重组人bFGF的原核表达及其高效价抗血清的制备

目的 以重组人碱性成纤维生长因子为免疫原，制备高效价抗hbFGF抗血清。方法通过PCR方法改造5’编码区的12个密码子，构建hbFGF’原核表达载体并在大肠杆菌(E．coli)中表达，以纯化的hbFGF、免疫新西兰兔，制备高效价抗血清，用于重组hbFGF、的免疫印迹分析。结果经过改造的hbFGF基因在E．coli中获得较高水平表达。从可溶性部分纯化得到纯度95％以上的重组hbFGF，以该重组蛋白免疫兔子，在二次加强后以间接ELISA检测抗血清效价可达1：512000。免疫印迹分析显示该抗血清与E．coli中表达的重组hbFGF、和标准hbFGF、均有特异性反应，但与某些细菌蛋白存在弱交叉反应，经E．coli菌体蛋白吸附的抗血清，与菌体蛋白的弱交叉反应消失。结论以纯化的重组hbFGF为免疫原制备了高效价的特异性抗血清，经菌体蛋白吸附可消除存在的交叉反应性。     

Expression of mucin genes in the human testis and its relationship to spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.     

Molecular profiling indicates avian branchiomotor nuclei invade the hindbrain alar plate

It is generally believed that the spinal cord and hindbrain consist of a motor basal plate and a sensory alar plate. We now have molecular markers for these territories. The relationship of migrating branchiomotor neurons to molecularly defined alar and basal domains was examined in the chicken embryo by mapping the expression of cadherin-7 and cadherin-6B, in comparison to genetic markers for ventrodorsal patterning (Otp, Pax6, Pax7, Nkx2.2, and Shh) and motoneuron subpopulations (Phox2b and Isl1). We show cadherin-7 is expressed in a complete radial domain occupying a lateral region of the hindbrain basal plate. The cadherin-7 domain abuts the medial border of Pax7 expression; this common limit defines, or at least approximates, the basal/alar boundary. The hindbrain branchiomotor neurons originate in the medial part of the basal plate, close to the floor plate. Their cadherin-7-positive axons grow into the alar plate and exit the hindbrain close to the corresponding afferent nerve root. The cadherin-7-positive neuronal cell bodies later translocate laterally, following this axonal trajectory, thereby passing through the cadherin-7-positive basal plate domain. Finally, the cell bodies traverse the molecularly defined basal/alar boundary and move into positions within the alar plate. After the migration has ended, the branchiomotor neurons switch expression from cadherin-7 to cadherin-6B. These findings demonstrate that a specific subset of primary motor neurons, the branchiomotor neurons, migrate into the alar plate of the chicken embryo. Consequently, the century-old concept that all primary motor neurons come to reside in the basal plate should be revised.     

The high incidence of meiotic errors increases with decreased sperm count in severe male factor infertilities

BACKGROUND:The high frequency of aneuploidy sperm raises concerns that there may be an increased incidence of aneuploid offspring in ICSI programmes. In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal and abnormal fertility. METHODS: To understand more clearly the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) patients, multi-colour fluorescence in situ hybridization was used to determine numerical chromosome abnormalities. RESULTS: Increased aneuploidy frequencies for chromosomes 13, 18, 21, X and Y were detected in sperm from OAT patients. The frequencies of diploidy also increased. There were no differences in non-disjunction at meiosis I compared to meiosis II. Sperm count inversely correlated with the frequencies of diploidy, aneuploidies for chromosomes 13 and 21 in OAT patients. Twenty-two cycles of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies. CONCLUSIONS: A higher incidence of meiotic errors and lower sperm counts was found in sperm from OAT patients.     

Heat-shock protein (HSP70-2) allelic frequencies in three distinct Mexican populations

The major histocompatibility complex (MHC) genes are highly polymorphic and therefore have been useful in population genetics and disease association studies. We analyzed restriction fragment length polymorphism of HSP70-2 alleles in healthy unrelated Mestizo, Mazatecan and Nahua populations. Both Indian groups, Mazatecans and Nahuas, were in Hardy-Weinberg equilibrium, while Mestizos were in disequilibrium (chi 2 = 0.399; P < 0.05). The Mazatecan Indians presented a high frequency of BB homozygosity (17.35%) compared to Mestizos (5%) (P = 0.01). Mexican ethnic groups present differences in distribution of BB genotype. The low frequency of BB genotype in Mestizos may be the result of a negative selection process.     

