Induction of amyloid precursor protein mRNA after heat shock in cultured human lymphoblastoid cells.

In an attempt to examine a possible relationship between heat shock stress and an induction of amyloid precursor protein, cultured lymphoblastoid cells established from 12 human subjects were treated with heat shock at 42 degrees C for 30 min. The levels of mRNA for amyloid precursor protein (APP), heat shock protein (HSP) 70, and actin were examined by Northern blot at 1, 3, 8, 24, and 48 h after the heat shock treatment. HSP70 mRNA was induced at 1 and 3 h, and became undetectable again by 8 h. APP mRNA was also induced at 3 and 8 h, and recovered to the steady level by 48 h. No induction was observed in actin mRNA. These results indicate that APP mRNA is induced by heat shock treatment after the induction of HSP70 mRNA, suggesting a role of heat shock response in an induction of APP.     

骨骼肌小热休克蛋白在离心运动后的表达

背景：研究证明离心方式的训练可使骨骼肌产生保护作用,避免离心运动引起的损伤,但是机械负荷引起的小热休克蛋白的保护作用至今却少有报道。 目的：观察骨骼肌小热休克蛋白家族中αB-晶体蛋白在离心运动后的表达,以此探讨机械负荷诱导的小热休克蛋白对骨骼肌细胞的保护作用机制。 方法：将Wistar大鼠随机分为安静对照组和运动训练组。运动训练组使用动物跑台进行6周间歇性离心运动训练,每周训练5 d,安静对照组正常喂养。训练6周休息48 h后,安静对照组与运动训练组随机选出6只大鼠做1次性大负荷离心运动。观察两组血清肌酸激酶变化;蛋白免疫印迹法检测两组腓肠肌αB-晶体蛋白含量变化,用免疫荧光组织化学法分析两组腓肠肌αB-晶体蛋白亚细胞表达特征。 结果与结论：大负荷离心运动后,安静对照组血清肌酸激酶与运动前相比显著增高(P< 0.05),说明骨骼肌细胞出现严重的损伤,而运动训练组这种损伤不明显。蛋白免疫印迹结果表明,运动训练组做一次性大负荷离心运动后αB-晶体蛋白表达水平比安静对照组增加(P < 0.05)。从免疫荧光组化切片可见,αB-晶体蛋白在细胞内发生了移位变化,从胞浆移位于Z盘和细胞膜。提示αB-晶体蛋白在离心运动训练后表达增多,并通过移位于肌细胞Z-盘和细胞膜发挥对骨骼肌细胞的保护作用。     

Expression of 60-kD heat shock protein increases during carcinogenesis in the uterine exocervix.

OBJECTIVES: The aim of the present study was to determine the presence and expression of the 60-kD heat shock protein (HSP60) in the dysplasia-carcinoma sequence in the uterine exocervix and to evaluate its diagnostic and prognostic significance. METHODS AND RESULTS: We performed Western blot and immunohistochemical analyses on biopsies from 40 cases, consisting of 10 normal exocervical biopsies, 10 low-grade squamous intraepithelial lesions (L-SIL), 10 high-grade squamous intraepithelial lesions (H-SIL) and 10 cancerous exocervices (G2 grade). The immunohistochemical results were quantified by computer-assisted image analysis. Western blot analysis showed that HSP60 was undetectable in normal tissues and that there was a gradual increase of protein expression from L-SIL to carcinoma. Immunostaining for HSP60 was negative in normal tissue and positive in basal and parabasal layers of L-SIL epithelium; H-SIL were markedly stained in all layers of epithelium, and carcinomas showed an even stronger positivity. The increasing expression correlated with the malignancy grade. Finally, koilocytes were mostly negative in L-SIL and positive in H-SIL. CONCLUSIONS: The increasing degree of expression of HSP60 from L-SIL to carcinoma and the different intraepithelial distribution between L-SIL and H-SIL could be used as a new diagnostic tool. Moreover, HSP60 could have a role in cervical carcinogenesis.     

