Objective: To investigate the expression of phosphoglycerate mutase 1 (PGAM1) in the mouse testis after exposure to single heat stress (SHS). METHODS: We randomly assigned 32 C57 male mice to an SHS (n = 16) and a control group (n = 16), the former bathed in water at 43 ℃ and the latter at 25 ℃ for 15 minutes. At 1 and 7 days after exposure, we harvested the testicular tissue for observation of the morphological changes of testicular cells by HE staining and determination of the location and expression of the PGAM1 protein by immunohistochemistry and Western blot. RESULTS: The testis volume of the mice were reduced significantly, the spermatogenic tubules were disorganized, and the cells were reduced in number after heat stress and basically disappeared after 7 days. Immunohistochemistry showed extensive expression of the PGAM1 protein in the testicular spermatogenic tubules of the SHS-exposed mice, significantly higher than in the control group at 1 day after exposure, which was down-regulated in the testis tissue at 7 days, but still markedly higher than that in the control. Western blot exhibited significantly up-regulated expression of the PGAM1 protein after heat stress compared with that in the control group. CONCLUSIONS The expression of the PGAM1 protein undergoes dynamic changes in the mouse testis after exposed to single heat stress, which is related to heat stress-induced proliferation and division of testicular spermatogenic cells.
Animals ; Heat-Shock Response ; Male ; Mice ; Phosphoglycerate Mutase ; Testis

heat shock response of mammalian cells. The heat shock response is a coordinated biochemical response that helps to protect cells from stresses of various forms. Several protective proteins, termed heat shock proteins (hsp) are produced as part of this response. To begin to understand the role of the stress response of osteoblasts during surgical manipulation of bone, the heat shock protein response was evaluated in osteoblastic cells.MATERIALS AND METHODS: With primary cell culture studies and ROS 17/2.8 osteoblastic cells transfected with hsp27 encoding vectors culture studies, the thermal stress response of mammalian osteoblastic cells was evaluated by immunohistochemistry and western blot analysis.RESULTS: Immunocytochemistry indicated that hsp27 was present in unstressed osteoblastic cells, but not fibroblastic cells. Primarily cultured osteoblasts and fibroblasts expressed the major hsp in response to thermal stress, however, the small Mr hsp, hsp27 was shown to be a constitutive product only in osteoblasts. Creation of stable transformed osteoblastic cells expressing abundant hsp27 protein was used to demonstrate that hsp27 confers stress resistance to osteoblastic cells.CONCLUSIONS: The demonstrable presence and function of hsp27 in cultured bones and cells implicates this protein as a determinant of osteoblastic cell fate in vivo.]]>
Blotting, Western ; Fibroblasts ; Heat-Shock Proteins ; Heat-Shock Response ; HSP27 Heat-Shock Proteins ; Immunohistochemistry ; Osseointegration ; Osteoblasts ; Primary Cell Culture ; Proteins ; Staphylococcal Protein A

Antigen-specific T cell activation requires interaction of the T cell with specialized antigen-presenting cells. Signaling through the TCR is necessary but not sufficient to induce antigen-specific T cell activation and cytokine secretion. This first signal, termed signal 1, is both antigen-specific and MHC-restricted. Signal 2, which is neither antigen-specific nor MHC-restricted, is necessary to induce cytokine secretion, cellular proliferation, and effector function. Recently immunological studies in T cell activation area are mainly focused on biological and molecular biological characterization of TCR/CD3 complex and accessary molecules providing costimulatory signal (signal 2). If signal 2 is not delivered, T cell enter a state of long term un-responsiveness to specific antigen-termed anergy. Monoclonal antibody technique has been especially involved in recognizing novel inducible cell surface antigens on T cell activation. This study was aimed to develop monoclonal antibodies recognizing novel cytoplasmic proteins present in activated T cells. We make 6 monoclones involved in changing pattern of T cell activated cytoplasmic proteins. Using these 6 monoclonal antibodies analyze to find novel molecules involved in T cell activation associated response, apoptosis, and/or heat shock response of the T cells in early T cell activation.
Antibodies, Monoclonal* ; Antigen-Presenting Cells ; Antigens, Surface ; Apoptosis ; Cell Proliferation ; Cytoplasm* ; Heat-Shock Proteins ; Heat-Shock Response ; Humans* ; T-Lymphocytes*

OBJECTIVEA CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock.

METHODSA competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro.

RESULTSThe in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene.

CONCLUSIONThe heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.

