人睾丸酵母双杂交cDNA文库的构建与鉴定

目的:构建应用于酵母双杂交系统的人睾丸组织cDNA文库。方法:从正常人睾丸组织总RNA中纯化出mRNA,以SMARTШTM和CDSШ为引物,合成两端具有同源臂的双链cDNA,后者与线性质粒pGADT7-Rec共转化入感受态酵母细胞Y187,两者在酵母细胞中同源重组成环形的cDNA文库质粒,收集在营养缺陷培养板上筛选出的所有克隆,即为人睾丸酵母双杂交cDNA文库。结果:构建了具有基因多样性和库容量足够大的人睾丸cDNA文库,共获得2×106个转化子,插入的双链cDNA片段的长度在0.3～4.0 kb之间。结论:利用ClontechSMART技术,成功构建了应用于酵母双杂交的人睾丸组织cDNA文库,为探讨人精子发生的分子机制奠定了基础。     

Primary structure of mouse and rat nephrin cDNA and structure and expression of the mouse gene

Nephrin is a central component of the glomerular podocyte slit diaphragm and is essential for the normal renal filtration process. This study describes the complete structure of the mouse nephrin gene, which was shown to be homologous to the human gene, the major difference being 30 exons in the mouse gene as opposed to 29 in human. The complete primary structure of mouse and rat nephrins was also determined. The sequence identity between the mouse and rat proteins was shown to be 93%, while both rodent proteins have only about 83% sequence identity with human nephrin. The availability of the three mammalian sequences is significant for the interpretation of sequence variants and mutations in the nephrin gene in patients with congenital nephrotic syndrome. In situ hybridization analyses of whole mouse embryos and tissues revealed high expression of nephrin in kidney glomeruli and, surprisingly, an intense and highly restricted expression in a set of cells in hindbrain and spinal cord. No expression was observed elsewhere. This expression pattern may explain occasionally occurring neural symptoms caused by inactivating mutations in the nephrin gene in patients with congenital nephrotic syndrome.     

Isolation and preliminary characterization of ACNU-resistant sublines of rat brain tumors in vivo.

Two variant cells lines resistant to the nitrosourea derivative ACNU ((1-4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride), namely C6/ACNU and 9L/ACNU, were selected in vivo from rat brain tumors. Stable resistance to ACNU proved to be a characteristic of these cell lines, whether they were grown in vivo or in vitro. These cell lines exhibited a different pattern of cross-resistance to a wide range of chemotherapeutic agents with dissimilar chemical structures and mechanisms of action as compared with that of other ACNU-resistant cell lines established in vitro. Distinct cross-resistance was observed in both the C6/ACNU and 9L/ACNU cell lines to chloroethyl-nitrosoureas such as BCNU (carmustine), CCNU (lomustine), and methyl CCNU and, additionally, to vincristine, vinblastine, Adriamycin (doxorubicin), and arabinosylcytosine, but not to bleomycin, methotrexate, cis-platinum, and 5-fluorouracil. This might point to a multifactorial mechanism of drug resistance in ACNU-resistant cell lines derived from rat C6 and 9L brain tumor cells.     

Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line

A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.     

乳鼠成骨细胞酵母双杂交cDNA文库构建

目的构建乳鼠成骨细胞的酵母双杂交cDNA文库。方法用TRIzol法提取乳鼠成骨细胞总RNA,按照SMART cDNA Library Construction Kit ( CLONTECH)说明书反转录合成双链cDNA.Spin Column去除短片段,然后用同源重组的方法将双链 cDNA和PGADT7~rec载体共转化到酵母细胞Y187中,建立文库并计算文库滴度。结果提取的总RNA的A 260/A 280为 1.99,琼脂糖凝胶电泳示28SrRNA、18SrRNA2条带,且28S与18S带亮度比值约为2。酵母文库库容为1. 68 x 107,重组率为 100%。插人片段PCR检测提示大小分布为1. 5 -4. 0 kb,平均长度约为3. 0 kb。结论乳鼠成骨细胞酵母双杂交cDNA文 库构建成功。     

ACNU-resistant mutants of 9L rat glioma cell line. Isolation and preliminary characterization of these subclones

Three ACNU-resistant subclones were isolated and characterized from a wild-typed 9L rat glioma cell line in culture. At an early stage after cloning, these ACNU-resistant subclones showed a high frequency of chromosomal aberrations compared with nonresistant 9L cells. These ACNU-resistant subclones revealed a cross resistance to BCNU, CCNU, methyl CCNU, nitrogen mustard, cyclophosphamide, and cis-platinum, which are alkylating agents. Further studies are necessary to clarify the mechanisms of ACNU-resistance from the aspect of repair of DNA alkylation damage.     

Preparation and characterization of a mouse osteoclast-like multinucleated cell population.

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.     

The experimental model of hydatidosis in rabbit testis. A preliminary study

OBJECTIVES: Although no part of the human anatomy is invulnerable to hydatid disease, it has been reported to occur mostly in vital organs such as liver, lungs and brain. Hydatid disease of the urinary tract is uncommon accounting for only 2% of all such cases. Testes are extremely rare sites for echinococcosis. To our knowledge there are only 3 cases of testicular hydatid cyst described. In this animal model, we studied echinococcosis in rabbit testis. METHODS: We directly infected the unilateral testis of 4 male rabbits with infective protoscolices via intratesticular injection and housed them under pathogen-free conditions for 10 weeks. All rabbits survived during the follow-up period and then, at the 10th week, all testes were removed for histopathologic investigation. RESULTS: Despite of the direct infection of the rabbit testes with Echinococcus granulosus, there was no demonstrable hydatid cyst after a 10-week period except from some fibrosis in the injection tract of the testicular tissue in 1 case. CONCLUSION: The testes are extremely rare anatomic locations for echinococcosis infection. The mechanism of this resistance should be another reason apart from blood-testicular barrier. We think that low temperature in the scrotum or different properties of the testicular tissue may be the reasons of this defense mechanism. If this hypothesis clarifies with the further studies, new treatment options may be defined in the medical literature for the hydatid cyst.     

Response of rat testis to localized induced hyperthermia.

A method is presented in which the rat testis is extensively mobilized through a low abdominal incision, but in which its blood supply is carefully preserved. Localized hyperthermia is induced in this mobilized testis by water bath immersion. The tissue temperature is measured during and after immersion by means of a thermocouple inserted into the tissue. The relative sensitivity of spermatogenic tissue to increased temperature is confirmed and the relative resistance of Sertoli and Leydig cells is noted. Minimal or absent inflammatory reaction to thermal destruction of testicular cells is found as long as the tubule is intact. A marked peritesticular inflammatory response is noted when the total testicular tissue is destroyed at the highest temperature tested.     

Iron-induced injury of rat testis

Summary. Male Wistar rats were intraperitoneally injected twice with 0.4 mmol kg⁻¹ FeSO₄. One, 2 and 4 days after the second Fe injection, Fe and malondialdehyde (MDA) content in testis was measured, the morphology studied by light and electron microscopy and the number of spermatids counted. After Fe injection, Fe and MDA content had increased in parallel. Light microscopic inspection on days 1 and 2 after Fe injection revealed numerous necroses in the different cell types of the germ epithelium. Four days after Fe injection, fewer alterations were found. Electron microscopic investigations revealed that some spermatids contained up to three nuclei and at least three axonemes. In some sperm tails up to 11 axonemes were found. In some midpieces two or three complexes of axonemes, outer dense fibres and mitochondria were observed. In other midpieces axonemes were absent and replaced by granular and filamentous material. The number of spermatids was reduced 4 days after Fe treatment. The increase in the number of axonemes was similar to that seen in Mg and Zn deficiency, indicating that the increase in Fe content and oxygen free radicals is the major reason for the biochemical and morphological alterations in Mg and Zn deficiency.     

Ultrastructural study of foetal and adult mouse renal tissue implanted in testis.

Kidneys of mice foetuses 15, 17, 19 days old, as well as kidneys of mice 1 and 4 weeks old of the pure C3H/SY species, were implanted into the right testis of 40 adult mice 1.5 to 2 months old, of the same species. The animals were sacrificed after 30 and 60 days and the evolution and development of the renal implants within the testis ware studied. The findings in the light as well as in the electron microscope, showed that the renal implants presented the histologic characters of nephroblastoma, which became clearer the more prolonged the time of the implantation was.     

Antioxidant enzyme activity in the maturing rat testis.

Developmental profiles of the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), and hexose monophosphate shunt (HMS) were measured in the rat testis and liver. The level of SOD in the testis was high at the age of 6 to 10 days, after which it dropped to approximately one third of that level by 20 days of age, and remained there up to 8 months of age. In the liver, SOD activity steadily increased from the neonatal to adult stage of life, reaching the same level as detected in the testis. The testicular activity of catalase was only 2% to 7% of that found in liver at all ages. It increased in both organs up to 6 weeks of age, whereafter the hepatic activity gradually decreased and no further changes were seen in the testis. The GSH-Px activity was low in the testis and declined slightly with age, whereas activity in the liver increased four-fold between birth and adulthood. The activity of GSH-Tr was similar in both organs studied: it increased after birth, showing a maximum in the liver at 1.5 months (ten-fold increase) and in the testis at 5 months of age (four-fold increase). The HMS activity was two to three times higher in the liver than in the testis, and decreased slightly with age in both organs. Thus, the basal levels and developmental profiles of antioxidant enzymes in the testis differ greatly from those in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries

OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).     

Expression of claudin-1 in mouse testis

In testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of the blood-testis barrier (BTB) and crucial for spermatogenesis. The present study aimed to find postnatal changes in the expression of claudin-1, one of the TJ genes in mouse testis. By semiquantitative RT-PCR, it was found that claudin-1 expression in testis increased up to a peak at 10 days after birth and decreased thereafter. Western blot analysis showed abundant expression of 21-kDa protein in testis, lung, and brain from the adult mouse. The developmental change in the expression of claudin-1 protein in testis coincided with that from the RT-PCR. Testosterone treatment significantly increased claudin-1 expression in immature Sertoli cells, suggesting the possible regulation of claudin-1 expression by androgen in mouse Sertoli cells. Claudin-1 expression appears to be developmentally regulated in the mouse testis.     

Trapeziometacarpal denervation. Description of surgical technique and preliminary results from a prospective series of 14 cases

INTRODUCTION: The main goal of first carpometacarpal arthritis surgical treatment is to relieve pain. The main disadvantages of the usual techniques (trapeziectomy, implant arthroplasty) are loss of strength or presence of a prosthetic device. It is difficult to propose such extensive surgery at an early stage of the disease. Selective denervation of the first carpometacarpal joint seems to be an interesting choice. We propose a new technique of denervation based on our previous anatomical investigations. TECHNIQUE: Two incisions are needed to cut all the articular branches derive from the superficial branch of the radial nerve, the palmar cutaneous branch of the median nerve, the thenar branch of the median nerve and the lateral ante brachial cutaneous nerve. MATERIAL: Fourteen patients were prospectively included in our study with a mean follow-up of 5 months. RESULTS: Pain relief was very satisfying in 12 cases (mean decrease 84%). An increase in grip and key pinch strength was noted. Complications were uncommon, excepted temporary paresthésia in the radial nerve area. DISCUSSION: This technique seems to be promising and a good indication for patients with no disabling deformity, but only long-term results will confirm the place of denervation in the treatment of first carpometacarpal arthritis.     

Temperature-dependent expression of a 95 kD protein in rat testis

Summary.  Testicular tissue from mature male Sprague-Dawley rats was maintained in culture at 33 °C or 37 °C. Detergent-extracted proteins were separated by polyacrylamide gel electrophoresis (PAGE) followed by staining or fluorography. Unilateral surgical cryptorchidism was performed as an in vivo model for testicular exposure to abdominal temperature. Testes were harvested at various time points, followed by protein analysis as performed for in vitro studies. Tissue incubated in vitro for 48 h at 37 °C demonstrated loss of a prominent actively synthesized 95 kD protein (p95) seen at 33 °C. No other temperature-dependent protein changes were observed. Liver, kidney, spleen and thymus failed to reveal p95 or any other temperature-sensitive proteins when incubated at 33 °C and 37 °C. Surgical cryptorchidism resulted in loss of p95 by 1 week in abdominal testes and normal p95 expression in sham-operated scrotal testes. p95 appears to be a temperature-sensitive protein in rat testis, with increased degradation accounting for its loss at abdominal temperature. The failure to identify similar protein changes in other tissues suggests a role for p95 in the temperature-dependent function of the testis.