Exogenous heat shock cognate protein Hsc 70 prevents axotomy-induced death of spinal sensory neurons

The objective of this study was to evaluate the individual and combined effects of Nacystelyn (NAL) and rhDNase in vitro on the rheological properties of cystic fibrosis (CF) sputum. Sputum samples were collected from 11 CF patients and subjected to the following protocols: 1) negative control sample without any treatment; 2) positive control sample incubated with 0.02 ml of normal saline; 3) incubation of CF sputum with 0.02 mL DNase (25 micrograms/mL in normal saline) at 37 degrees C to achieve 2.5 micrograms/g final sputum concentration (approximately 100 nM); 4) incubation of CF sputum with 0.02 mL NAL (30.9 micrograms/mL in normal saline) at 37 degrees C to achieve 3.09 micrograms/g final sputum concentration (10 microM); and 5) combination of protocols 3 and 4 with half the concentration of each drug. The samples in protocols 2 through 5 were incubated for 30 minutes at 37 degrees C. For each protocol, spinnability by filancemeter and viscoelasticity (log G*) by magnetic microrheometer were measured at baseline and 30 minutes. Treatment of the sputum with rhDNase alone or NAL alone decreased spinnability more than control treatment with saline. Combining NAL with rhDNase at half the concentration of each drug significantly decreased spinnability more than either treatment by itself. There were no significant changes in log G* or the derivative parameters, mucociliary clearability index (MCI) and cough clearability index (CCI). The enhanced reduction in sputum spinnability by the combination of NAL and rhDNase indicates additative effects between these two mucolytic treatments. These results suggest that combined treatment with rhDNase and NAL should be considered as a potential therapy for CF patients.     

The effect of physical exercise on the response to exertion in the elderly

BACKGROUND: A decrease in adaptation to exertion has been observed as age progresses. Although this decline may also be affected by factors such as health conditions and age, physical inactivity related to sedentary behaviour plays a dominant role. METHODS: In order to evaluate the influence of physical activity on cardiovascular response to exertion in the elderly, 4 groups of 22 subjects each were submitted to maximal electrocardiographic exercise test on a cycloergometer (multistage program with 30 Watts x 3 min. steps). All subjects were male. The composition of the groups was as follows: 1) veteran long distance runners (mean age: 71 +/- 5.4); 2) sedentary veterans (mean age: 69.8 +/- 3.9); 3) young long distance runners (mean age: 25.4 +/- 4.3); 4) sedentary young adults (mean age: 25.8 +/- 3.9). The endurance athletes, well fitted to competition, had been practicing sport activity for at least 3 years. RESULTS: Heart rate, arterial systolic and diastolic blood pressure were recorded; mean blood pressure and double product were calculated at baseline and at the climax of the stress test; furthermore, total and maximal watts were recorded. For each of the parameters, Student's t test for non-paired observations were used to evaluate statistical differences amongst the four groups. The most interesting result arises in the comparison between veteran long distance runners and sedentary young adults: between the two groups no statistically significant differences in workload, expressed as total watts (1649.55 +/- 296.32 vs 1650.00 +/- 446.32; p = NS) and maximal watts (175.91 +/- 19.19 vs 173.18 +/- 24.38; p = N.S.), were observed. On the contrary, highly significant differences in both total (p < 0.01) and maximal (p < 0.01) watts were noticed by comparing long distance runners and sedentary subjects of the same age. CONCLUSIONS: These data support the hypothesis that the progressive reduction in physical activity, which is usually observed in aging, is the major determinant of exercise deconditioning in the elderly.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed.     

Heat shock proteins and heat adaptation of the whole organism

Adaptation to heat may occur through acclimatization or thermotolerance; however, the linkage of these phenomena is poorly understood. The importance of heat shock proteins (HSPs) in thermotolerance and differences in their accumulation in organisms adapted to the heat suggest a role for HSPs in acclimatization as well. The role of HSPs in heat adaptation of the whole organism and the interrelationships among heat adaptation, endotoxin tolerance, and cytokine resistance through HSPs are reviewed.     

