A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.

Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

Precipitation of kidney myosin IIA and IIB by freezing

Actomyosin precipitation is a critical step in the purification of myosins. In this work, the objective was to precipitate rat kidney actomyosin and isolate myosin by freezing and thawing the soluble fraction. Kidney was homogenized in imidazole buffer, centrifuged at 45000 g for 30 min, and the supernatant was frozen at -20°C for 48 h. The supernatant was thawed at 4°C, centrifuged at 45000 g for 30 min and the precipitate washed twice with imidazole buffer pH 7.0 (with and without Triton X-100, respectively). The resulting precipitate presented a polypeptide profile in SDS/PAGE characteristic of actomyosin and expressed Mg- and K/EDTA-ATPase activity. The actomyosin complex was solubilized with ATP and Mg, and the main polypeptide, p200, was purified in a DEAE-Sepharose column. p200 was marked with anti-myosin II, co-sedimented with F-actin in the absence, but not in the presence, of ATP and was identified by MS/MS with a high Mascot score for myosin IIA. The analysis identified peptides exclusive of myosin IIB, but detected no peptides exclusive of myosin IIC.     

Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.     

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.     

Inhibition of intracellular sorting and processing of lysosomal cathepsins H and L at reduced temperature in primary cultures of rat hepatocytes

Our recent studies with pulse-chase kinetic analysis in primary cultures of rat hepatocytes suggest that newly synthesized lysosomal cathepsins H and L are initially synthesized as larger proform enzymes, and then the precursor molecules are subsequently converted to the mature enzymes by limited proteolysis during the intracellular sorting process. This proteolytic maturation of procathepsins appears to proceed within an acidic environment, and these processing events are closely connected with the activation of enzymes. To further characterize the intracellular processing site for lysosomal cathepsins H and L, the pulse-chase kinetic study was carried out at 20 degrees C in cultured rat hepatocytes, because the transport of the procathepsins was expected to be blocked at the trans-Golgi compartment at 20 degrees C. We show here that the newly synthesized procathepsins are accumulated intracellularly and the processing for lysosomal cathepsins is completely arrested at 20 degrees C along the sorting pathway. The procathepsins thus accumulated in the cell are presumed to be transported to the Golgi complex, since the oligosaccharide moieties of these polypeptides appear to be phosphorylated. When the cells were shifted to 37 degrees C after an incubation for 4 h at 20 degrees C, a gradual increase of the mature forms was found. However, the processing kinetics generating the mature enzymes were slow compared to those in control cells at 37 degrees C. When the NH4Cl was present in the cells after the temperature shift to 37 degrees C, the intracellular processing of procathepsins was considerably retarded and the release of intracellular procathepsins into the extracellular medium was observed. These results indicate that NH4Cl might exert the inhibitory effect on the mannose 6-phosphate receptor-mediated intracellular targeting mechanism for the lysosomal cathepsins. Hence, the intracellular location of procathepsins accumulated at 20 degrees C is considered to be in proximity to the trans-Golgi compartment. Taken together, the present observations suggest that the propeptide-processing step for procathepsins, which is a critical step for generating the active enzymes, proceeds within the prelysosomal compartment or the lysosomes after the enzymes leave the trans-Golgi compartment.     

Effects of hypothermia on the development of ischemic lesions of skeletal muscles in rats]

The influence of hypothermia on the development of the ischemic disorders was studied using allotransplantation of the rat skeletal muscle (m. lumbricalis) to the anterior chamber of the eye after different period of ischemia. The morphological and immunohistochemical (monoclonal antibodies to heavy chain of the fast myosin, PAP-method) data were found confirming that hypothermia (2-4 degrees C) prolongs the period of the ischemic disorders first appearance by 5 h (from 6 to 11 h) if compared with development of ischemia in the muscle at 21-23 degrees C.     

In vitro biosynthesis of rat sperm outer dense fiber components

Conditions were established for in vitro culture of seminiferous tubules of adult rat testis. Tubules fragments were able to incorporate radioactive amino acids for up to 6 hours of incubation at 32 degrees C in a modified Eagle's minimum media, indicating biosynthetic activity. Addition of D-glucose (11 Mm) increased the incorporation of either [3H] Leucine or [35S] Methionine four-fold in the protein components of seminiferous tubules. Polyclonal antibodies against outer dense fibers (ODF) polypeptides, which represent approximately 30% of the total sperm proteins, immunoprecipitated 5% of the total radioactivity from cultures carried out either in the presence or absence of D-glucose. Moreover, antibodies specific for the 27-30 kilodalton polypeptides of ODF immunoprecipitated 2% of the total radioactivity, showing no differences in the presence and absence of D-glucose. This study indicates that ODF polypeptides can be synthesized in vitro at 32 degrees C with and without D-glucose.     

