HSP70 Immunoreactivity Research Articles

Investigation of the heat shock protein 70 activity in intestine cells of goats with coccidiosis

ABSTRACT Coccidiosis is one of the most common infectious diseases in goat farming. The disease causes major economic loss in the world. In this study, we aimed to investigate the activity of heat shock protein 70 in intestine cells of goats with coccidiosis. We used total of twenty-seven goats for this purpose. Gross findings were diarrhoea, cachexia, and dehydration. In the microscopical examination, we observed proliferative enteritis with Eimeria. parasites. Immunohistochemical examinations revealed moderate to severe Hsp70 immunoreactivity in intestines. Considering Hsp70 is a stress protein with anti-apoptotic and immune regulatory features, Hsp70 immunoreactivity attributed to the stress caused by infection and anti-apoptotic activity of the protein along with immune regulatory effects of Hsp70.

Impact of maternal protein restriction on Hypoxia-Inducible Factor (HIF) expression in male fetal kidney development.

Kidney developmental studies have demonstrated molecular pathway changes that may be related to decreased nephron numbers in the male 17 gestational days (17GD) low protein (LP) intake offspring compared to normal protein intake (NP) progeny. Here, we evaluated the HIF-1 and components of its pathway in the kidneys of 17-GD LP offspring to elucidate the molecular modulations during nephrogenesis. Pregnant Wistar rats were allocated into two groups: NP (regular protein diet-17%) or LP (Low protein diet-6%). Taking into account miRNA transcriptome sequencing previous study (miRNA-Seq) in 17GD male offspring kidneys investigated predicted target genes and proteins related to the HIF-1 pathway by RT-qPCR and immunohistochemistry. In the present study, in male 17-GD LP offspring, an increased elF4, HSP90, p53, p300, NFκβ, and AT2 gene encoding compared to the NP progeny. Higher labeling of HIF-1α CAP cells in 17-DG LP offspring was associated with reduced elF4 and phosphorylated elF4 immunoreactivity in LP progeny CAP cells. In 17DG LP, the NFκβ and HSP90 immunoreactivity was enhanced, particularly in the CAP area. The current study supported that the programmed reduced nephron number in the 17-DG LP offspring may be related to changes in the HIF-1α signaling pathway. Factors that facilitate the transposition of HIF-1α to progenitor renal cell nuclei, such as increased NOS, Ep300, and HSP90 expression, may have a crucial role in this regulatory system. Also, HIF-1α changes could be associated with reduced transcription of elF-4 and its respective signaling path.

Shear stress induces monocyte/macrophage-mediated inflammation by upregulating cell-surface expression of heat shock proteins

The loss of endothelial cells is associated with the accumulation of monocytes/macrophages underneath the surface of the arteries, where cells are prone to mechanical stimulation, such as shear stress. However, the impact of mechanical stimuli on monocytic cells remains unclear. To assess whether mechanical stress affects monocytic cell function, we examined the expression of inflammatory molecules and surface proteins, whose levels changed following shear stress in human THP-1 cells. Shear stress increased the inflammatory chemokine CCL2, which enhanced the migration of monocytic cells and tumor necrosis factor (TNF)-α and interleukin (IL)− 1β at transcriptional and protein levels. We identified that the surface levels of heat shock protein 70 (HSP70), HSP90, and HSP105 increased using mass spectrometry-based proteomics, which was confirmed by western blot analysis, flow cytometry, and immunofluorescence. Treatment with HSP70/HSP105 and HSP90 inhibitors suppressed the expression and secretion of CCL2 and monocytic cell migration, suggesting an association between HSPs and inflammatory responses. We also demonstrated the coexistence and colocalization of increased HSP90 immunoreactivity and CD68 positive cells in atherosclerotic plaques of ApoE deficient mice fed a high-fat diet and human femoral artery endarterectomy specimens. These results suggest that monocytes/macrophages affected by shear stress polarize to a pro-inflammatory phenotype and increase surface protein levels involved in inflammatory responses. The regulation of the abovementioned HSPs upregulated on the monocytes/macrophages surface may serve as a novel therapeutic target for inflammation due to shear stress.

