Region-specific constitutive gene expression in the adult porcine meniscus.

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.     

Hemoglobin constant spring defined by specific oligonucleotide hybridization and hemoglobin D Punjab (beta 121----Gln) in a Batak Indonesian family

A Batak Indonesian from North Sumatra with hemoglobin (Hb) D Punjab (alpha 2 beta 2 121----Gln) and hemoglobin Constant Spring (Hb CoSp) is described. The 24-year-old man did not have clinical symptoms, and his hematological indices were normal. However, he had a persistent slight elevation of fetal hemoglobin level. His mother and his brother were heterozygous for Hb D Punjab; his father had Hb CoSp trait. A sister did not have any abnormal hemoglobin. To show the exact molecular defect leading to the synthesis of Hb CoSp in this family, genomic DNA from the father was analyzed by hybridization with synthetic oligonucleotides. Genomic DNA was digested with Sst I and Hind III producing a 1.05-kb fragment from the 3' end segment of the alpha 2-globin gene, including the termination codon. Two nonadecamers were synthesized to serve as probes: one, entirely homologous to the normal 3' end of alpha 2A-globin gene sequence, including the termination codon TAA, the other different from it by a replacement of the T in the termination codon TAA with C, changing it to CAA, the codon for the amino acid glutamine. DNA from normal controls gave a positive signal with the normal alpha 2TAA oligonucleotide probe but negative with the alpha 2 CAA probe. The father of propositus who had Hb CoSp trait gave a positive signal with the normal alpha 2TAA oligonucleotide probe as well as with the alpha 2CAA oligonucleotide probe, showing him to be heterozygous for the alpha 2CAA-globin gene. This result shows that the Hb CoSp in the Batak family is indeed due to a replacement of T by C in the TAA termination codon of the alpha 2-globin gene changing it to CAA the condon for glutamine. This explains the resulting readthrough of the untranslated sequence of the mRNA.     

Polymorphisms in ADAM33 are associated with allergic rhinitis due to Japanese cedar pollen

BACKGROUND: A recent report provided evidence that a disintegrin and metalloprotease domain 33 (ADAM33), a member of the ADAM family, is a novel susceptibility gene in asthma linked to bronchial hyper-responsiveness. However, there has been no investigation of the genetic role of ADAM33 variants in nasal allergy. OBJECTIVE: The purpose of this study was to test the association between ADAM33 polymorphisms and Japanese cedar pollinosis (JCPsis), a most common seasonal allergic rhinitis in Japan. METHODS: We conducted a case-control association study among a Japanese population, involving 95 adult individuals with JCPsis and 95 normal healthy controls. A total of 22 single-nucleotide polymorphisms (SNPs) in ADAM33 were genotyped using PCR-based molecular methods. RESULTS: Six SNPs of ADAM33 gene, three in introns (7575G/A, 9073G/A and 12540C/T) and three in the coding region (10918G/C, 12433T/C and 12462C/T), were strongly associated with JCPsis (P = 0.0002-0.022 for absolute allele frequencies) and most of the SNPs were in linkage disequilibrium with each other. A higher frequency of the common alleles of these SNPs was noted for the subjects with JCPsis in comparison with healthy controls. We also identified a haplotype associated with the disease susceptibility. In addition, associations were found between ADAM33 polymorphisms and various cedar pollinosis phenotypes including clinical severity, eosinophil counts in nasal secretion and allergen-specific IgE levels in sera, but not total serum IgE levels. CONCLUSION: These results indicate that polymorphisms in the ADAM33 gene are associated with susceptibility to allergic rhinitis due to Japanese cedar pollen, but the functional relationship still needs clarification.     

Striatal delivery of CERE-120, an AAV2 vector encoding human neurturin, enhances activity of the dopaminergic nigrostriatal system in aged monkeys.

Neurturin (NTN) is a potent survival factor for midbrain dopaminergic neurons. CERE-120, an adeno-associated virus type 2 (AAV2) vector encoding human NTN (AAV2-NTN), is currently being developed as a potential therapy for Parkinson's disease. This study examined the bioactivity and safety/tolerability of AAV2-NTN in the aged monkey model of nigrostriatal dopamine insufficiency. Aged rhesus monkeys received unilateral injections of AAV2-NTN into the caudate and putamen, with each animal therefore serving as its own control. Robust expression of NTN within the nigrostriatal system was observed 8 months postadministration. (18)F-fluorodopa imaging using positron emission tomography revealed statistically significant increases in (18)F-fluorodopa uptake in the injected striatum compared with the uninjected side at 4 and 8 months. In addition, at 8 months postadministration, a significant enhancement in tyrosine hydroxylase immunoreactive fibers and an increase in the number of tyrosine hydroxylase immunoreactive cells was observed in the AAV2-NTN injected striatum compared with the uninjected side. Robust activation of phosphorylated extracellular signal-regulated kinase immunoreactivity in the substantia nigra was also observed. Histopathological analyses revealed no adverse effects of AAV2-NTN in the brain. Collectively, these results are consistent with the neurotrophic effects of NTN on the dopaminergic nigrostriatal system and extend the growing body of evidence supporting the concept that AAV2-NTN may have therapeutic benefit for Parkinson's disease.     

