Inducible 70-kDa Heat Shock Protein Research Articles

Abstract WMP74: Cofilin-Actin Rod Formation in Experimental Stroke is Attenuated by Therapeutic Hypothermia and Overexpression of the Inducible 70-kDa Heat Shock Protein (Hsp70)

Background and Purpose: Cofilin-actin rods are covalently linked aggregates of cofilin -1 and actin. Under ischemic conditions, these rods have been observed in neuronal processes, but their significance is unknown. Here, we explored a potential role of these rods in two different models of neuroprotection from experimental stroke—therapeutic hypothermia and the 70-kDa heat shock protein (Hsp70). Methods: Male C57/BL6 mice were subjected to distal middle cerebral artery occlusion (dMCAO), and treated with hypothermia. Cooling (31°C, 2hrs) was begun at the onset of dMCAO. A neuroprotective effect of hypothermia was validated by functional assessments and infarct volume measurement. Cofilin-actin rod formation was assessed by histological analysis at 4 and 24hrs after dMCAO. Its expression was analyzed in three different regions, infarct core (the center of the infarct), penumbra (area salvaged by intervention and a fixed distance from the midline), and ischemic borderzone (border of ischemic lesion). In addition, Hsp70-overexpressing transgenic (Tg) mice and Hsp70-deficient (Ko) mice were also subjected to dMCAO, and cofilin-actin rod expression was assessed in same manner. Results: As shown previously by our lab, both hypothermia and Hsp70 Tg mice had smaller lesion sizes and improved neurological outcomes compared to normothermic and wildtype (Wt) mice. Hsp70 Ko mice had larger lesion sizes and worsened neurological outcomes. Following dMCAO, cofilin-actin rods were increased, but were reduced by hypothermia in the ischemic core (24hrs, p<0.05), penumbra (4 and 24hrs, p<0.05), and ischemic borderzone (4 and 24hrs, p<0.05). Among Hsp70 Tg mice, cofilin-actin rod formation was decreased in the ischemic borderzone (4 and 24hrs, p<0.05), while Hsp70 Ko mice showed increased rod formation in the penumbra (4 and 24hrs, p<0.05). Conclusions: Cofilin-actin rod formation was suppressed under conditions of improved neurological outcome, and increased under circumstances where outcome was worsened. This suggests that cofilin-actin rods may act to participate in or exacerbate ischemic pathology, and warrants further study as a potential therapeutic target.

Membrane Localization of HspA1A, a Stress Inducible 70-kDa Heat-Shock Protein, Depends on Its Interaction with Intracellular Phosphatidylserine.

HspA1A is a cytosolic molecular chaperone essential for cellular homeostasis. HspA1A also localizes at the plasma membrane (PM) of tumor and stressed cells. However, it is currently unknown how this cytosolic protein translocates to the PM. Taking into account that HspA1A interacts with lipids, including phosphatidylserine (PS), and that lipids recruit proteins to the PM, we hypothesized that the interaction of HspA1A with PS allows the chaperone to localize at the PM. To test this hypothesis, we subjected cells to mild heat-shock and the PM-localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that HspA1A’s membrane localization increased during recovery from non-apoptotic heat-shock. Next, we selectively reduced PS targets by overexpressing the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, and determined that HspA1A’s membrane localization was greatly reduced. In contrast, the reduction of PI(4,5)P2 availability by overexpression of the PLCδ-PH biosensor had minimal effects on HspA1A’s PM-localization. Implementation of a fluorescent PS analog, TopFluor-PS, established that PS co-localizes with HspA1A. Collectively, these results reveal that HspA1A’s PM localization and anchorage depend on its selective interaction with intracellular PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response.

Properties of Epithelial Cells and Vaginal Secretions in Pregnant Women When Lactobacillus crispatus or Lactobacillus iners Dominate the Vaginal Microbiome

