Effect of tacrolimus and partial hepatectomy on transthyretin metabolism in rats

Liver transplantation, which serves as treatment of familial amyloidotic polyneuropathy (FAP), and domino liver transplantation, which utilizes resected livers from patients with FAP for treatment of liver diseases, may induce changes in transthyretin (TTR), a pathogenic FAP-related protein. To evaluate this possibility, we performed a 70% hepatectomy or administered tacrolimus to Dark Agouti (DA) rats for 7 days and then measured changes in liver TTR mRNA levels and changes in serum TTR concentrations. After hepatectomy, TTR mRNA levels decreased by 77%; at day 3, they returned to preoperative levels. Except for slightly elevated serum TTR concentrations 12 h after operation, serum TTR levels remained unchanged. Thus, partial hepatectomy did not influence serum TTR concentrations. After tacrolimus administration, TTR mRNA declined by 56% 12 h after the experiment started; however, after day 3, a rebound phenomenon occurred until day 7. Tacrolimus may facilitate serum TTR degradation, although production of TTR in the liver also increased. This finding -- that TTR, the source of FAP-inducing amyloid, did not increase after transplantation -- may help post-transplantation treatment of patients who have FAP and other liver diseases.     

A novel lamina lucida component of epithelial and endothelial basement membranes detected by LH39 monoclonal antibody.

The murine monoclonal antibody, LH39 was characterized in this study and appeared to bind to a novel basement membrane epitope. This antigen was expressed in the epithelial basement membrane of human tissue derived from all three germ cell layers and in basement membranes surrounding small blood vessels within the stroma of all organs examined. LH39 antigen could be first detected in fetal skin at the dermo-epidermal junction at 7 weeks estimated gestational age but was not present in the dermal vasculature until 16 weeks. When tested against tissue from a range of lower mammalian species, LH39 antigen appeared to be primate-specific. The epithelial basement membrane zone in organotypical cultures, where there is de novo synthesis of basement membrane components, contained abundant LH39 antigen in contrast to other basement membrane components, type IV collagen, laminin, and type VII collagen. Ultrastructural localization of LH39 epitope, using immunogold electron microscopy on unfixed freshly frozen tissue, was to the lamina lucida. No cross-reactivity could be detected between LH39 and laminin, fibronectin, and collagens I, III, IV, and V using the ELISA assay. In vitro studies with a range of proteolytic enzymes suggested that the antigen was non-collagenous in nature. LH39 precipitated a polypeptide with a molecular weight of 185 kD from extracts of metabolically labelled cultured keratinocytes, and polypeptides of 185 and 200 kD from the culture medium. The tissue distribution of LH39 antigen suggested that it may be an epitope within anchoring filaments. Potential applications of this antibody include the study of benign and malignant human vascular disorders, diseases and tumours associated with angiogenesis, epithelial neoplasms, and conditions of tissue regeneration and repair, such as wound healing.     

Hypogonadism of male prolactinomas: Relation to pulsatile secretion of LH: Hypogonadisms des Mannes mit Prolaktinomen: Beziehungen zur pulsatilen LH-Sekretion

In order to investigate whether a hypothalamic disorder cause hypogonadism in male prolactinomas, LH pulsatile secretion was studied in 13 male patients. Serum PRL levels ranged from 186 to 45,000 ng ml-1 before treatment, and all the tumors were macroadenomas. Reduced LH secretion was revealed in 5 of 13 patients, and FSH was reduced in 1 of 13. Serum testosterone (T) levels were lower than the normal limit in all the patients. HCG tests in 3 patients showed good responses, but the peak values of T were lower than those of normal men. LH pulsatilities were examined in 5 hyperprolactinemic patients before treatment, in 4 hyperprolactinemic patients after operation, and in 8 normoprolactinemic patients after operation and/or bromocriptine treatment. There was no significant difference of the mean LH values, the frequencies of LH pulses, and amplitudes among the hyperprolactinemic patients before operation (n = 5), the normoprolactinemic patients after operation (n = 8), and normal men (n = 7). From these results, it was evident that the hypothalamus and pituitary function of male prolactinomas were well preserved, in spite of higher serum PRL levels and larger tumor size than those reported in females. It is suggested that the main cause of hypogonadism in these patients is due to testicular dysfunction resulting from excessive serum PRL.     