Nurr1基因转染人脐血间充质干细胞诱导分化为多巴胺能神经元的实验研究

目的探讨外源性Nurrl基因修饰的人脐血间充质干细胞在基因重组成纤维细胞生长因子-8(FGF8)和音猬因子(Shh)诱导下体外分化为多巴胺能神经元的情况。方法间充质干细胞被随机分为A组(对照组)、B组(Nurrl组)、C组(FGF8+Shh组)及D组(Nurrl+FGF8+Shh),采用脂质体法将pcDNA3.1-Ntrr1转染B、D组间充质干细胞,Western blot观察Nurr1基因表达情况,并在神经元条件培养液中进行增殖培养和诱导分化,间接免疫荧光染色法鉴定细胞性质,高效液相色谱法测定多巴胺含量。结果Western blot结果显示转染后Nurr1蛋白表达明显增高。A组和B组未发现酪氨酸羟化酶(TH)阳性细胞,而C组和D组TH阳性细胞分别为10．12％±2．65％及25．36％±3．13％,多巴胺含量分别为(43．6±2．1)nmol／L及(83．2±3．5)nmol／L,差异均有显著性意义(P< 0．01)。结论人脐血间充质干细胞在FGF8和Shh诱导下可以分化为多巴胺能神经元,外源性Nurr1基因修饰后,分化为多巴胺能神经元的数量增加。     

sFGFR1基因的克隆与在RTS系统中的高效表达

目的：克隆sFGFRl(soluble fibroblast growth factors receptor-1)基因，并在RTS(rapid translation system)系统中高效表达相应蛋白．方法：培养Swiss rat 3T3 fibroblast细胞株，提取总RNA，用RT—PCR方法获取鼠sFGFR1 cDNA片段，酶切后克隆到pIVEX2．3d载体并进行序列分析；采用Roche RTS ProteinMaster500系统，高效表达sFGFR1蛋白并用Western Blot鉴定表达的蛋白．结果：克隆了sFGFR1基因，测序证实序列正确；Western Blot证实sFGFR1蛋白在RTS系统中高效表达．结论：克隆了sFGFR1基因并在RTS系统获得高效表达．     

肿瘤坏死因子凋亡相关配体基因的克隆和表达

目的：克隆、表达和鉴定肿瘤坏死因子凋亡相关配体(TRAIL)．方法：从培养的人外周血淋巴细胞中提取总RNA，经RT-PCR获得TRAIL基因．将该基因克隆到pGEM-Teasy载体中，测序鉴定．将TRAIL基因插入pBV220表达载体中，42℃诱导表达4～5h，进行SDS-PAGE分析．免疫印迹法鉴定TRAIL蛋白的表达．结果：DNA测序证明，获得了TRAIL基因，其序列与GenBank中报道序列完全一致．SDSPAGE分析表明，TRAIL蛋白获得高效表达，分子质量为17ku，表达量约占菌体总蛋白的300k，．免疫印迹法鉴定显示，该蛋白可与鼠抗人TRAIL mAb产生阳性反应．结论：成功克隆和表达了TRAIL基因．     

No support for a truncated interferon-α17 allele as risk factor for MS

Cytokines have a central role in multiple sclerosis (MS) pathogenesis and may contribute to the aetiology of MS. A polymorphism in the IFNA17 gene with an allele carrying a pre-mature stop codon has been suggested to convey a 26-fold increased risk for MS. We investigated the possible association between this polymorphism and MS using population-based samples from a genetically well-characterized population. The IFNA17 gene variant was found in 2.8% of 327 MS cases and 3.3% of 698 referents ( P  = 0.64). Thus, our study does not support an association between the IFNA17 allele and risk for MS.     

Two novel gastric cancer-associated genes identified by differential display

AIM To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783. In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.     

Responses of periodontal nerve terminals to experimentally induced occlusal trauma in rat molars: an immunohistochenmical study using PGP 9.5 antibody

The response of periodontal nerves to experimentally induced occlusal trauma in rat molars was assessed by immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels, and by computerized image analysis. The occlusal surface on the left upper first molar of 8-wk-old male Wistar rats was raised approximately 1 min under ether anaesthesia. The rats were perfusion-fixed on d 1, 2, 3, 4, and 7 after bite-raising and then decalcified for 2–3 wk. Frozen sagittal cryostat sections were stained by the avidin-biotin complex method. By the second day after bite-raising many Ruffini endings were swollen and their outline unclear at the light microscopic level. Transmission electron microscopy disclosed PGP 9.5 reaction products within Ruffini endings that had unusually long cytoplasmic projections extending through enlarged slits of the Schwann sheaths and also diffuse extracellular PGP 9.5-immunoreactivity near the Ruffini endings. From d 2 to 4, thin nerve fibres on the pressure side of the periodontal ligament were orientated irregularly and had a prominent beaded appearance. An increase in beaded nerve terminals occurred at d 2–4 post elevation, and decreased later. These results suggest that occlusal trauma induces specific changes in the distribution and shape of nerve terminals in the periodontal ligament.