Our objective was to determine differences in properties of vaginal epithelial cells and the composition of vaginal secretions when Lactobacillus crispatus or Lactobacillus iners are numerically dominant in the vaginal microenvironment of pregnant women. The vaginal microbiomes of 157 first-trimester pregnant women were identified by classifying partial 16S gene sequences amplified from the V1 to V3 region of bacterial ribosomal 16S RNA genes. The extent of autophagy and cell stress in vaginal epithelial cells was determined by measuring the intracellular levels of p62 and the inducible 70-kDa heat shock protein (hsp70). Vaginal secretions were analyzed using a colorimetric assay for d- and l-lactic acid and by enzyme-linked immunosorbent assay for matrix metalloproteinase 8, neutrophil gelatinase-associated lipocalin, α-amylase, hyaluronan, calprotectin, S100A8, and extracellular matrix metalloproteinase inducer (EMMPRIN). L. crispatus was dominant in 69 (43.9%) women, while L iners dominated in 23 (14.6%) women. The median epithelial p62 levels were 0.41 and 4.26 ng/mL in women with L crispatus or L iners dominance, respectively ( P = .0035). The corresponding median hsp70 levels were 4.24 and 14.50 ng/mL, respectively ( P < .0001). The d-lactic acid concentration in vaginal fluid was highest in association with L crispatus dominance, while all other vaginal fluid compounds except for EMMPRIN were highest when L iners was dominant ( P< .03). Epithelial cells exhibit a higher level of autophagy, lower induction of stress-related hsp70, and release lower level of mediators when L crispatus is most abundant as compared to when L iners dominates the vaginal microbiota.

BGP-15 improves contractile function of regenerating soleus muscle.

This study investigated the effect of the heat shock protein inducer O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic amidoxime (BGP-15) on the morphology and contractile function of regenerating soleus muscles from mice. Cryolesioned soleus muscles from young mice treated daily with BGP-15 (15mg/Kg) were evaluated on post-cryolesion day 10. At this time point, there was a significant decrease in the cross-sectional area of regenerating myofibers, maximal force, specific tetanic force, and fatigue resistance of regenerating soleus muscles. BGP-15 did not reverse the decrease in myofiber cross-sectional area but effectively prevented the reduction in tetanic force and fatigue resistance of regenerating muscles. In addition, BGP-15 treatment increased the expression of embryonic myosin heavy chain (e-MyHC), MyHC-II and MyHC-I in regenerating muscles. Although BGP-15 did not alter voltage dependent anion-selective channel 2 (VDAC2) expression in cryolesioned muscles, it was able to increase inducible 70-kDaheat shock protein(HSP70) expression. Our results suggest that BGP-15 improves strength recovery in regenerating soleus muscles by accelerating the re-expression of adult MyHC-II and MyHC-I isoforms and HSP70 induction. The beneficial effects of BGP-15 on the contractile function of regenerating muscles reinforce the potential of this molecule to be used as a therapeutic agent.

The composition of the vaginal microbiome in first trimester pregnant women influences the level of autophagy and stress in vaginal epithelial cells

Epithelial cells lining the vagina are major components of genital tract immunity. The influence of the vaginal microbiome on properties of host epithelial cells is largely unexplored. We evaluated whether differences in the most abundant lactobacilli species or bacterial genera in the vagina of first trimester pregnant women were associated with variations in the extent of stress and autophagy in vaginal epithelial cells. Vaginal swabs from 154 first trimester pregnant women were analyzed for bacterial composition by amplification and sequencing of the V1–V3 region of bacterial 16S rRNA genes. Vaginal epithelial cells were lysed and autophagy quantitated by measurement of p62. Intracellular levels of the inducible 70kDa heat shock protein (hsp70), an indicator of cell stress and an autophagy inhibitor, were determined. When Lactobacillus crispatus was the most abundant member of the vaginal microbiota, epithelial p62 and hsp70 levels were lowest as compared to when other bacterial taxa were most abundant. The highest concentrations of p62 and hsp70 were associated with Streptococcus and Bifidobacterium abundance. The p62 level associated with Gardnerella abundance was lower than that observed when lactobacilli other than L. crispatus were most abundant. In conclusion, in the first trimester of pregnancy the abundance of different bacterial taxa is associated with variations in autophagy and magnitude of the stress response in vaginal epithelial cells.

Group B streptococcus alters properties of vaginal epithelial cells in pregnant women

Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage.

807: Autophagy and inducible 70kDa heat shock protein influences in pregnancy, parturition and postpartum

807: Autophagy and inducible 70kDa heat shock protein influences in pregnancy, parturition and postpartum

Interaction between the inducible 70-kDa heat shock protein and autophagy: effects on fertility and pregnancy.

