神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究

目的：在原核表达系统中表达对凋亡神经元具有保护作用的重要蛋白Bcl-XL与蛋白质转导序列（PTD）的融合蛋白，并检测重组蛋白对重金属离子所诱导的细胞凋亡的保护作用。方法：通过RT-PCR的方法，用Bcl-XL特异引物从乳腺癌细胞系MCF-7细胞的总RNA中扩增出Bcl-XL基因，构建相应的原核表达载体，体外表达的TAT-Bcl-XL融合蛋白经镍亲和层析介质纯化后，通过免疫荧光方法检测其转导293T细胞的能力；并用流式细胞仪检测融合蛋白抑制细胞凋亡的能力。结果：经RT-PCR从MCF-7细胞总RNA中得到相应的TAT-Bcl-XL基因片段，并将其克隆入pCRT7/CT-TOPO载体中，重组质粒pTBTOPO转化大肠杆菌后，在SDS-PAGE和Western blot的结果中出现了与预期分子量相同的蛋白条带和阳性信号，纯化的TAT-Bcl-XL重组蛋白经免疫荧光检测，主要分布于细胞的细胞质中。流式细胞仪的检测结果显示融合表达蛋白可以有效地抑制Zn2＋离子所诱导的细胞凋亡，使细胞的存活率提高40%。结论：TAT-Bcl-XL融合蛋白在大肠杆菌中获得高效表达，初步的功能性检测表明融合蛋白具有抑制细胞凋亡的功能。     

Premature ovarian failure

Premature ovarian failure (POF) causing hypergonadotrophic hypogonadism occurs in 1% of women. In majority of cases the underlying cause is not identified. The known causes include: (a) Genetic aberrations, which could involve the X chromosome or autosomes. A large number of genes have been screened as candidates for causing POF; however, few clear causal mutations have been identified. (b) Autoimmune ovarian damage, as suggested by the observed association of POF with other autoimmune disorders. Anti-ovarian antibodies are reported in POF by several studies, but their specificity and pathogenic role are questionable. (c) Iatrogenic following surgical, radiotherapeutic or chemotherapeutic interventions as in malignancies. (d) Environmental factors like viral infections and toxins for whom no clear mechanism is known. The diagnosis is based on finding of amenorrhoea before age 40 associated with FSH levels in the menopausal range. Screening for associated autoimmune disorders and karyotyping, particularly in early onset disease, constitute part of the diagnostic work-up. There is no role of ovarian biopsy or ultrasound in making the diagnosis. Management essentially involves hormone replacement and infertility treatment, the only proven means for the latter being assisted conception with donated oocytes. Embryo cryopreservation, ovarian tissue cryopreservation and oocyte cryopreservation hold promise in cases where ovarian failure is foreseeable as in women undergoing cancer treatments.     

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.     

UNILATERAL BRAIN DAMAGE AND BILATERAL SKIN CONDUCTANCE LEVELS IN HUMANS

Left and right, palmar and dorsal skin conductance levels (SCLs) were obtained from hospital controls, left hemisphere lesion Ss, right hemisphere lesion Ss, and diffuse or bilateral lesion Ss during several experimental conditions involving rest, passive auditory stimulation, motor reactions, and simple “perception”. The unilateral lesion groups generally displayed significantly higher palmar SCLs on the side contralateral to their lesion. Such “laterality” was not demonstrated in dorsal recordings or in the hospital controls or diffuse lesion group. These unilateral lesion groups had higher palmar SCLs during passive stimulation than during rest, motor, or perception phases. Results were discussed in terms of possible neural mechanisms underlying the phenomena.     

密质骨损伤机理的实验研究

用Ｉｎｓｔｒｏｎ试验机对新鲜夫股骨密质骨试样进行一维拉伸实验研究，利用实验结果对由平行粘弹性杆系统建立的密质骨损伤本构模型待定参数进行拟合，获得较高的相关系数和较好的显著性水平。     