Seasonal variation in the expression pattern of heat shock protein 70 and 90 in Common carp (Cyprinus carpio) from Karataş Lake, Burdur, Türkiye.

Organisms have evolved defense mechanisms to protect themselves from stressful conditions. The expression of heat shock proteins is considered a valid indication of protection from the adverse effects of hostile conditions. In this study, we used immunohistochemistry to investigate the seasonal effects of some abiotic factors on heat shock protein 70 and 90 (HSP70 and HSP90) expression in the liver, gills, and muscle tissues of 24 Common carp (Cyprinus carpio) caught in Karataş Lake (Burdur, Türkiye) using gillnets of various mesh sizes. We also measured some physicochemical parameters on-site at sampling time and took water samples for further analyses of other physicochemical parameters and heavy metals. Immunostaining for HSP90 was stronger than for HSP70 in both liver and gill samples. Liver and gill structures exhibited significant seasonal differences in HSP70 and HSP90 immunoreactivity, and the same was true for immunostaining for HSP70 and HSP90 in muscle samples. Some physicochemical properties seemed to vary considerably between seasons, with Fe, Mn, and Zn levels tending to exhibit changes throughout the seasons. However, these levels were considered acceptable for human health. In conclusion, this study suggests that substantial changes in HSP70 and HSP90 expression may be essential for seasonal adaptation and tolerance. Further research on fish HSPs would greatly contribute to aquaculture, which is essential for meeting food requirements.

The relationship between SOD1 and Hsp70 expression in broiler ileum throughout post-hatching development

Heat shock proteins (Hsps) are molecular chaperones that play critical functions in the survival and development of cells. Hsps influence adaptive and innate immune responses and may promote cross-talk between the two systems. Superoxide dismutases (SODs) are metalloenzymes that play an essential role in the body's defense against oxidative stress by efficiently removing excess reactive oxygen species. This study is an experimental study that was conducted to determine the relationship between SOD1 and Hsp70 expression in the ileum during the post-hatching development of the broiler. In the study, samples were taken from ileum tissue of 0-, 21- and 42-day-old broilers were used as material. While the Hsp70 immunoreactivity observed in the epithelial cells was specific to a few cells on day 0, it was detected in more villus epithelial cells on days 21 and 42. The Hsp70 expression in the ileum increased from the age 0 to up to day 42, especially in villus epithelial cells. In sections stained by SOD1, the ileum's villus epithelial cells and smooth muscle cells showed an intracytoplasmic reaction. From day 21 to day 42, a regular increase in SOD1 expression was detected in the crypt and villus epithelial cells. As a remarkable finding, a more intense intracytoplasmic staining was detected in villus epithelial cells located at the apex of intestinal villi. In conclusion, it was observed that SOD1 and Hsp70 expression increased in the ileum tissue throughout post-hatching development in broilers with a positive correlation with age. Based on the histological findings, it can be concluded that SOD1 and Hsp70 play a critical protective role in the small intestine after hatching and contribute to the rapid development of the intestine.

ODP543 Chemotherapy-induced extracellular HSP70 enhances pro-tumorigenic effects of macrophages in breast cancers.