Platelet adhesion onto segmented polyurethane film surfaces modified by addition and crosslinking of PEO-containing block copolymers

Polyethylene oxide (PEO) surfaces were prepared by the addition of PEO-containing amphiphilic block copolymers as surface modifying additives and of dicumyl peroxide (DCP) as a crosslinking agent in segmented polyurethane (PU). PEO-polypropylene oxide-PEO triblock copolymers (Pluronics) with different PEO chain length (from 0 to 98) were used as the surface modifying additives. The PEO additives in the PU film were then crosslinked to be stably entrapped in the PU matrix. The crosslinking was done by free radicals produced from the decomposition of DCP in the film through heating (120 degrees C) or ultraviolet irradiation (254 nm). The surface properties of the PEO additive-entrapped PU films were investigated by the measurement of water contact angles and electron spectroscopy for chemical analysis. The bulk properties such as water absorption, long-term film stability, and tensile strength and elongation at break, were also investigated. It was observed that addition of a small amount (5 wt% based on PU) of the PEO additives resulted in a considerable change of surface characteristics. The PEO additives were stably entrapped in the PU films by crosslinking of them, without significant changes of bulk properties of the films. From the platelet adhesion test on the prepared PEO additive-containing film surfaces, it was observed that the platelet adhesion on the surfaces decreases with increase in PEO chain length of PEO additives. The film surface containing additive with long PEO chains (chain length of 98) was particularly effective in preventing platelet adhesion. The crosslinking of the PEO additives in PU films did not affect the behavior of platelet adhesion on the surfaces; the films with crosslinked PEO additives showed similar platelet adhesion on the surfaces to the films with uncrosslinked ones.     

Expression and changes of HSP70 in the rat forebrain subjected to gamma knife (100Gy) irradiation targeted on the caudate putamen and survived for different times

Forebrain heat shock protein 70 (HSP70) immunohistochemical reactivity was investigated in rats subjected to gamma knife irradiation focusing on the right caudate putamen nucleus. The forebrain sections of all experimental animals were processed with anti-HSP70 antiserum and then by avidin-biotin peroxidase complex immunohistochemistry after gamma ray irradiation with a dose of 100Gy and they each survived for different times (from 30 min to 30 days). Some neurons, glial cells, and endothelial cells were HSP70-like immunoreactivity (HSP70-LI) positive. HSP70-LI was mainly distributed in the target area of irradiation, as well as in non-target regions, e.g. the cortex, hippocampus, and hypothalamus, etc. The expression and change of HSP70-LI from 3 h to 30 days after irradiation followed the following rules: (1) Within 3 to 24 h, the dilated vessels with HSP70-LI endothelial cells were found at first, and a few lightly stained HSP70-LI neurons and glias were observed in the target and non-target regions; (2) In 3-7 days, darkly stained HSP70-LI neurons and glias were apparently increased and formed an expression peak. From 14 to 30 days, HSP70-LI cells were distinctly decreased and became weakly stained or negative. These results suggested that although the irradiation target of the gamma knife was localized, the response to irradiation occurred extensively.     

Sonographic findings of breast hamartoma: emphasis on compressibility

The characteristic features of hamartoma in terms of discrepancies in mammographic and sonographic shapes of the mass were evaluated. We reviewed 16 pathologically proven breast hamartomas, which had undergone preoperative mammography and ultrasonography. All masses were analyzed according to ACR-BIRADS on mammography. On sonography, each mass was analyzed for size, shape, margin, internal echogenicity, and posterior acoustic enhancement. We also analyzed the echogenicity of halo, and compared the characteristic changes in the shape of hamartomas attributable to compression in mammograms and sonograms. The most common sites were at 12 o'clock in the right breast and 2 o'clock in the left. The most common mammographic findings of the hamartomas were a round shape (11/16), a circumscribed margin (13/16), internal fat densities (D4)(16/16) and radiolucent halos (14/16). The most common sonographic findings of the hamartomas were an oval shape (16/16), circumscribed margins (10/16), heterogeneous internal echogenicity (14/16), echogenic (7/16) or echolucent halos (5/16), and posterior enhancements (12/16). The characteristic feature of hamartomas was a change of the mammographic round shape mass into an elongated oval shape mass by sonography (11/11), suggesting the compressibility of hamartomas. Three of the hamartomas contained a pathologically proven internal calcification. The presence of a hamartoma was suggested by a change in a mammographic round mass with a radiolucent halo into an oval heterogeneous mass surrounded by an echogenic or echolucent halo on the sonogram. This characteristic difference between the mammographic and sonographic findings was attributed to the hamartoma compressibility, and was associated with the over-proliferation of fat containing mature normal breast tissue.