P2 receptor stimulation induces amyloid precursor protein production and secretion in rat cortical astrocytes

Amyloid precursor protein (APP) is ubiquitously expressed in a variety of tissues but is predominantly expressed in the brain. The expression of APP has been well studied in neurons but little is known about its presence in astrocytes. The study presented here shows that purinergic signaling is involved in the production and secretion of APP in primary cultures of rat cortical astrocytes. Extracellular ATP caused an increase in APP production and release in a time- and concentration-dependent manner and was inhibited by antagonists of P2 receptors. Further agonist and antagonist studies revealed involvement of P2Y2 and P2Y4 receptors in nucleotide-stimulated production and release of APP. In addition, signaling studies with various protein kinase inhibitors demonstrated that blockade of mitogen-activated protein kinases, but not Akt, inhibited nucleotide-stimulated APP expression and release. These results indicate that APP production and secretion can be regulated by activation of P2Y2/4 receptors coupled to protein kinase signaling pathways and suggest that astrocytes can be a potential source of APP.     

大鼠角膜碱烧伤后热休克蛋白70的表达

背景：角膜碱烧伤后损伤修复受许多因素的影响,热休克蛋白可促进变性、损伤蛋白质的迅速恢复或清除。 目的：观察大鼠角膜碱烧伤后热休克蛋白70的表达及其与角膜损伤修复的关系。 方法：检查大鼠眼无炎症及其他病变后,奥布卡因滴眼液点眼2次,棉签吸除结膜囊液体,将统一规格直径5 mm的滤纸片浸泡于1 mol/L NaOH溶液中10 s,然后置于大鼠角膜中央30 s制作大鼠角膜碱烧伤模型。分别于碱烧伤后6 h,1,3,7,14,21 d取材。 结果与结论：RT-PCR、免疫组织化学染色、Western blot结果均显示热休克蛋白70 mRNA和蛋白在角膜碱烧伤后1 d即开始升高,7 d时达高峰,14 d后开始下降。苏木精-伊红染色及电镜观察显示角膜损伤在烧伤后6 h即较明显,烧伤后7 d逐渐恢复。提示大鼠角膜碱烧伤后热休克蛋白70的表达与碱烧伤后角膜损伤修复过程一致,参与了大鼠角膜碱烧伤后细胞的自我保护及修复过程。     

热休克蛋白47和热休克蛋白60在小鼠胚胎器官形成过程中的表达

目的：研究小鼠胚胎器官形成过程中热休克蛋白47（Hsp47）和热休克蛋白60（Hsp60）的表达情况。方法：于GD11～GD18,取得小鼠胚胎脑、眼、心、肺、胃、肝、四肢,于GD13-GD18取得肾,利用RT-PCR方法半定量检测Hsp47和Hsp60在各器官的表达丰度。结果：Hsp47在GD11～GD12和GD18的脑、GD11～GD12和GD17～GD18的眼、GD11～GD12和GD16～GD18的肺、GD11-GD18的肝脏不表达,在其余时间和其余器官均有表达;Hsp60在CD11～GD18时段胚胎的脑、眼、心、肺、胃、肝、肾（GD13～GD18）、四肢中均有表达。结论：Hsp47和Hsp60在小鼠胚胎器官形成过程中有不同的表达丰度,其表达模式也不一致;Hsp47在小鼠胚胎发育过程中可能有重要功能,Hsp60则具有广泛表达的特点。     

Regulation of amyloid precursor protein processing by Abeta in human glioma cells

Amyloid precursor protein (APP) is cleaved to neurotoxic/proinflammatory amyloid beta protein (Abeta) or to the neuroprotective secreted alpha-APPs. A balance in APP metabolism may influence the outcome between toxicity and protection to central nervous system (CNS) neurons in Alzheimer's disease. Treatment of U-373 MG astrocytoma cells with aggregated Abeta (1-40) decreases APP secretion into the medium to 10-30% of control values. This decreased secretion appears to be specific for APP since Abeta treatment causes an approximately 2-fold increase in interleukin-8 (IL-8) secretion. Abeta treatment also causes a 4- to 9-fold increase in total cell-associated APP. This increase is due to cellular retention of alpha secretase-cleaved APP and a 2-fold increase in mature full-length APP. These data suggest that deposition of aggregated Abeta may contribute to Alzheimer's-associated neurotoxicity by altering the metabolism of the APP protein. Abeta may exert harmful effects by decreasing the secretion of neuroprotective or neurotrophic APP and, in addition, by increasing intracellular full-length APP; thereby providing increased substrate for generation of amyloidogenic peptide within astrocytes.     

Infertility: Expression of heat shock protein 70 kDa in human endometrium of normal and infertile women

The expression of heat shock protein (hsp) 70 was investigatedin endometrial samples from patients with unexplained infertilityassociated (n = 5)or not associated (n = 10) with endometriosis,and compared with a control group consisting of fertile women(n= 27) with reported menstrual disturbance. The expression ofhsp, and in particular hsp 70, is up-regulated in response tomany physico-biochemical insults as well as infection and possiblyoncogenic transformation and is a good indicator of a biologicalsystem under stress. A significant over-expression of hsp 70was found in the infertile groups (P < 0.001), suggestingthat a stress response may be involved in the aetiology of unexplainedinfertility irrespective of the presence of endometriosis.     

脑死亡致肺损伤家兔热休克蛋白70的表达与作用

背景：脑死亡供体已成为当前器官移植的主要来源。研究有效的方法与手段,保护供体器官,提高器官质量至关重要。 目的：观察热休克蛋白70在脑死亡状态致肺损伤中的作用。 方法：60只家兔随机均分为3组,即正常对照组：不行手术;假手术组：行股动脉插管、气管插管及颅骨钻孔置管术,不行颅内加压脑死亡术;脑死亡组：行股动脉插管、气管插管、颅骨钻孔置管及颅内加压脑死亡术,呼吸机维持脑死亡状态。各组均在术后2,4,6,8 h记录动脉血压及心率的变化。苏木精-伊红染色光镜观察肺脏结构改变,RT-PCR和免疫组化检测各组肺脏热休克蛋白70的mRNA及蛋白表达。 结果与结论：脑死亡组较假手术组血压与心率尚未发现显著变化(P > 0.05)。假手术组各时间点肺脏组织损伤不明显。2-6 h内,随着时间延长,脑死亡组家兔肺脏损伤逐渐加重(P < 0.05),但8 h出现一定程度好转。热休克蛋白70 mRNA与蛋白表达呈时间依赖性增高(P < 0.05),始于2 h,于8 h最高。结果证实,热休克蛋白70可能介入脑死亡状态诱导的肺脏损伤,发挥自身防御性保护作用。     

Coexistence of Alzheimer's amyloid precursor protein and amyloid protein in cerebral vessel walls

Polyclonal antibodies to synthetic peptides homologous to amino acid residues 45-62, 597-624, and 676-695 of the predicted sequence of Alzheimer's amyloid precursor protein (APP) were used to investigate the site of origin of APP, and the relationship between APP and amyloid protein in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D). Cortical sections as well as homogenates of isolated leptomeningeal and cortical microvessels from three patients with AD, two patients with HCHWA-D, and two nondemented controls were probed. In vessel extracts of both groups of patients and the controls, APP was detected as a set of proteins with electrophoretic mobility of 105 to 135 kilodaltons. In cortical sections of all subjects, APP immunoreactivity was found in leptomeningeal and cortical vessel walls. In patients with AD and HCHWA-D, APP and amyloid fibrils coexisted in the same vessels. Moreover, APP immunoreactivity was found in association with 50% of senile plaques in AD brains, but was not evidenced in parenchymal amyloid deposits in patients with HCHWA-D. These data suggest that the vascular system is a source of APP and that the processing of APP into insoluble fibrils in AD and HCHWA-D may take place in situ.     