Chromatin Assembly and Disassembly ; genetics ; Genes, Reporter ; genetics ; HSP90 Heat-Shock Proteins ; genetics ; Heat-Shock Response ; genetics ; Humans ; Transcription, Genetic

AIMTo explore the effects of gentamicin ototoxicity on the expression of heat shock protein 70 in guinea pigs cochlea.

METHODSWe used immunohistochemistry staining and image quantitative analysis system, combined with auditory brainstem response (ABR) measurement to investigate the change on the expression of HSP70 in guinea pigs cochlea of gentamicin ototoxicity.

RESULTSThe levels of HSP70 immunoreactivity in guinea pigs cochlea of experimental animals were high including Corti's organ, stria vascularis, medial spiral limbus, spiral ganglion cells and the threshold of ABR was in high correlation with the expression of HSP70 ([ r] > 0.8, P < 0.01).

CONCLUSIONGentamicin can induce expression of HSP 70 in guinea pigs cochlea and protect hearing function.

Animals ; Cochlea ; drug effects ; physiopathology ; Gentamicins ; toxicity ; Guinea Pigs ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; drug effects

OBJECTIVE: Our aim was to explore whether heat stress protein (HSP) 9 preferentially expresses under heat stress and affects the expression of other heat stress proteins as well as to explore the effect of HSPB9 overexpression and knockdown on apoptosis in DF-1. METHODS: We used gene cloning to construct an overexpression vector of the target gene, and synthesized the target gene interference fragment to transfect the chicken fibroblast cell line. Gene and protein expression, as well as apoptosis, were detected by RT-qPCR, Western blot, and flow cytometry. RESULTS: Chicken DF-1 cells showed an early state of apoptosis in the early stages of HSPB9 overexpression. In the later stages, as HSPB9 expression increased, the cells showed inhibition of apoptosis. When the cells were under heat stress, HSPB9 expression was much higher and earlier than the expression of HSPB1 and HSPA2. In addition, high expression of HSPB9 had a negative effect on HSPB1 and HSPA2 expression. This negative feedback decreased the percentage of early stages of apoptotic cells and promoted cell survival. CONCLUSION HSPB9 expression, although rapid, is detrimental to cell survival early during its overexpression. In heat stress, HSPB9 overexpression, while inhibiting the expression of HSPA2 and HSPB1, is beneficial to cell survival.
Animals ; Apoptosis ; genetics ; Avian Proteins ; genetics ; Cell Line ; Chickens ; Heat-Shock Proteins ; genetics ; Heat-Shock Response ; genetics

BACKGROUND: The heat shock protein (HSP) 70 families are known to protect cells against the irreversible tissue injury induced by stress and to induce the recovery of cell function during stress. Heat pretreatment was reported to decrease the acute lung injury(ALI) of rats induced by lipopolysaccharide (LPS). However the role of heat shock with LPS co-treatmenton ALI is unclear. The purpose of this study was to investigate the effect of heat treatment, which was given immediately after the beginning of ALI induced by LPS intratracheally administered in rats. METHODS: Either saline (saline group) or LPS was intratracheally instilled without heat treatment (LPS group). In addition, heat was conducted 18 hours prior to the instillation of LPS (pre-treatment group) and conducted immediately after instillation of LPS (co-treatment group). Six hours after the LPS or saline treatment, blood, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. The myeloperoxidase (MPO) activity and the heat shock protein expression in the lung tissue, the differential counts of the polymorphonuclear leukocytes (PMN) in the BAL fluids, and the LDH, protein, IL-1beta, TNF-alpha and IL-10 levels in BAL fluid and serum were measured. RESULTS: 1)The MPO activity, the differential PMN counts in the BAL fluid, BAL fluid and serum cytokines were higher in the LPS, the heat pre-treatment and co-treatment group than those of the saline group (p value <0.05). 2)The MPO activity and the protein level in the BAL fluid from the heat co-treatment group were similar to those of the LPS group. 3) The serum TNF-alpha level of the heat co-treatment group was significantly higher than that of the LPS group (p=0.01) . CONCLUSIONS: Heat shock response administered immediately after a LPS instillation did not attenuate the ALI in this model.
Acute Lung Injury* ; Animals ; Bronchoalveolar Lavage ; Cytokines ; Heat-Shock Proteins ; Heat-Shock Response ; Hot Temperature* ; Humans ; Interleukin-10 ; Lung ; Neutrophils ; Peroxidase ; Rats* ; Shock ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the protective effects and mechanism of heat shock response (HSR) on circulatory collapse induced by hyperthermia.