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

Sheep testicular gonadotropin binding sites: characterization and changes with surgical shortening of the scrotum

Gonadotropin receptors in previously frozen (-70 degrees C) sheep testicular tissue were characterized, and methods of assessment of receptor binding activity were established and applied to an investigation of testicular function in the short scrotum ram. Binding of 125I-labelled ovine luteinizing hormone (125I-oLH) and 125I-labelled ovine follicle-stimulating hormone (125I-oFSH) to testicular membranes was highly specific and saturable. Uptake of labelled gonadotropins was proportional to the amount of membrane protein, with 125I-oFSH showing greater specific binding. Initial association of 125I-oLH with binding sites was comparable at 4, 25, and 34 degrees C; with prolonged incubation, maximal binding occurred at 4 degrees C. Equilibrium was achieved in 8 h at 34 degrees C and in 16 h at 25 and 4 degrees C. In contrast, the temperature-dependent association of LH with rat testicular membranes was greater at 25 than at 4 degrees C. The rate of association of 125I-oFSH to binding sites was proportional to incubation temperature, with equilibrium being achieved in 2 h at 34 degrees C and in 16 h at 25 degrees C; binding at 4 degrees C; was slow and still increasing by 48 h. Binding of radioactive and nonradioactive oLH and oFSH was hormone specific and increased in a dose-dependent manner until saturation occurred. Shortening the scrotum of adult rams led to reductions (p less than 0.05) in testicular weight (60%) and in the number of LH (55%) and FSH (90%) binding sites per testis, with no apparent change in serum testosterone concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Induction of cellular DNA synthesis in G0-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses

tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

Neurochemical and thermal control of surfactant secretion by alveolar type II cells isolated from the marsupial, Sminthopsis crassicaudata

Pulmonary surfactant is synthesised in alveolar type II cells and secreted into the lining of the lung in response to ventilation, temperature changes and autonomic neurotransmitters. Type II cells were isolated from the heterothermic marsupial, Sminthopsis crassicaudata. The neurotransmitters, isoproterenol and carbamylcholine chloride significantly increased phosphatidylcholine secretion at 37 degrees C (basal: 14.2%, isoproterenol: 20.1%, carbamylcholine: 17.0%). Temperature reduced the rate of secretion from dunnart type II cells (e.g. basal: 14.2% at 37 degrees C; 7.2% at 18 degrees C). However, the change in secretory rate between 37 degrees C and 18 degrees C was less than expected if due to temperature alone (Q10= 1.4). The surfactant secretory pathway is therefore modulated by factors other than and in addition to, temperature. The response of dunnart type II cells to the agonists remained the same at both temperatures. Basal secretion was higher in dunnart type II cells (14.2% in 4 h) than has been reported in rat type II cells (1.9% in 3 h) and consequently, the agonist-stimulated increases in secretion from dunnart type II cells (41% above basal in 4 h) were much lower than observed for rat type II cells (200% above basal in 1.5 h).     

Effect of temperature and duration of hyperthermia on HSP72 induction in rat tissues

The aim of the present study was to determine whether heat shock protein 72 (HSP72) is induced in a heated rat model at rectal temperatures below 42 degrees C. Rats were divided into a control group and six groups (n = 6) heated to different rectal temperatures: 39 degrees C for 1 h (39), 40.0 degrees C for either 15 min (40S) or 1 h (40L), 41.0 degrees C for either 15 min (41S) or 1 h (41L) and 42.0 degrees C for 15 min (42). Tissues were sampled 4 h after heating. Following 1 h at 40.0 degrees C, HSP72 was significantly elevated in heart (p < 0.005), but not in gut or liver tissue. In all three tissues, HSP72 was significantly elevated under the conditions 41L and 42 compared to control tissue (p < 0.005). Marked differences were found in the amount of HSP72 induced in different tissues in response to the same heat stress. Duration of heating was important in modulating HSP72 induction, with a significantly greater induction of HSP72 following 1 h compared to 15 min at 41 degrees C in all three tissues (p < 0.02). A correlation was found between thermal load and HSP72 content in liver, heart (both p < 0.01) and gut (p < 0.001) for the rats heated to 41 and 42 degrees C. These data show that HSP72 is induced at temperatures below 42 degrees C, with striking differences between tissues.     

alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress

Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise.