Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.     

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts

It is now well recognized that a disorder of left ventricular filling can be sufficient to account for congestive heart failure. Furthermore, evaluation of heart disease would not be complete if it did not include assessment of left ventricular filling, improvement of which probably ensures better control of the heart disease. An efficient and reliable tool for the study of diastolic function is therefore essential. The authors review the current state of knowledge and the more recent developments in Doppler echocardiography in the evaluation of left ventricular diastolic function. After revising the pathophysiology, the methods of studying ventricular filling are described. The recording technique is described, taking into account recent developments in transthoracic and transoesophageal approaches. This investigation provides parameters allowing semiquantitative estimation of filling pressures (mean left atrial pressure, end-diastolic pressure) and reliable evaluation of overall diastolic performance.     

Characteristic expression of 105-kDa heat shock protein (HSP105) in various tissues of nonstressed and heat-stressed rats

BACKGROUND: Transient lower esophageal sphincter relaxations (TLESRs) are the major mechanism permitting gastroesophageal reflux (GER). Little information is available on how anti-reflux surgery affects reflux mechanisms, especially TLESRs. We evaluated the effects of partial fundoplication (Belsey Mark IV) on reflux mechanisms. METHODS: Sixteen patients were prospectively studied before and after Belsey Mark-IV operation by endoscopy, 24-h esophageal pH-metry, and simultaneous recording of pH and lower esophageal sphincter (LES) characteristics by sleeve manometry. RESULTS: The operation was successful in 14 of 16 patients (87%). Fasting and postprandial reflux decreased significantly (P < 0.01) after the operation. Partial fundoplication significantly (P < 0.05) decreased the number of TLESRs per hour in the fasting and postprandial period from 3.2+/-0.4 and 5.6+/-0.5 to 1.7+/-0.3 and 2.8+/-0.4, respectively. The percentage of TLESRs associated with reflux also decreased significantly (P < 0.05). Basal LES pressure increased from 14.7+/-2.1 mmHg to 17.9+/-2.6 mmHg (not significant). CONCLUSIONS: Partial fundoplication controls GER through a reduction in the number of TLESRs and by decreasing the number of relaxations associated with reflux.     

Expression of the heat shock protein HSP27 in human ovarian cancer

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.     

A small influence of HSP90 levels on the trehalose and heat shock element inductions of the yeast heat shock response

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.     

Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.     

Constitutive and heat-inducible expression of HSP105 in neurons and glial cells in culture

The constitutive and heat-inducible expression of HSP105 was investigated in newborn mouse brain cell cultures by Northern blotting, Western blotting and immunocytochemistry. HSP105 was expressed most abundantly in the brain among the various tissues examined. HSP105 mRNA and protein were both present at substantial levels in brain cell cultures under unstressed conditions and up-regulated greatly during 3-48 h following exposure to heat stress (43 degrees C/20 min). HSP105 was expressed in nearly all neurons, oligodendrocytes, microglia and astrocytes with its location of both cytoplasmic and nuclear regions under unstressed and heat-stressed conditions. HSP105 expression was significantly down-regulated in astrocytes following treatment with IL-beta or TNF-alpha (50 ng/ml for 6 days), both of which are known growth-stimulatory cytokines for astrocytes. These results indicate that HSP105 is constitutive and heat-inducible HSP in neurons and glial cells in which its expression is under the control of both stressful stimuli and growth-regulatory factors.     

Effect of ischemia--reperfusion on heat shock protein 70 and 90 gene expression in rat liver: relation to nutritional status

p53 gene mutations occur in most human cancers and result in an altered protein product that accumulates within the cell. Although the observed endogenous human CTL response to p53 is weak, high-affinity, human p53-specific CTLs have been generated from HLA A2.1 transgenic mice immunized with human CTL epitope peptides. In this study, we examine the ability of HLA A2.1-restricted and human p53-specific CTLs from HLA A2.1 transgenic mice to suppress the growth of p53-overexpressing human tumors in severe combined immunodeficient (SCID) mice. In vitro, murine p53(149-157)-specific CTLs selectively lysed the p53-overexpressing pancreatic carcinoma cell line Panc-1 but did not recognize HLA A2.1- tumor cells or HLA A2.1+ normal human fibroblasts. Furthermore, in vivo, the growth of established human tumor xenografts in SCID mice was significantly reduced and survival was prolonged after the administration of p53-specific CTLs but not after the administration of control CTLs or PBS alone. Following treatment with p53(149-157)-specific CTLs, regressing Panc-1 tumors were infiltrated by the CD8+ CTLs, as demonstrated by immunohistochemistry. These findings suggest that p53(149-157)-specific and HLA A2.1-restricted murine CTLs suppress the growth of established Panc-1 tumors following adoptive transfer into SCID hosts and prolong their survival.     

Whole body hyperthermia selectively induces heat shock protein 72 in neurons of the rat spinal cord

Rabbits with lesions of either medial prefrontal cortex (mPFC) or amygdala central nucleus (ACN) were compared with sham-lesioned animals during differential and reversal classical conditioning of the eyeblink (EB) and heart rate (HR) response. Lesions of the mPFC, but not ACN, produced a severe impairment in EB reversal conditioning, but neither lesion affected original discrimination. However, both mPFC and ACN lesions produced a severe attenuation of accompanying HR decelerations during both initial differentiation and reversal. These results suggest that mPFC processing of Pavlovian conditioning contingencies affects not only the autonomic component of learning but preservative somatomotor conditioning as well, whereas ACN processing affects only the autonomic component.     

Intestinal expression of human heat shock protein 90 in patients with Crohn's disease and ulcerative colitis

Heat shock proteins are induced by several stress factors and are potential antigens in autoimmune disorders. Expression of heat shock protein 90 (HSP 90) was investigated in patients with inflammatory bowel disease and normal controls. We combined western blot analysis with laser densitometry for quantitation. Localization of HSP 90 was investigated by immunohistochemistry. Western blots showed a significant mucosal expression of HSP 90, which was comparable in patients and controls. There was also no difference between normal and inflamed mucosa in inflammatory bowel disease. In immunohistochemical staining studies, HSP 90 was detected in epithelial cells, mononuclear cells, giant cells, nerve cells, and endothelial cells of small vessels. There was no difference in the intensity of staining or localization in patients with inflammatory bowel disease compared to controls. These findings render a potential protective or immunogenic function of HSP 90 in inflammatory bowel disease unlikely.     

Influence of chronic variable stress (CVS) on the association of glucocorticoid receptor with heat-shock protein (HSP) 90 in rat hippocampus

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.