A consequence of hsp70 (HSPA1A) induction is the inhibition of autophagy. Evidence of autophagy involvement in all aspects of the reproductive process is reviewed, and possible consequences of hsp70 induction at each developmental stage are postulated. It is proposed that aberrant external or internal stimuli that result in high levels of hsp70 production interfere with normal autophagy-related functions and lead to a decrease in the number of functional ova and spermatozoa, impaired pre- and post-implantation embryo development, and increased susceptibility to premature labor and delivery. The purpose of this review is to increase understanding of hsp70-autophagy interactions during reproduction. Interventions to modulate this interaction will lead to development of novel protocols to improve fertility and pregnancy outcome.

Human Epididymis Protein 4 and Secretory Leukocyte Protease Inhibitor in Vaginal Fluid: Relation to Vaginal Components and Bacterial Composition

Human epididymis protein 4 (HE4) is a protease inhibitor and a recently identified serum biomarker for ovarian cancer. Properties of HE4 in the genital tract of healthy women have not been evaluated. We evaluated associations between HE4 and a second vaginal protease inhibitor, secretory leukocyte protease inhibitor (SLPI), with vaginal concentrations of innate immune mediators or proteases and with the types of vaginal bacterial communities. Vaginal secretions were collected from 18 healthy reproductive age women and assayed by enzyme-linked immunosorbent assay for concentrations of HE4, SLPI, kallikrein 5, cathepsin B, interleukin 1β (IL-1), IL-1 receptor antagonist (IL-1 ra), mannose-binding lectin (MBL), the inducible 70-kDa heat shock protein, and matrix metalloproteinase (MMP)-8. The species composition of vaginal bacterial communities in 16 women was characterized by sequencing amplicons derived from 16S bacterial ribosomal RNA genes. Correlations between any 2 assays were analyzed by the Spearman rank correlation tests. Differences in the concentrations of HE4 and SLPI, and between soluble components and vaginal community types, were analyzed by the Mann-Whitney U tests. Vaginal HE4 concentrations, but not SLPI levels, were positively correlated with levels of IL-1β (P = .0152), IL-1ra (P = .0061), MBL (P = .0100), and MMP-8 (P = .0315). The median vaginal HE4 level, as well as concentrations of MBL, IL-1β, IL-1ra, and MMP-8, was highest when Gardnerella vaginalis dominated a vaginal community. The association between HE4, elevated levels of proteases, immune mediators and high proportions of G vaginalis strongly suggests that HE4 is a component of the proinflammatory immune response in the female genital tract.

Hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.

Hsp70 and a Novel Axis of Type I Interferon-Dependent Antiviral Immunity in the Measles Virus-Infected Brain

The major inducible 70-kDa heat shock protein (hsp70) is host protective in a mouse model of measles virus (MeV) brain infection. Transgenic constitutive expression of hsp70 in neurons, the primary target of MeV infection, abrogates neurovirulence in neonatal H-2(d) congenic C57BL/6 mice. A significant level of protection is retained after depletion of T lymphocytes, implicating innate immune mechanisms. The focus of the present work was to elucidate the basis for hsp70-dependent innate immunity using this model. Transcriptome analysis of brains from transgenic (TG) and nontransgenic (NT) mice 5 days after infection identified type I interferon (IFN) signaling, macrophage activation, and antigen presentation as the main differences linked to survival. The pivotal role of type I IFN in hsp70-mediated protection was demonstrated in mice with a genetically disrupted type I IFN receptor (IFNAR(-/-)), where IFNAR(-/-) eliminated the difference in survival between TG and NT mice. Brain macrophages, not neurons, are the predominant source of type I IFN in the virus-infected brain, and in vitro studies provided a mechanistic basis by which MeV-infected neurons can induce IFN-β in uninfected microglia in an hsp70-dependent manner. MeV infection induced extracellular release of hsp70 from mouse neuronal cells that constitutively express hsp70, and extracellular hsp70 induced IFN-β transcription in mouse microglial cells through Toll-like receptors 2 and 4. Collectively, our results support a novel axis of type I IFN-dependent antiviral immunity in the virus-infected brain that is driven by hsp70.