Abstract Breast cancer is one of the most common types of malignancies in women worldwide. Cytotoxic chemotherapy has improved the prognosis of aggressive phenotype of breast cancer patients including triple-negative breast cancers. However, resistance to chemotherapy is an important problem. Tumor-associated macrophages (TAMs) are primary components of tumor microenvironment and promote tumor progression by releasing various cytokines. Pro-tumorigenic effects of TAMs are known to induce chemoresistance in breast cancers. On the other hand, we previously showed that pro-tumorigenic effects of TAMs were enhanced in breast cancer tissues following chemotherapy by immunohistochemical study. Here, we demonstrated that breast carcinoma cells exposed by chemotherapy induced pro-tumorigenic effects of TAMs. THP-1 human monocyte cell-derived macrophages were interacted with MDA-MB-231 and MDA-MB-453 human breast cancer cells treated with epirubicin (EPI), and conditioned media from them significantly promoted proliferation and migration of breast cancer cells. We found that the expression of heat shock protein (HSP) 70 in the cytoplasm of MDA-MB-231 cells was upregulated following EPI treatment. HSP70 is an intracellular molecular chaperone and recently described with extracellular actions in stressful conditions. However, extracellular HSP70 action on TAMs in breast cancer has remained largely unknown. MDA-MB-231 cells treated with EPI and EPI-resistant MDA-MB-231 subline promoted secretion of HSP70, especially exosomal HSP70. Then, HSP70 knockdown in MDA-MB-231 cells were performed using siRNA. Macrophages cultured with HSP70 suppressed conditioned media from MDA-MB-231 cells showed decreased expression of CD163, a marker of pro-tumorigenic macrophages. Furthermore, HSP70 suppressed conditioned media downregulated the expression of transforming growth factor (TGF)-beta and colony stimulating factor (CSF)-1 in MDA-MB-231 cells, which are known to increase pro-tumorigenic effects of macrophages. Then, we performed immunohistochemistry for HSP70 in 116 breast carcinoma tissues. HSP70 immunoreactivity in the cytoplasm of breast carcinoma cells was correlated with stage, lymph node metastasis, pathological T factor, histological grade, Ki-67 LI, and negatively correlated with ER and progesterone receptor (PR). Furthermore, immunoreactivity of HSP70 was increased in breast cancer patients who had received neoadjuvant chemotherapy. When we correlated HSP70 with clinical outcome, HSP70 immunoreactivity was correlated with increased risk of recurrences and breast-cancer-specific mortality. Importantly, macrophage infiltration was correlated with increased risk of recurrences only in the HSP70 positive group, but not in the HSP70-negative group, which indicates that HSP70 may be associated with the pro-tumorigenic effects of TAMs in breast cancers. Taken together, breast carcinoma cells promoted secretion of extracellular HSP70 following chemotherapy and it might induce chemoresistance by upregulating pro-tumorigenic effects of macrophages, directly or indirectly via regulation of cytokines expression in breast carcinoma cells. Extracellular HSP70 may be an important target to improve the prognosis of breast cancer patients. Presentation: No date and time listed

Lysosomal and cytoskeletal events in epithelial salivary tumours as assessed by imunohistochemistry for CD63 and HSP27

Little attention has been paid to immunohistochemically assessed, lysosomal activities in salivary neoplasia. In an attempt to remedy this, the present investigation applied immunohistochemistry for CD63 antigen (a lysosomal membranous protein) and HSP27 (a molecular chaperone with roles in intracellular homeostasis) to archival paraffin-embedded surgical specimens of 101 benign and malignant, epithelial salivary tumours. Diffuse cytoplasmic CD63 immunoreactivity was seen in serous cells in acinic cell carcinoma, and mucous cells in mucoepidermoid carcinoma, pleomorphic adenoma (PA) and Warthin tumour. Apical rims or bands of CD63 immunoreactivity were also seen in simple cells lining tubular structures in PA, acinic cell carcinoma, mucoepidermoid carcinoma and polymorphous (low-grade) adenocarcinoma; and, occasionally, oncocytic luminal cells in Warthin tumour. HSP27 immunoreactivity was usually seen in non-luminal cells of PA, basal cells of oncocytic tumours, epidermoid cells of mucoepidermoid carcinoma and PA, and cells outlining aggregates or pseudolumina of adenoid cystic carcinoma. Expression of CD63 is preferentially associated with differentiated or simple luminal cell phenotypes in epithelial salivary tumours and possibly reflects autophagy of secretory granules or absorption of luminal material. Expression of HSP27 is preferentially associated with non-luminal cells and possibly reflects remodelling of the cytoskeleton.

Expression of heat shock protein HSP-70 in the retrosplenial cortex of rat brain after administration of (R,S)-ketamine and (S)-ketamine, but not (R)-ketamine

