beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.     

Tamoxifen treatment failure in cancer and the nonlinear dynamics of TGFbeta

The process of cancer invasion involves a complex interplay between cell-cell and cell-medium adhesion, proteolytic enzyme secretion, cell birth and death processes, random and directed motility, and immune response, as well as many other factors. The growth factor TGF beta is known to have a complex effect on this process. It inhibits mitosis and promotes apoptosis in a concentration-dependent manner in vitro, and it is for this reason that its secretion is thought to be helpful in inhibiting tumour growth. However, recent in vitro and in vivo results have shown a significant effect of this growth factor in promoting the sensitivity of malignantly transformed cells to gradients of extracellular matrix proteins--an effect which tends to increase invasiveness. The drug tamoxifen has been demonstrated to be therapeutically effective in the treatment of patients with breast cancer; however, it is known also that many patients become resistant to the effect of this drug after a few years, and the reasons for this remain controversial. In this work we take our established model of cancer invasion (J. Theor. Biol. 216(1) (2002) 85), and extend it to include the effect of TGF beta. In so doing we demonstrate that a tamoxifen-stimulated upregulation of the secretion of TGF beta may give rise to a tumour which has a smaller number of cells but which has a greater invasiveness, greater metastatic potential, and a tumour histology which is known to correlate with a poorer prognosis. These data suggest that tamoxifen-stimulated secretion of TGF beta might explain treatment failure in some patients.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Antifibrotic effect of the Chinese herbs, Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis

Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects. METHODS: Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n = 7) or enalapril 10 mg/kg/d (n = 7). Normal control rats (n = 7) received saline rather than puromycin, and an untreated control group (n = 7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-beta1 (TGFbeta1), osteopontin, ED-1-positive cells, and alpha-actin. TGFbeta1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and alpha-actin positive cells and expression of osteopontin compared to untreated controls. The combination of Astragalus and Angelica retarded the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFbeta1 and osteopontin, reduction of infiltrating macrophages, and less activation of renal intrinsic cells [corrected].     

TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

非天然氨基酸β-Homo天冬酰胺的合成研究

以α-氨基酸天冬酰胺为原料,经多步反应合成了β-氨基酸β-Homo天冬酰胺,中间产物和目的产物经熔点、红外、核磁、质谱以及元素分析,其结构得到证实。     

1型糖尿病葡萄糖代谢与胰岛素信号传导途径异常导致大鼠海马Alzheimer样tau蛋白过度磷酸化修饰

Tau蛋白过度磷酸化是Alzheimer病(Alzheimer disease, AD)的一个重要病理特征.采用 I 型糖尿病大鼠模型,研究胰岛素信号传导途径及葡萄糖代谢失调对tau蛋白过度磷酸化的形成机制进行探讨.以同龄Wistar大鼠做对照（CTL）,胰腺大部分切除造低胰岛素组（PX）,STZ较大剂量一次性注射造1型糖尿病模型即低胰岛素高血糖组（T1DM）.葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,蛋白质印迹分析海马内总tau蛋白及tau蛋白上部分位点（Ser199、Thr212、Ser214、Ser396及Ser422）的磷酸化及神经细胞膜上葡萄糖转运子3（Glucose transport 3,GLUT3）水平.γ-32P-ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合成酶激酶-3β（Glycogen synthase kinase-3β, GSK-3β）活性.发现3组大鼠海马回总tau蛋白水平无显著差异,但以高血糖、低胰岛素血症为特征的T1DM组在tau蛋白Ser199、Thr212、Ser214、Ser396及Ser422位点上,呈现过度磷酸化状态,以低胰岛素血症为特征而血糖正常的PX组在位点Ser199、Thr212及Ser396上磷酸化程度比CTL组显著上升, 在位点Ser214及 Ser422上的磷酸化程度的改变不显著;T1DM及PX组大鼠海马 GSK-3β活性显著高于CTL组, 而GLUT3水平在T1DM和PX组均降低, 尤以T1DM组降低更显著.研究结果显示,胰岛素水平低下可能通过激活GSK-3β和下调细胞内葡萄糖代谢的双重作用引起脑内tau蛋白过度磷酸化.