Regulation of heat shock protein 27 expression of prostatic cells in response to heat treatment

BACKGROUND: Reading acuity as well as reading speed are good predictors of everyday visual function. As visual acuity tests are poor predictors of the real-world function, performance-based tests, e.g., reading speed measurements, can be used for the determination of visual function. Thus, a German reading chart was developed in order to evaluate reading acuity as well as reading speed. METHODS: Print size is defined as the height of a lower case x and progresses logarithmically from one phrase to another (factor: 1.25). Reading acuity is determined in LogRAD (Reading Acuity Determination). 32 short German phrases were created, comparable concerning grammatical difficulty as well as in number (n = 14), length and position of words. The reading speed parameters measured with a stop-watch in 160 persons (aged: Phi = 21a +/- 3.8a) were calculated in words per minute (w/min). Out of the 32 phrases the 24 most similar ones were selected statistically and used for the reading charts (Radner Reading Charts). With these reading charts a reading acuity score (LogRAD-score) can be calculated considering reading errors in words of different length. Reading speed can be determined at the same time. Reading acuity (LogRAD-Score) was measured in 32 normal eyes of 16 students and compared to the angular visual acuity (LogMAR). RESULTS: The mean reading speed of the test persons was 211.8 +/- 34.1 w/min. 24 phrases fulfilled the test item criteria for the reading chart: mean +/- 0.25 x SD. The reliability analyses yielded an overall Cronbach's alpha coefficient of 0.98! The mean visual acuity measured in 32 eyes was -0.115 +/- 0.097 LogMAR and the mean reading acuity score was +0.026 +/- 0.091 LogRAD. The mean difference was +0.104 +/- 0.066 and the correlation between LogMAR and LogRAD was good (r = 0.59). CONCLUSIONS: With these reading charts it is for the first time possible to simultaneously determine reading acuity as well as reading speed in German. The high reliability of the 24 phrases and the high correlation between LogMAR and LogRAD leads us to expect a good reproducibility of the reading acuity evaluations. For the "Radner Reading Charts" we have shown that print size is the main reason for changes of reading speed.     

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.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

Expression of heat shock protein 27 in chromomycosis

We report on a 58-year-old woman with long-lasting (36 years) chromomycosis on the foot and secondary self-inoculation from foot to hand 4 years ago. Mycological classification was performed after culture on Sabouraud glucose agar. We used haematoxylin and eosin and Giemsa staining and an antibody to heat shock protein (HSP) 27 (Stress Gen, Clone G3.1) on paraffin-embedded and cryostat specimens of chromomycosis. The mycological culture revealed the fungus Fonsecaea pedosoi. Histopathology revealed dermal fibrosis with persistent fungi (Medlar bodies), numerous mast cells and pseudoepitheliomatous hyperplasia. Immunohistochemically, HSP 27 was positively identified in F. pedrosoi. Moreover, in differentiating keratinocytes in the pseudoepitheliomatous lesions of chromomycosis, HSP 27 was increasingly expressed from basal layers to stratum spinosum in the epidermis but not in keratinocytes directly bordering Medlar bodies. In chromomycosis, HSP 27 is expressed, in accordance with its role as a marker of differentiation and proliferation, in keratinocytes and also in F. pedrosoi. It remains unknown if these results might explain the therapeutic efficacy of hyperthermic treatment.     

A novel 29-kDa chicken heat shock protein

The family of small heat shock proteins is the more variable among the highly conserved superfamily of heat shock proteins (HSP). Using a metabolic labeling procedure with tissue explants, we have detected in chickens a new member of the small HSP family with an apparent molecular weight of 29-kDa. This protein was induced in broiler chickens' heart muscle and lungs following an in vivo heat stress. The 29-kDa band appears after 3 h of heat stress, much later than the induction of HSP 90, HSP 70, and HSP 27. The late onset of induction suggests that HSP 29 plays a more specific role of a "second stage defense protein".     