The N-methyl-d-aspartate receptor (NMDAR) antagonist (R,S)-ketamine has robust antidepressant effects in depressed patients although it has detrimental side effects such as psychotomimetic and dissociative symptoms. (R,S)-Ketamine is known to cause the expression of heat shock protein HSP-70 (a marker for neuronal injury) in the retrosplenial cortex of rat brain, suggesting that the neuropathological changes may play a role in the detrimental side effects of (R,S)-ketamine. This study was undertaken to examine whether (R,S)-ketamine and its two enantiomers, (R)-ketamine and (S)-ketamine, causes the expression of HSP-70 in the rat retrosplenial cortex after a single administration. The HSP-70 immunohistochemistry in the rat brain was performed 24 h after intraperitoneal administration of saline (1 ml/kg), (+)-MK-801 (or dizocilpine: 1.0 mg/kg), (R,S)-ketamine (100 mg/kg), (S)-ketamine (25, 50, or 75, mg/kg), or (R)-ketamine (25, 50, or 75 mg/kg). Marked expression of HSP-70 immunoreactivity in the retrosplenial cortex was detected after administration of dizocilpine or (R,S)-ketamine (100 mg/kg). Higher does (50 and 75 mg/kg) of (S)-ketamine, but not low dose (25 mg/kg), caused expression of HSP-70 in this region. In contrast, all doses of (R)-ketamine did not induce the expression of HSP-70 in this region. These findings suggest that marked expression of HSP-70 in the retrosplenial cortex after a single dose of (R,S)-ketamine or (S)-ketamine may have detrimental side effects in the rat brain. Therefore, it is likely that (R)-ketamine is a safer compound in humans than (R,S)-ketamine and (S)-ketamine.

HOXC13 and HSP27 Expression in Skin and the Periodic Growth of Secondary Fiber Follicles from Longdong Cashmere Goats Raised in Different Production Systems.

This experiment was conducted to identify periodic changes in secondary follicles (SFs) over one year of extensive (grazed) and intensive (housed and fed supplement) farming of Longdong cashmere goats. One-year old goats (n=24, 12 extensively fed and 12 intensively fed) were studied. The diameter, depth, density, activity, and ultra-structural features of SFs were assessed using light microscopy and transmission electron microscopy. HOXC13 and HSP27 expression were studied using immunohistochemistry and immunofluorescence method. The anagen stage in the extensively grazed goats was from April to September, but was longer (April to October) in the housed, supplementary fed group. The depth and activity of the SF in anagen and catagen differed (P<0.05) between the groups. HSP27 and HOXC13 protein were present in both the epidermis and dermis, with HSP27 immunoreactivity highest in the hair shaft (HS), outer root sheath (ORS) and inner root sheath (IRS). HOXC13 expression was prominent in both the eprdermis and ORS. HSP27 and HOXC13 expression were prominent during anagen, and less so during catagen and telogen. In anagen, HSP27 expression in the HS, IRS, and ORS of the extensively fed group was higher than in the intensively fed group (p<0.05). In contrast, HOXC13 expression in HS, IRS and ORS of the extensively fed group was lower than in the intensively fed group (P<0.05). This suggests the growth of cashmere is influenced by nutrition and that housed goats could be used in cashmere production. Anat Rec, 301:742-752, 2018. © 2017 Wiley Periodicals, Inc.

Increased expression of phosphorylated forms of heat-shock protein-27 and p38MAPK in macrophage-rich regions of fibro-fatty atherosclerotic lesions in the rabbit.

We aimed to assess the expression and distribution of Hsp27, pHsp27 (Ser82), p38MAPK and p-p38MAPK in fibro-fatty atherosclerotic lesions and the myocardium of hypercholesterolaemic rabbits. Male New Zealand white rabbits were fed a high-cholesterol diet for 18 weeks, maintaining serum cholesterol at approximately 20 mmol/l over this period. Aortic arch and myocardial tissues were analysed by Western blot, immunohistochemistry and double immunofluorescence. Plasma Hsp27 levels were measured by ELISA. There was a significant increase in the expression of monomeric and dimeric forms of Hsp27, together with pHsp27 (Ser82), p38MAPK and p-p38MAPK in the fibro-fatty atherosclerotic lesions (P < 0.01; P < 0.05; P < 0.001; and P < 0.001, respectively) and the myocardial tissues (P < 0.001) from the cholesterol-fed rabbits compared with equivalent tissues from controls when the plasma concentration was low. Immunohistochemical analysis of the fibro-fatty lesions showed marked increases in Hsp27 and pHsp27 (Ser82) immunoreactivity. Double immunostaining showed intense expression of pHsp27 and p-p38MAPK in regions that were rich in macrophages, suggesting a close association with these inflammatory cells, whereas, in regions rich in smooth muscle cells, only p-p38MAPK was found to be strongly expressed. An increased expression of pHsp27 (Ser82) was spatially associated with increased p-p38MAPK within fibro-fatty atherosclerotic lesions and was colocalized to regions rich in macrophages. The initial increase in plasma Hsp27 levels may reflect the increase in systemic inflammation and oxidative stress in the early phases of disease. The falling concentrations subsequently may be coincident with the development of the advanced atherosclerotic lesions.