Caspase-cleaved amyloid precursor protein in Alzheimer's disease

Caspase-3 mediated cleavage of the amyloid precursor protein (APP) has been proposed as a putative mechanism underlying amyloidosis and neuronal cell death in Alzheimer's disease (AD). We utilized an antibody that selectively recognizes the neo epitope generated by caspase-3 mediated cleavage of APP (alphadeltaC(csp)-APP) to determine if this proteolytic event occurs in senile plaques in the inferior frontal gyrus and superior temporal gyrus of autopsied AD and age-matched control brains. Consistent with a role for caspase-3 activation in AD pathology, alphadeltaC(csp)-APP immunoreactivity colocalized with a subset of TUNEL-positive pyramidal neurons in AD brains. AlphadeltaC(csp)-APP immunoreactivity was found in neurons and glial cells, as well as in small- and medium-size particulate elements, resembling dystrophic terminals and condensed nuclei, respectively, in AD and age-matched control brains. There were a larger number of alphadeltaC(csp)-APP immunoreactive elements in the inferior frontal gyrus and superior temporal gyrus of subjects with AD pathology than age-matched controls. AlphadeltaC(csp)-APP immunoreactivity in small and medium size particulate elements were the main component colocalized with 30% of senile plaques in the inferior frontal gyrus and superior temporal gyrus of AD brains. In some control brains, alphadeltaC(csp)-APP immunoreactivity appeared to be associated with a clinical history of metabolic encephalopathy. Our results suggest that apoptosis contributes to cell death resulting from amyloidosis and plaque deposition in AD.     

Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer's disease (AD).     

The amyloid precursor protein of Alzheimer disease in human brain and blood.

Studies of the metabolism and function of the amyloid precursor protein (APP) and its proteolytic fragment A beta in cultured cells, transgenic mice, and post-mortem brain tissue have advanced our understanding of Alzheimer disease (AD). However, the molecular pathogenesis of the disease is still not clear, and we are a long way from finding a cure for the disease. Studies carried out on human platelets and leukocytes have also helped shed light on APP and A beta metabolism and function. Platelet and leukocyte APP isoforms are processed using mechanisms similar to those in neuronal cells to generate A beta and soluble forms of APP. The activation of platelets and leukocytes leads to the secretion of APP and A beta, resulting in higher levels of these proteins in serum. APP and A beta in the circulation may be involved in the regulation of platelet function and in the modulation of immune responses. Because human platelets and lymphocytes produce all forms of APP and secrete amyloidogenic A beta peptides, these tissues may be useful in monitoring responses to therapeutic interventions directed at APP metabolism. Although not of neuronal origin, further studies on the more accessible ex vivo tissues, including platelets and leukocytes and other blood components, may reveal potential peripheral markers for AD and will further our understanding of the molecular pathogenesis of AD.     

Expression of potentially amyloidogenic derivatives of the Alzheimer amyloid precursor protein in cultured 293 cells

The expression of the carboxyl-terminal 100 (C-100) residues of the amyloid precursor protein (APP) may provide a model for studying the processing of APP to the 42–43 residue β-amyloid peptide (βA4) implicated in Alzheimer's disease. Expression of human C-100 in mammalian cells reportedly causes ‘toxicity’ and amyloid-like fibrils. We have expressed the C-100 fragment in human embryonic kidney cells (293 cells) in a transient assay and compared it to the expression of transfected wild type and mutant (Swedish familial Alzheimer's disease) full length APP. Products were characterized by Western blot analysis using antibodies to the carboxyl-terminal region of APP.     