Modulation of osteopontin in proteinuria-induced renal interstitial fibrosis

Proteinuria is associated with macrophage-dependent interstitial fibrosis (IF). Osteopontin (OPN), a macrophage chemoattractant, may be involved in the transition of proteinuria to IF but protective properties have also been reported. To elucidate whether OPN may be involved in the proteinuria-induced cascade of tubulointerstitial damage, renal expression of OPN was studied during the development of proteinuria-induced renal damage and during anti-proteinuric intervention with ACE inhibition (ACEi). First, the temporal relationships between proteinuria, interstitial OPN induction, and IF in adriamycin nephrosis (AN), a model of chronic proteinuria-induced renal damage, were studied. Second, the effect of anti-proteinuric treatment on OPN expression was investigated. The time course of OPN induction and markers of renal damage was studied in rats with unilateral AN at 6-week intervals until week 30. In a second study, a renal biopsy was taken 6 weeks after induction of bilateral AN; subsequently, rats were treated with ACEi until termination (week 12). In unilateral AN, proteinuria developed gradually and stabilized at week 10. In proteinuric kidneys, OPN expression was induced from week 12 onwards. Simultaneously, a progressive increase in interstitial macrophages, alpha-smooth muscle actin (alpha-SMA), collagen type III, and focal glomerulosclerosis (FGS) was observed. In bilateral AN, ACEi reduced proteinuria and OPN protein and stabilized fibrosis. In untreated animals, OPN mRNA increased, with stable OPN protein and fibrosis and increased FGS. Thus, in AN, development of proteinuria is followed by up-regulation of OPN along with markers of renal damage. The up-regulation of OPN is reversible by anti-proteinuric treatment without a corresponding reduction in fibrosis. Whereas these data are consistent with a role for OPN in the cascade of transition from proteinuria to fibrosis, intervention with ACEi showed that reduction of OPN does not attenuate established fibrosis.     

Antistressor effect of dalargin in rats with immobilization stress

Laboratory of Experimental Surgery, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR M. I. Kuzin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 6, pp. 617–619, June, 1991.     

Analysis of 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation

In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20-60 Gy of gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA.     

Increase in ferric and ferrous iron in the rat hippocampus with time after kainate-induced excitotoxic injury

The present study aimed to elucidate the distribution of ferric and ferrous iron in the hippocampus after kainate-induced neuronal injury. A modified Perl's or Turnbull's blue histochemical stain was used to demonstrate Fe3+ and Fe2+ respectively. Very light staining for iron was observed in the hippocampus, in normal or saline-injected rats and 1-day post-kainate-injected rats. At 1 week postinjection, a number of Fe3+-positive, but very few Fe2+-positive, cells were present, in the degenerating CA fields. At 1 month postinjection, large numbers of Fe3+-positive glial cells, and some Fe2+-positive blood vessels, were observed. At 2 months postinjection, large numbers of Fe3+- and Fe2+-positive glial cells were present. The labeled cells had light and electron microscopic features of oligodendrocytes, and were double labeled with CNPase, a marker for oligodendrocytes. The observation of an increasing number of Fe3+- and Fe2+-positive cells in the degenerating hippocampus with time is consistent with the results of a nuclear microscopic study, in which an increasing amount of iron was detected in the degenerating hippocampus after kainate injection. In addition, the present study showed a shift in the oxidation state of the accumulated iron, with more cells becoming Fe2+ at a late stage. A possible consequence of the high amounts of Fe2+ in the hippocampus after kainate injection is that it could promote free radical damage in the lesioned areas.     

Enhanced sprouting of retinotectal fibers after early superior colliculus lesions in hamsters treated with gangliosides

Summary The effects of exogenous gangliosides on sprouting of optic tract axons was studied in hamsters which, after a right tectal lesion on the day after birth (P1), had an abnormal retinotectal projection from the left eye to the left superior colliculus (SC). Sprouting of these axons was induced by removing the competing input by right eye removal on postnatal day 9 (P9). Intraperitoneal G_M1, given daily and started on P9, significantly stimulated the sprouting response. This was demonstrated by Fink-Heimer silver staining of anterograde axonal degeneration three days after the left eye was removed on P36. Terminal fields in the left SC were, in average, twice as large compared to controls. An estimate of the total number of terminals (silver stained particles) revealed a value of 7.9×10⁶ for G_M1 and 3.2×10⁶ for control hamsters, respectively. Diencephalic structures which also receive collateral input from the sprouting optic tract did not show any alterations in the size of the terminal field due to G_M1-treatment, suggesting that, in vivo, gangliosides fail to initiate sprouting in areas that have not previously been denervated. Unexpectedly, G_M1-treated hamsters also had significantly smaller right SC damage and less left damage near the midline. Subsequent reanalysis of the data based on a lesion-matching procedure indicates that effects on reducing atrophy were independent of the G_M1-enhanced sprouting of retinofugal axons. These findings provide the first direct evidence that exogenous G_M1 stimulates lesion-induced axon sprouting in the mammalian brain.