Differential induction responses of delta-aminolevulinate synthase mRNAs during erythroid differentiation: use of nonradioactive in situ hybridization.

Expression of mRNAs encoding the erythroid-specific delta-aminolevulinate synthase (ALAS-E) and the nonspecific delta-aminolevulinate synthase (ALAS-N) were examined in murine Friend virus-transformed erythroleukemia (MEL) cells using nonradioactive in situ hybridization. Following dimethyl sulfoxide (DMSO) treatment, ALAS-E mRNA increased markedly, while ALAS-N mRNA did not increase in wild-type MEL cells. In contrast, in a DMSO-resistant clone of MEL cells, ALAS-E was not detectable before and after DMSO treatment. These findings suggest that ALAS-E and ALAS-N mRNAs are under separate controls and that the expression of ALAS-E mRNA is a critical event in erythroid differentiation.     

用SYBR Green I实时逆转录-聚合酶链反应定量检测人、小鼠精子中CatSper1 mRNA

目的:建立并优化SYBR GreenI实时RT-PCR体系,定量检测人、小鼠成熟精子中的CatSper1 mRNA。方法:用TRIzol分别提取人、小鼠成熟精子中的总RNA,逆转录后用SYBR GreenI实时PCR定量检测CatSper1 mRNA。SYBR GreenI实时PCR采用普通PCR试剂,加入SYBR GreenI染料,优化退火温度、Mg2+浓度及上、下游引物比例,并在PCR循环时采用四步法以消除引物二聚体的影响。优化完成后用不同浓度的精子cDNA为模板做标准曲线,以检测SYBR GreenI实时PCR的扩增效率。结果:定量检测CatSper1 mRNA的SYBR GreenI实时PCR体系适宜退火温度、Mg2+浓度及上、下游引物比例分别为63℃、3.0mmol/L和1∶1,四步法中采集荧光的温度为88℃。优化后用人和小鼠精子cDNA为模板做标准曲线分别为Y=-3.402log(X)+25.99和Y=-3.409log(X)+24.09,扩增效率分别为96.8%和96.5%,可定量检测人、小鼠成熟精子中的CatSper1 mRNA。结论:用普通的逆转录及PCR系统和试剂,建立了一种方便、廉价、可靠的SYBR GreenI实时荧光定量RT-PCR系统,可用于人、小鼠精子中CatSper1 mRNA定量检测。     

降调节对卵子质量的影响

近10余年来，在体外受精一胚胎移植（IVF-ET）周期中应用促性腺激素释放激素激动剂（GnRH-a）进行控制性卵巢刺激（COS）已成为普遍采用的方法，其主要原因是GnRH-a可以促进卵巢内多个卵泡同步发育和成熟，抑制内源性黄体生成素（LH）峰，阻遏卵泡过早黄素化及卵子早熟。由于GnRH-a在IVF-ET进行降调节COS中的应用不同，可将COS方案分为长方案和短方案。[第一段]     

Differential Cellular Gene Expression in Ganglioglioma

Summary:  Purpose:  Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and that these changes would yield insights into seizure onset and lesion formation.
Methods:  Candidate gene expression was defined in single immunohistochemically labeled neurons and astrocytes microdissected from GG specimens compared with neurons and astrocytes microdissected from morphologically intact cortex adjacent to the GG or normal control cortex.
Results:  Differential expression of 16 genes including glutamate transporter (EAAC1) and receptor (NMDA2C, mGluR5), growth factor (hepatocyte growth factor), and receptor (platelet derived growth factor receptor β, fibroblast growth factor receptor 3) mRNAs was detected in GG neurons compared with control neurons. In astrocytes, altered expression of p75NGF, mGluR3, TGFβ3 and Glt-1 mRNAs was detected. Nestin mRNA, a gene that exhibits enhanced expression in balloon cell cortical dysplasia, was increased in GG neurons. Because of the morphological similarities between GG and cortical dysplasia, we show that there is activation of the mTOR cascade in GG as evidenced by enhanced expression of phospho-p70S6kinase and phosphoribosomal S6 proteins.
Conclusion:  We find differential candidate gene expression in neurons and astrocytes in GG compared with adjacent cortex and show that there is activation of the mTOR pathway. These changes highlight pathways that may be pivotal for epileptogenesis and lesion growth.