Reduction in heat shock protein 90 correlates to neuronal vulnerability in the rat piriform cortex following status epilepticus

In the present study, we addressed the question of whether the distinct patterns of heat shock protein (HSP) 70 and HSP90 expressions in the brain region represents the regional specific responses to status epilepsticus (SE) in an effort to better understand the role of HSPs in epileptogenic insult. HSP70 immunoreactivity was increased in CA3 pyramidal cells as well as dentate granule cells at 12h–1week after SE. HSP70 immunoreactivity was transiently increased in neurons within the piriform cortex (PC) following SE. Linear regression analysis showed no correlation between the intensity of NeuN and that of HSP70. In contrast to HSP70, HSP90 immunoreactivity was decreased in CA1–3 pyramidal cells at 4days–4weeks after SE. In addition, HSP90 immunoreactivity was decreased in PC neurons at 12h–4weeks after SE. linear regression analysis showed a direct proportional relationship between the intensity of NeuN and that of HSP90. Therefore, these findings suggest that HSP90 degradation may be closely related to neuronal vulnerability to SE insult.

Lack of cell stress markers in fibrous cap cells in the left main coronary artery.

Fibrous cap formation is a key aspect of preventing clinical events but animal models to study this are limited and cellular stress plays a fundamental role in fibrous cap formation. To characterise cellular stress markers in an established animal model to study coronary artery fibrous cap formation. Male New Zealand White rabbits were fed a diet containing 0.5% cholesterol and 1% methionine for 4 weeks, then 9 weeks of normal diet to induce fibrous cap formation. Immunohistochemistry was used to detect CHOP, GRP78, nitrotyrosine HSP70, HSP90, iNOS and HSP32. The core within the left main coronary artery atherosclerosis contained vast amount of foamy macrophages which readily stained for all markers. However, the smooth muscle cells within the formed fibrous cap were negative for all markers. The endothelium overlying the fibrous cap was positive for CHOP, GRP78, nitrotyrosine, iNOS and HSP32, however it was difficult to detect positive endothelial HSP70 or HSP90 immunoreactivity. Serial sectioning and immunohistochemistry for all factors showed clear dual iNOS+ / HSP32 + / HSP70- / HSP90- single cells within the fibrous cap formed. Smooth muscle cells within fibrous caps appear 'stress free', however isolated single smooth muscle cells within caps and within the core show positive immunoreactivity for stress markers. This model could be used to understand the role of cellular stress in fibrous cap formation in the coronary artery.

Expression of heat shock proteins in premalignant and malignant urothelial lesions of bovine urinary bladder

Abnormal heat shock protein (HSP) levels have been observed in a number of human tumours, where they are involved in all hallmarks of cancer. Since bovine urothelial tumours share striking morphological and biochemical features with their human counterparts, the aim of this study was to evaluate the immunohistochemical levels of Hsp27, Hsp60, Hsp72, Hsp73 and Hsp90 in 28 normal bovine urinary bladders and 30 bovine papillomavirus-positive urothelial tumours (9 in situ carcinomas, 9 low-grade and 12 high-grade carcinomas) and adjacent premalignant lesions obtained from cows suffering from chronic enzootic haematuria, in order to investigate the role of these proteins in the process of urothelial carcinogenesis. A semi-quantitative method was used for the analysis of the results. Western blot analysis was also used to confirm HSP expression in normal controls. All investigated HSPs were expressed in normal bovine urothelium, showing characteristic patterns of immunolabelling throughout urothelial cell layers, which usually appeared to be conserved in urothelial hyperplasia and dysplasia. On the other hand, gradual loss of Hsp27 immunostaining resulted to be significantly associated with increasing histological grade of malignancy (P < 0.01). As well, a significantly reduced immunosignal of Hsp73 and Hsp90 was observed in high-grade and low-/high-grade carcinomas, respectively (P < 0.01). In contrast, Hsp60 (P < 0.01) and Hsp72 (P < 0.05) immunoreactivity appeared to be significantly increased both in premalignant and malignant lesions when compared to that observed in normal urothelium, thus suggesting an early involvement of these proteins in neoplastic transformation of urinary bladder mucosa.

