent protein in the cytoplasm of Purkinje cell somata and dendrites.     

Population survey of CCR5 delta32, CCR5 m303, CCR2b 64I,and SDF1 3'A allele frequencies in indigenous Chinese healthy individuals,and in HIV-1-infected and HIV-1-uninfected individuals in HIV-1 risk groups

The aim of this study is to determine in indigenous Chinese ethnic groups the frequencies of the chemokine (SDF1 3'A) and chemokine receptors (CCR5 delta32, CCR5 m303, and CCR2b 64I) HIV-1/AIDS restriction alleles. The study includes two cohorts; the first comprised 3165 indigenous healthy subjects representing eight ethnic groups: Han (n = 1406), Uygur (n = 316), Mongolia (n = 134), Hui (n = 386), Tibetan (n = 330), Zhuang (n = 378), Dai (n = 101), and Jingbo (n =114). The second cohort consisted of 330 HIV-1-infected (86 subjects infected by sexual transmission and 198 subjects infected by HIV-1-contaminated blood or by sharing injection equipment; the remaining 46 subjects said nothing about HIV-1 transmission) and 474 HIV-1-uninfected Han Chinese belonging to one of two HIV-1 high-risk groups: intravenous drug users (n = 215) and individuals with sexually transmitted diseases (n = 259). Genotypes for the four genes were obtained using PCR (CCR5 delta32) or PCR-restriction fragment length polymorphism. Randomly selected amplified PCR products were further confirmed by direct DNA sequencing. The variant allele frequencies were determined to be 0% to 3.48% for CCR5 delta32, 0% for CCR5 m303, 16.23% to 28.79% for CCR2b 64I, and 17.70% to 27.76% for SDF1 3'A in Chinese healthy individuals from eight ethnic groups. These findings show that allele frequencies differ among the eight Chinese ethnic groups for CCR5 delta32, CCR2b 64I, and SDF1 3'A and that the CCR5 m303 and CCR5 delta32 mutant alleles were absent or infrequent in Chinese, which may be helpful for studies of specific anti-HIV-1 vaccine trials and coreceptor inhibitor drug targets in Chinese populations. Furthermore, we observed no significant differences in allele or genotypic frequencies between HIV-1-infected and HIV-1-uninfected groups from the Han ethnic group. Our finding is the first reporting that there is likely no effect of the examined polymorphisms in our study on HIV-1 transmission in the Chinese Han population, However, the genetic effects of these and other AIDS-modifying polymorphisms on the pathogenesis and clinical outcome of HIV-1/AIDS diseases is under investigation in Chinese populations.     

MMA/MPEOMA/VSA copolymer as a novel blood-compatible material: effect of PEO and negatively charged side chains on protein adsorption and platelet adhesion

This study was designed to evaluate the effect of polyethylene oxide (PEO) and negatively charged side chains on blood compatibility. For this, novel copolymers (MMA/MPEOMA/VSA copolymers) with both PEO and negatively chargeable side chains were synthesized by random copolymerization of methyl methacrylate (MMA), methoxy PEO monomethacrylate (MPEOMA; PEO mol wt 1000), and vinyl sulfonic acid sodium salt (VSA) monomers of different compositions. MMA/MPEOMA copolymer (with PEO side chains) and MMA/VSA copolymer (with negatively chargeable side groups) also were synthesized for purposes of comparison. The synthesized copolymers were characterized by 1H-nuclear magnetic resonance spectroscopy and gel permeation chromatography. They were coated onto polyurethane (PU) or polymethyl methacrylate (PMMA) films by spin coating. The surface properties of MMA/MPEOMA/VSA copolymers were compared by water contact angle and zeta potential with those of MMA/MPEOMA and MMA/VSA copolymers of similar MPEOMA or VSA composition. Using electron spectroscopy for chemical analysis and scanning electron microscopy, respectively, the behaviors of the adsorption of blood proteins (albumin, gamma-globulin, fibrinogen, and plasma proteins) and the adhesion of platelets on the copolymer-coated surfaces also were compared. Among the copolymers, the MMA/MPEOMA/VSA copolymer with a monomer molar ratio 8:1:1 was observed to be particularly effective in preventing both protein adsorption and platelet adhesion on the surfaces, probably owing to the combined effects of highly mobile, hydrophilic PEO side chains and negatively charged side groups in aqueous solution.     

The effect of dopamine D2, D5 receptor and transporter (SLC6A3) polymorphisms on the cue-elicited heroin craving in Chinese.

Heroin dependence is resulted from the interaction between multiple genetic and environmental factors. Subjective craving is considered to be a central phenomenon, which contributes to the continuation of drug use in active abuser and the occurrence of relapse in detoxified abusers. Dopamine pathway has been implicated in the cue-elicited craving for a variety of addictive substances. The objective of this study was to test the hypothesis that heroin addicts carrying specific variants in dopamine-related genes would have higher levels of craving following exposure to a heroin-related cue. Craving induced by a series of exposure to heroin-related cue was assessed in a cohort of Chinese heroin abuser (n = 420) recruited from natural abstinence center at Shanghai. Significantly stronger cue-elicited heroin craving was found in individuals carrying D2 dopamine receptor gene (DRD2) TaqI RFLP A1 allele than the non-carriers (P < 0.001). Furthermore, we did not observed significant association of cue-elicited craving with the nine-repeat allelic variants in dopamine transporter gene (DAT) SLC6A3 and with the dinucleotide repeat polymorphism (DRP) 148bp allele in D5 dopamine receptor gene (DRD5). The results of our study suggest that human dopamine pathway be involved in cue-induced heroin craving, and indicate a potential genetic risk factor for persistent heroin behavior and relapse.     