METHODSTwo experiments were carried out: (1) Protective effects of HSR. Rats were divided into 2 groups: heat shock (HS) group, sham control (SC) group. After HS group was pretreated with heat shock and recovered for 20 h at room temperature, both groups were exposed to heat till death, and blood pressure, electrocardiogram were measured continuously during exposure. Mean arterial pressure (MAP), survival time etc were acquired through Chart software. (2) Mechanism of effects. Rats were divided into 3 groups: HS group, SC group and normal control (NC) group. The treatment in HS and SC groups was identical with that in the first experiment, but it would be terminated at 73 min after heat exposure. Systolic pressure (Ps), diastolic pressure (Pd) etc were recorded and content of NO and HSP70 in myocardium were measured.

RESULTS(1) The survival time in HS group [(102.3 +/- 11.4) min] was longer than that in SC group [(87.9 +/- 7.7) min] and shock revealed later (P < 0.01); (2) During early heat exposure MAP in HS group was not different from that in SC group, but after 60 min MAP in HS group were higher than that in SC group; (3) MAP, Ps, Pd, HR and HSP70 in HS group were significantly higher but content of NO was lower than those in SC group (P < 0.01, P < 0.05).

CONCLUSIONHSR may induce upregulation of HSP70 and inhibit excessive production of NO in myocardium, thus result in relief of circulatory collapse induced by hyperthermia.

Animals ; Heat-Shock Proteins ; analysis ; Heat-Shock Response ; physiology ; Hot Temperature ; Male ; Nitric Oxide ; analysis ; Rats ; Rats, Sprague-Dawley ; Shock ; metabolism ; physiopathology ; Time Factors

The heat shock response is an important homeostatic mechanism that enables cells of animals, plants, and bacteria to survive a variety of environmental stresses. In all organisms a small set of proteins(heat shock proteins, hsps) is preferentially synthesized in response to stress. By seeking the expression of mRNA of hsps to tissue ischemia-reperfusion injury, a guideline can be provided in its application. A total of 100 Sprague-Dawley rats weighing about 200 to 300 gm were used. The rats were divided into three groups; group1: sham operation group, group 2: ischemia induced only, and group 3: ischemia-reperfusion group. The ischemia group(group 2) was subdivided according to the duration of ischemia. 1, 2, 4, 8, and 12 hours each. Ischemia-reperfusion group(group 3) was subdivided according to the duration of ischemic insult lasting 1, 2, and 4 hours each(group 3-1, 2, 3). Samples were taken from both subgroups 15 min, 30 min, 1, 2, 4, 8 hours after reperfusion. Creatine phosphokinase levels were measured and mRNA expression was observed using in situ hydridization histochemistry. The elevation of creatine phosphokinase was correlated to the duration of ischemic insult and to the duration of reperfusion time. This postulated the fact that the amount of muscle injury had direct connection with time of ischemia and the injury continued even after reperfusion. The overall mRNA of hsp in the ischemia-reperfusion group showed faster and stronger expression compared to that of the ischemia-only group. Among the ischemia-reperfusion subgroups, as the ischemic time was increased, the mRNA expression demonstrated faster but with decreased amount. This findings suggest that there can be a limit in expression of hsp based on the extent of ischemic insult. This can be helpful in clinical applications. When the gastrocnemius and soleus muscle are compared, white muscle group(gastrocnemius) revealed faster and stronger expression of mRNA of hsps. This is most likely due to the fact that red muscle group has abundant blood supply and mitochondria, and implies decreased injury after ischemia-reperfusion. Although the precisive mechanism of hsp is not yet known, heat shock induced protection increases flap survival dramatically. But by demonstrating the pattern of expression based on ischemia time and reperfusion, this paper suggests a possible limit of hsp against ischemia- reperfusion injury.
Animals ; Bacteria ; Creatine Kinase ; Heat-Shock Proteins* ; Heat-Shock Response ; Hot Temperature* ; Ischemia ; Mitochondria ; Muscle, Skeletal* ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; Reperfusion Injury ; RNA, Messenger* ; Shock

OBJECTIVETo compare the difference of protein expression in the supernatant of heat injured keratinocytes (KC) and normal KC.

METHODSA model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software.

RESULTS(1) Average protein spots were 1,898 +/- 113, 1,877 +/- 97 in the supernatant of normal and heat injured KC and 1,118 protein spots could be used for statistical analysis. (2) Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. (3) 16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic 2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth l in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily member 10, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injured KC.

CONCLUSIONSThe result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC. These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.

Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Heat-Shock Response ; Hot Temperature ; Humans ; Keratinocytes ; metabolism ; Proteome ; metabolism