The Distribution of the Stress Protein HSP70 in the Cerebellum of Patients with Schizophrenia

Data accumulated from neuro-imaging, clinical and morphological studies suggest that the cerebellum is involved in cognitive functions and thus may be important in the etiopathogenesis of schizophrenia, since patients show cognitive abnormalities. In the present study, we have attempted to localize cellular metabolic dysfunctions applying the immunohistochemical and Western blot method to demonstrate the expression of the stress protein HSP70, which is a marker of cellular metabolic dysfunction in the brain. We studied the post mortem brains’ cerebellum of 12 normal controls and 10 schizophrenics. We have used the polyclonal antibody rabbit anti-HSP70 on paraffin sections as well as on nitrocellulose membranes. Bound antibody was detected using the indirect method of streptavidin-peroxidase-DAB. The results in the cerebellum of controls showed intense HSP70 immunoreaction in the synaptic glomeruli of the granular cell layer, in the cytoplasm and dendrites of Purkinje cells. In the same areas of the cerebellum of schizophrenics the HSP70 immunoreactivity was minimal. These results suggest that the reduced levels of HSP70 in the cerebellum are likely to contribute synergistically to the cognitive dysfunction in schizophrenia. This may suggest abnormality of protective neural mechanisms in such pathological conditions.

Heat shock protein expression in canine osteosarcoma

Abnormal levels of heat shock proteins have been observed in a number of human neoplasms and demonstrate prognostic, predictive and therapeutic implications. Since osteosarcoma (OSA) in dogs provides an important model for the same disease in humans, the aim of this study was to evaluate the immunohistochemical expression of Hsp27, Hsp72, Hsp73 and Hsp90 in 18 samples of canine appendicular OSA, in relation to histological grade and overall survival (OS), in order to investigate their potential prognostic, predictive and/or therapeutic value. A semiquantitative method was used for the analysis of the results. Hsp27, Hsp73 and Hsp90 showed a variably intense, cytoplasmic and nuclear immunoreactivity that was not associated with histological type or grade. On the other hand, a high percentage of Hsp72 immunostaining was significantly associated with grade III (P < 0.01) and a lack of immunolabelling was significantly correlated to a longer OS (P = 0.006). Neoplastic emboli were occasionally positive for Hsp27, faintly immunoreactive for Hsp72 and intensely immunolabelled by Hsp73 and Hsp90. In conclusion, absence of Hsp72 immunosignal appears to be associated with a favourable prognosis whilst the widespread Hsp90 immunoreactivity detected in all tumour cases as well as in neoplastic emboli, suggests this protein could be targeted in the therapy of canine OSA, and likewise in its human counterpart.

The 27-kDa heat shock protein (HSP27) is a reliable hippocampal marker of full development of pilocarpine-induced status epilepticus

The pilocarpine-induced status epilepticus (SE) in rodents provides a valuable animal model of temporal lobe epilepsy. Since not all animals enter SE following pilocarpine injection, we aimed to find a biomarker for full development of pilocarpine-induced SE using a proteomic approach. Two-dimensional gel electrophoresis and Western blot analysis were performed with protein extracts from hippocampal tissue taken from four different groups of animals: pilocarpine-treated rats with full development of SE, pilocarpine-treated rats without seizures, pilocarpine-treated rats with seizures but without SE, and saline-injected rats. 2D gel electrophoresis revealed two protein spots which were only present after full development of pilocarpine-induced SE, but neither in gels from pilocarpine-treated rats without seizures nor in those of saline-injected animals. The protein in both spots was identified as the small 27-kDa heat shock protein (HSP27) by MALDI-TOF mass spectrometry. Subsequent Western blot analyses confirmed that HSP27 immunoreactivity was only observed with the hippocampal protein extracts in the status epilepticus group, but in none of the other groups. Furthermore, immunocytochemistry showed that HSP27 expression following pilocarpine-induced SE was localized in astrocytes. We propose that HSP27 is a highly sensitive and specific hippocampal marker for full development of pilocarpine-induced status epilepticus.