Myocardial accumulation and localization of the inducible 70-kDa heat shock protein, Hsp70, following exercise

Exercise increases the 70-kDa heat shock protein (Hsp70) in the myocardium, and this exercise-induced increase is associated with significantly improved cardiac recovery following insult. However, while heat shock has been shown to elevate Hsp70 primarily in the cardiac vasculature of the myocardium, the localization following exercise is unknown. Male Sprague-Dawley rats performed continuous treadmill running at 30 m/min for 60 min (2% incline) on either 1 or 5 consecutive days. At 30 min and 24 h following exercise, hearts were extirpated, and the left ventricle was isolated, OCT-cork mounted, and sectioned for immunofluorescent analysis. Whereas immunofluorescent analysis revealed little to no Hsp70 in control hearts and 30 min postexercise, the accumulation of Hsp70 24 h after a single exercise bout or 5 days of training was predominantly located in large blood vessels and, in particular, colocalized with a marker of smooth muscle. Furthermore, higher core temperatures attained during exercise led to more abundant accumulation in smaller vessels and the endothelium. It is concluded that the accumulation of myocardial Hsp70 following acute exercise predominantly occurs in a cell type-specific manner, such that changes in the cardiac vasculature account for much of the increase. This accumulation appears first in the smooth muscle of larger vessels and then increases in smaller vessels and the endothelium, as core temperature attained during exercise increases. This finding supports the observations after heat shock and further suggests that the vasculature is a primary target in exercise-induced cardioprotection.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7days and subsequently released from immobilization and evaluated after 7days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

Measles Virus Neurovirulence and Host Immunity

Measles virus is highly neuroinvasive, yet host immune responses are highly effective at limiting neurovirulence in humans. We know that neurons are an important target of infection and that both IFN-γ and -β expression are observed in the measles virus-infected human brain. Rodent models can be used to understand how this response is orchestrated. Constitutive expression of the major inducible 70-kDa heat-shock protein is a feature of primate tissues that is lacking in mice. This article examines the importance of addressing this difference when modeling outcomes of brain infection in mice, particularly in terms of understanding how infected neurons may activate uninfected brain macrophages to produce IFN-β and support T-cell production of IFN-γ, a mediator of noncytolytic viral clearance. New and historical data suggest that the virus heat-shock protein 70 relationship is key to a protective host immune response and has potential broad relevance.

Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study

Toxoplasma gondii and Plasmodium falciparum are apicomplexan parasites responsible for serious diseases in humans. Many studies have focused on the post-translational modifications (PTMs) found in the two protists including phosphorylation, acetylation or SUMOylation but only a few of these are concerned with the nuclear and cytosolic-specific glycosylation O-GlcNAcylation. O-GlcNAcylation is a highly dynamic PTM-regulated by the ON and OFF enzymes: O-GlcNAc transferase and O-GlcNAcase-that can compete with phosphorylation but its function remains unclear. In this work, we directly prove the O-GlcNAcylation in T. gondii using antibodies specifically directed against the modification and we strongly suggest its occurrence in P. falciparum. We found that the inducible 70kDa-Heat Shock Protein is O-GlcNAcylated, or associated with an O-GlcNAc-partner, in T. gondii. Using anti-OGT antibodies we were able to detect the expression of the glycosyltransferase in T. gondii cultured both in human foreskin fibroblast and in Vero cells and report its putative sequence. For the first time the presence of O-GlcNAcylation is unequivocally shown in T. gondii and suspected in P. falciparum. Since the O-GlcNAcylation is implicated in many biological fundamental processes this study opens a new research track in the knowledge of apicomplexans' life cycle and pathogenic potential.

The decay phase of Ca2+ transients in skeletal muscle: regulation and physiologyThis paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process.

Cytosolic Ca2+ transients associated with contraction and relaxation cycles in skeletal muscle are primarily dependent on the kinetics of Ca2+ release and Ca2+ uptake by the sarcoplasmic reticulum (SR). In humans, sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are solely responsible for the removal of Ca2+ from the cytosol following muscle contraction. There are several signalling systems involved in the acute regulation of SERCAs required to achieve a given Ca2+ transient during muscle contraction-relaxation cycles. Cyclic-AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase signalling activate SERCAs through the regulation of the endogenous SERCA-regulatory proteins, phospholamban and sarcolipin, both of which are highly expressed in human skeletal muscle. Recent studies on the regulation of SERCA2b in arterial smooth muscle and work from my laboratory on the interaction between SERCAs and the inducible 70-kDa heat shock protein suggests a novel role for redox signalling in regulating SERCA activity. In the absence of fatigue, activation of these signalling systems in response to repeated muscle activity serves to increase the rate of cytosolic free Ca2+ ([Ca2+]f) decay (i.e., SR Ca2+ uptake) and the speed of muscle relaxation.