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology.

To develop improved reagents for mumps serology a high-level yeast expression system was employed to produce recombinant mumps nucleoprotein (rNP). The rNP was purified by CsCl gradient centrifugation and yielded approximately 15 mg/l of yeast culture. Electron microscopy of the rNP revealed characteristic herring-bone structures. The electrophoretic mobility of rNP in yeast cells was similar to native NP in SDS-PAGE. Monoclonal antibodies to rNP reacted with native mumps virus nucleoprotein by immunofluorescence assay. A monoclonal antibody to native mumps virus NP reacted with rNP by Western blot assay. The rNP was investigated as antigen in an IgM capture enzyme immunoassay (EIA) using a horseradish peroxidase conjugate of monoclonal antibody to the rNP. Eighteen sera previously found to be positive by IgM capture radioimmunoassay (MACRIA) and 30 sera that were mumps IgM negative by MACRIA were tested by mumps IgM capture EIA. The results for the two test were concordant. In addition, 26 rheumatoid factor positive sera and 35 sera that were IgM positive for measles, rubella or parvovirus B19 were tested. Fifty-nine sera were negative by mumps IgM capture EIA but two sera collected from two infants 3 and 6 weeks after mumps, measles and rubella vaccination were positive. Mumps MACRIA confirmed these results. Compared to MACRIA the overall sensitivity was 100% (20/20) and specificity was 96.8% (30/31). The yeast expressed rNP was highly immunogenic and suitable for use in IgM capture EIA for the diagnosis of mumps.     

Identification of p46 Shc expressed in the nuclei of hepatocytes with high proliferating activity: Study of regenerating rat liver

The adaptor molecule Shc is a proto-oncogene product, and it is known to be associated with cell proliferation. However, the role of Shc in the proliferation and regeneration of hepatocytes remains unknown. In the present study, we report that p46 Shc is specifically expressed in the nuclei of proliferative (or regenerative) hepatocytes, suggesting that p46 Shc protein plays a role in hepatocellular proliferation. The expression of Shc was analyzed in liver tissue after partial hepatectomy (PH) or sham operation in Wistar rats by using immunohistochemistry and/or Western blot analysis. In addition, the expression of various cell cycle-related proteins, such as Cdk4, cyclin D1, PCNA, and Cdk1 was analyzed in the tissues of regenerating rat liver. Furthermore, the tyrosine phosphorylation of Shc was studied in liver tissue after PH or sham operation by immunoprecipitation using a monoclonal phosphotyrosine antibody. Although the protein levels of p52 Shc were unchanged in liver tissues after PH or sham operation, tyrosine phosphorylation was detected only in the regenerating rat liver after PH. The levels of p46 Shc protein were markedly increased in liver tissues during the liver regenerative process. In contrast, p66 Shc was not detected in the liver tissues after PH or sham operation. Western blotting and immunohistochemistry showed that the main location of p46 Shc was in the nuclei of proliferating hepatocytes after PH. These data suggest that p46 Shc expressed in hepatocellular nuclei may be closely related to the proliferation of hepatocytes. Therefore, it is suggested that p46 Shc expressed in hepatocellular nuclei may be a useful marker for detecting hepatocytes with high proliferative activity.     

Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa

The activation of group I metabotropic glutamate receptors (mGluRs) produces a long-term potentiation of sensory transmission in the substantia gelatinosa (SG) region of the spinal cord (Prog. Brain Res. 129 (2000) 115). The mechanism(s) responsible for the induction of this potentiation is not known. Using rat spinal cord slice preparation and patch-clamp recordings, here we show, that the activation of the group I mGluRs by (S)-3,5-dihydroxyphenylglycine (DHPG, 1 microM), the mGluR1/5 agonist, increased the frequency of both activity-dependent spontaneous EPSCs, and activity-independent miniature EPSCs (mEPSCs). However, DHPG did not affect amplitude of mEPSCs. The effects of DHPG were not seen in the presence of the preferential mGluR1 antagonist CPCCOEt (10 microM). On the other hand, 2-methyl-6-(phenylethynyl)-pyridine (10 microM), a selective mGluR5 antagonist, blocked the DHPG facilitation present during the wash-out of the drug. This novel facilitating effect of the group I mGluR activation on glutamate release is the first report of a direct facilitatory action of both mGluR1 and mGluR5 subtypes on sensory transmission in the spinal cord SG region. These results indicate the potential contribution of synaptic activation of these facilitatory autoreceptors in plasticity of primary afferent neurotransmission.     

Chemical, structural properties, and osteoconductive effectiveness of bone block derived from porcine cancellous bone

The purpose of this study is to determine the efficacy of bioactive calcium phosphate obtained from porcine cancellous bone for the treatment of bone defects and nonunion. Porcine cancellous bone blocks were heat treated at 1300 degrees C for 2 h. The chemical composition, calcium-to-phosphate ratio, and microstructure of the porcine bone blocks were examined. For in vivo implantation, bone defects were created on the anteromedial aspect of the proximal tibia in seven beagle dogs and the xenograft bone blocks were placed into these defects. Plain radiographs were taken at 2-week intervals for roentgenographic evaluation. At 12 weeks, the specimens were stained with hematoxylin and eosin (H&E). The composition and morphology of heat-treated porcine cancellous bone were found to be similar to heat-treated human cancellous bone. Radiographs showed union between the host bone/bone-block interfaces. At 12 weeks, uniform and substantial new bone formation was observed. It is concluded that heat-treated porcine cancellous bone demonstrated effective osteoconductivity. This high-temperature heat-treatment technique has several advantages, including decreased risk of disease transmission and immunoreactivity, while also offering excellent biocompatibility.