Heat shock protein 90 and its cochaperone, p23, are markedly increased in the aged gerbil hippocampus

In the present study, we compared HSP90 and its co-chaperone, p23, immunoreactivity and their protein levels in the hippocampus between adult (postnatal month 6) and aged (postnatal month 24) gerbils using immunohistochemistry and western blot analysis. HSP90 immunoreactivity was markedly increased in pyramidal cells in the hippocampus proper and in polymorphic cells in the dentate gyrus of the aged group compared to the adult group. p23 immunoreactivity was slightly increased in pyramidal cells of the hippocampus proper and in granule cells of the dentate gyrus in the aged group. In addition, HSP90 and p23 protein levels in the aged hippocampus were much higher than the adult hippocampus. These results indicate that HSP90 and p23 immunoreactivity and protein levels in the hippocampus are distinctively increased in the aged gerbils compared to the adult gerbils.

Expression of Heat Shock Protein 70 in the Periodontal Ligament During Orthodontic Tooth Movement

Heat shock proteins (HSPs) play an important role in maintaining protein homeostasis within the cell, and they are also related to inflammation. However, it is not clear how HSPs regulate inflammatory cytokines in periodontal ligament (PDL) cells during mechanical stress. The present study focuses on the expression of HSP70 in the PDL during experimental tooth movement in rats. A total of 35 6-week-old male Wistar rats were subjected to orthodontic force of 10 g to induce mesial tipping movement of the upper first molars. Experimental tooth movement was accomplished for 14 days. We determined the expression of HSP70 proteins in the PDL during orthodontic tooth movement by immunohistochemistry. HSP70 immunoreactivity was detected in PDL fibroblasts on the compressive side by an orthodontic force of 10 g. On day 1 after tooth movement, the immunoreactivity of HSP70 was weak. On days 2, 3 and 4, we identified a greater positive reaction for HSP70 in the nucleus of fibroblasts than that recognized on the first day. The HSP70-positive reaction further increased on days 7 and 14. From our quantitative evaluation, the rate of HSP70-positive PDL fibroblasts significantly increased compared with control fibroblasts, and this occurred in a time-dependent manner. Therefore, HSP70 may function as a homeostatic factor to compensate for PDL-cell changes that occur during orthodontic force.

The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant ( abc−) for the Arabidopsis thaliana MBF1 genes AtMBF1a , AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc− mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc− was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc− phenotype was partially or completely rescued by AtMBF1c cDNA over-expression ( abc− +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc− mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

Heat shock protein 27 upregulation and phosphorylation in rat experimental autoimmune encephalomyelitis

Following stress or inflammation, the 27-kDa heat shock protein (HSP27) is induced in various cell types, where it promotes cell survival and inhibits inflammatory reactions. We examined the expression of HSP27 and phosphorylated HSP27 (p-HSP27) in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blotting analysis revealed low levels of HSP27 and p-HSP27 in the normal spinal cords and significantly higher levels in EAE-affected spinal cords. Immunohistochemistry revealed that HSP27 was expressed constitutively in the neurons and some fibrous astrocytes of the spinal cords of normal rats. However, in EAE-affected spinal cords, HSP27 immunoreactivity was higher and located primarily in the fibrous astrocytes of the white matter, whereas few of the inflammatory cells were immunopositive for HSP27. Immunoreactivity for p-HSP27 was detected predominantly in the fibrous astrocytes of the normal controls and was markedly increased in EAE-affected spinal cords. Therefore, the levels of HSP27 expression and phosphorylation of HSP27 were increased primarily during reactive astrogliosis of spinal white matter affected by EAE. These observations suggest that in rat EAE, the increased expression and elevated activation of HSP27 modulate host cell activity, survival, and inflammation to counter the autoimmune inflammatory injury. Our results also suggest that HSP27 plays a role in spontaneous recovery from EAE-induced paralysis.