Effects of ovarian sex hormones and downhill running on fiber-type-specific HSP70 expression in rat soleus

This study examined the influence of the ovarian sex hormones, estrogen and progesterone, on the fiber-type-specific response of the inducible 70-kDa heat shock protein (HSP70) to damaging exercise in rat soleus. Ovariectomized female rats were divided into three treatment groups (n = 16 per group): sham (S), progesterone (P; 25 mg pellet), and estrogen (E; 0.25 mg pellet). Each treatment group was divided into control and exercised groups. After 8 days of sham or hormone treatment, animals ran downhill intermittently for 90 min (17 m/min, -13.5 degrees grade) on a treadmill, and soleus muscles were removed 24 h postexercise. HSP70 expression was assessed in whole muscle homogenates by Western blotting and in individual muscle fiber types by immunohistochemical analysis of serial cross sections of soleus samples. Comparisons between control groups showed that HSP70 expression in soleus was increased (P < 0.05) in E compared with both S and P. No difference (P > 0.05) was observed between S and P. Following downhill running, HSP70 content in soleus was increased (P < 0.05) compared with control in S and P, but not (P > 0.05) in E. As a result, soleus HSP70 content following downhill running was not different (P > 0.05) between any of the treatment groups. Under all conditions, HSP70 content was higher in type I vs. type II fibers, and the effects of both estrogen and exercise on HSP70 expression in soleus were also more pronounced in type I vs. type II fibers. These results demonstrate that 1) estrogen regulates HSP70 expression in skeletal muscle, increasing basal HSP70 expression and preventing further increases in HSP70 in response to exercise; 2) progesterone is not involved in the regulation of HSP70 expression in skeletal muscle; and 3) the effects of estrogen and exercise on HSP70 expression in skeletal muscle are fiber type specific.

Intracellular and extracellular expression of the major inducible 70kDa heat shock protein in experimental ischemia-reperfusion injury of the spinal cord

Inflammatory responses exacerbate ischemia-reperfusion (IR) injury of spinal cord, although understanding of mediators is incomplete. The major inducible 70kDa heat shock protein (hsp70) is induced by ischemia and extracellular hsp70 (e-hsp70) can modulate inflammatory responses, but there is no published information regarding e-hsp70 levels in the cerebrospinal fluid (CSF) or serum as part of any neurological disease state save trauma. The present work addresses this deficiency by examining e-hsp70 in serum and CSF of dogs in an experimental model of spinal cord IR injury. IR injury of spinal cord caused hind limb paraplegia within 2–3 h that was correlated to lumbosacral poliomalacia with T cell infiltrates at 3 d post-ischemia. In this context, we showed a 5.2-fold elevation of e-hsp70 in CSF that was induced by ischemia and was sustained for the following 3 d observation interval. Plasma e-hsp70 levels were unaffected by IR injury, indicating e-hsp70 release from within the central nervous system. A putative source of this e-hsp70 was ependymal cells in the ischemic penumbra, based upon elevated i-hsp70 levels detected within these cells. Results warrant further investigation of e-hsp70's potential to modulate spinal cord IR injury.

Biological Heterogeneity of the Peptide-binding Motif of the 70-kDa Heat Shock Protein by Surface Plasmon Resonance Analysis

70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.

Adaptive and Maladaptive Cardiac Hypertrophy: What Is the Effective Role of Heat Shock Transcription Factor 1?

To the Editor: In the recent interesting article by Sakamoto et al,1 the authors found that rats undergoing exercise training showed lower degree of left ventricular (LV) hypertrophy, higher expression and activity of heat shock transcription factor 1 (HSF1), and preserved LV systolic function in comparison with rats exposed to sustained pressure overload by surgical constriction of the transverse aorta (TAC). They also observed that transgenic mice expressing constitutively active HSF1 showed less evident hypertrophic response and better systolic function than their wild-type littermates 5 weeks after TAC. Conversely, HSF1-deficient mice showed similar degree of LV hypertrophy but worse systolic function in comparison with their wild-type littermates after 4 weeks of exercise or 1 week after TAC. These data support the intriguing hypothesis that HSF1 upregulation in cardiac myocytes may play a key role in preventing LV systolic impairment in the exercise-induced hypertrophy model, and in determining the adaptive or maladaptive nature of the hypertrophic response. An important …