Expression of the heat shock protein 47 in gentamicin-treated rat kidneys

Heat-shock proteins (HSPs) are rapidly synthesized in cells in response to various cytotoxic agents. Although several stress proteins are actively involved in the gentamicin-induced renal damages, the possible role of HSP47 in this condition is not yet clear. In this study, the expression of HSP47 in the gentamicin nephrotoxicity was examined by immunohistochemistry. Twenty male Wistar rats were sacrificed at day 0, 3, 7, 14 and 28 after subcutaneous injection of gentamicin. Gentamicin treatment causes tubular necrosis at day 3, followed by tubular regenerative changes and interstitial fibrosis, which was most prominent at day 14. The renal structures returned to almost normal architectures at day 28. By immunohistochemistry, HSP47 was weakly expressed in most of the glomeruli and occasionally in interstitial cells in the control rat kidneys. In contrast, strong immunostaining for HSP47 was noted in the tubular epithelial cells and interstitial cells in gentamicin treated rat kidneys, and strongest staining was observed at day 7. The immunostaining for HSP47 then gradually decreased, and returned to the normal level at day 28. In the whole experimental period, staining pattern of HSP47 in the glomeruli was not changed. In addition, phenotypically altered tubulointerstitial cells including regenerative tubular epithelial cells (immuno-positive for vimentin) and interstitial cells (immuno-positive for α-smooth muscle actin) were found in gentamicin nephrotoxicity. Expression of type III collagen increased in the areas of interstitial fibrosis. By double immunostaining, the regenerated and phenotypically altered tubulointerstitial cells were found to express HSP47 in and around interstitial fibrosis. It is concluded that overexpression of HSP47 by phenotypically altered renal cells might play a significant role in the development of gentamicin nephrotoxicity.     

Cytokine induction of heat shock protein in human granulosa-luteal cells

The infiltration of leukocytes is a characteristic feature ofluteolysis in humans. Leukocytes are known to generate physiologicalinducers of cell stress such as cytokines which have been implicatedas mediators of functional luteal regression. In cells exposedto stress, a response characterized by an increase in heat shockprotein (HSP) synthesis occurs. Recently, the induction of HSP-70in rat luteal cells has been shown to inhibit luteinizing hormone(LH) and cAMP-sensitive progesterone production, possibly byinterfering with the translocation of cholesterol to the mitochondrialcytochrome P450_SCC. We therefore investigated whether HSP-70is induced in human granulosa-luteal cells and its relationshipto steroidogenesis. [³⁵S]Methionine labelling showed an increasein a 70 kDa protein after heat treatment which was demonstratedto be HSP-70 by Western analysis using monoclonal antibodiesagainst the constitutive and inducible forms of HSP-70. Inductionof HSP-70 in human granulosa-luteal cells was also seen withinterferon (IFN) (10 ng/ml), tumour necrosis factor (TNF)-     

Expression of heat shock proteins in heavy metal-provoked inflamed human skin

The expression of heat shock protein (hsp) 65, 72 and 90 was studied in human inflamed skin after heavy metal salt treatment, by using epicutaneous patch-testing procedure and immunohistochemistry. There was a slight immunoreactivity to hsp 65 and hsp 72, but not to hsp 90 in keratinocytes in normal skin. An increased immunoreactivity to hsp 72 was obtained in apically located keratinocytes in a statistically significant (p<0.01) increased number of reactions to cobalt chloride and gold chloride compared to control skin. These metal salts also induced the highest degree of expression of keratinocyte intercellular adhesion molecule (ICAM)-1. There were epidermal dendritic cells as well as macrophage-like cells in the dermis, which expressed hsp 65 and hsp 90, in biopsies from metal salt-treated and control skin, while expression of hsp 72 in macrophage-like cells was found only in the dermis.     

NMDA receptor/amyloid precursor protein interactions: a comparison between wild-type and amyloid precursor protein mutations associated with familial Alzheimer's disease

Two recent reports showed that amyloid precursor protein (APP) may contribute to postsynaptic mechanisms via the regulation of the surface trafficking of excitatory N-methyl-D-aspartate (NMDA) receptors. Here we have investigated the interactions and surface trafficking of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptor subtypes with three APP mutations linked to familial Alzheimer's disease, APP695(Indiana), APP695(London) and APP695(Swedish). Flag-tagged mutated APP695s were generated and shown to be expressed at equivalent levels to wild-type APP695 in mammalian cells. Each APP mutant co-precipitated with NR1-1a/NR2A and NR1-1a/NR2B receptors following co-expression in mammalian cells. Further, as found for wild-type APP695, each enhanced NMDA receptor surface expression with no concomitant increase in total NR1-1a, NR2A or NR2B subunit expression. Thus these three familial APP mutations behave as wild-type APP695 with respect to their association with assembled NMDA receptors and their APP695-enhanced receptor cell surface trafficking.