Differential transcript induction of parsley pathogenesis-related proteins and of a small heat shock protein by ozone and heat shock

Cyclophosphamide (CP) is a widely used antineoplastic drug. It tests positive in several genotoxicity assays, including those with endpoints such as chromosomal aberrations in mammalian cells, mitotic recombination in Drosophila melanogaster, and dominant lethal mutations in rodents. We have explored the effects of CP on genome stability of mouse (Mus domesticus) spermatogenic cells, using a recombination-based transgenic assay called MUSCATEER. In this system, intrachromosomal gene conversion events between two mutually defective lacZ genes generates beta-galactosidase activity in spermatids. The frequency of gene conversion events is determined by scoring spermatids stained with the lacZ substrate, X-gal. A dose-dependent induction of lacZ-positive spermatids was observed following single intraperitoneal CP exposures of 10, 100, and 200 mg/kg. At 200 mg/kg, there was a 25-fold increase over baseline. Treatment of a control transgenic line containing only a frame-shifted lacZ transgene provided an indication that CP also induced reversion mutations. The timing of the response indicated that the induction of recombination and/or mutation occurred primarily in meiotic stage cells. These results demonstrate potent germline mutagenicity of CP, and validate the utility and sensitivity of genetic recombination as a rapid indicator of genotoxicity in whole animals.     

A new heat shock protein that binds nucleic acids

Familial acromegaly/gigantism occurring in the absence of multiple endocrine neoplasia type I (MEN-1) or the Carney complex has been reported in 18 families since the biochemical diagnosis of GH excess became available, and the genetic defect is unknown. In the present study we examined 2 unrelated families with isolated acromegaly/gigantism. In family A, 3 of 4 siblings were affected, with ages at diagnosis of 19, 21, and 23 yr. In family B, 5 of 13 siblings exhibited the phenotype and were diagnosed at 13, 15, 17, 17, and 24 yr of age. All 8 affected patients had elevated basal GH levels associated with high insulin-like growth factor I levels and/or nonsuppressible serum GH levels during an oral glucose tolerance test. GHRH levels were normal in affected members of family A. An invasive macroadenoma was found in 6 subjects, and a microadenoma was found in 1 subject from family B. The sequence of the GHRH receptor complementary DNA in 1 tumor from family A was normal. There was no history of consanguinity in either family, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all affected and unaffected screened subjects. Five of 8 subjects have undergone pituitary surgery to date, and paraffin-embedded pituitary blocks were available for analysis. Loss of heterozygosity on chromosome 11q13 was studied by comparing microsatellite polymorphisms of leukocyte and tumor DNA using PYGM (centromeric) and D11S527 (telomeric), markers closely linked to the MEN-1 tumor suppressor gene. All tumors exhibited a loss of heterozygosity at both markers. Sequencing of the MEN-1 gene revealed no germline mutations in either family, nor was a somatic mutation found in tumor DNA from one subject in family A. The integrity of the MEN-1 gene in this subject was further supported by demonstration of the presence of MEN-1 messenger ribonucleic acid, as assessed by RT-PCR. These data indicate that loss of heterozygosity in these affected family members appears independent of MEN-1 gene changes and suggest that a novel (tissue-specific?) tumor suppressor gene(s) linked to the PYGM marker and expressed in the pituitary is essential for regulation of somatotrope proliferation.     

Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin

alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alphaB-crystallin are modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alphaB-crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein.     

Structure-function analysis of the Escherichia coli GrpE heat shock protein

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.     

Phagocytosis of Pseudomonas aeruginosa fails to elicit heat shock protein expression in human monocytes

Phagocytosis represents a powerful stress for the phagocytic cells. Phagocytosis of Staphylococcus aureus induces a stress response associated with the synthesis of specific heat shock/stress proteins (HSP). Here we investigated the stress response of human monocyte-macrophages (m phi) to Pseudomonas aeruginosa, a bacterium found, as for S. aureus, in the airways of patients suffering cystic fibrosis. P. aeruginosa activated in m phi the production of both extra- and intracellular O2-; increased Interleukin-1 beta and actin, but failed to induce host HSP. Neither S. aureus' exotoxins nor the scavenging property of P. aeruginosa's alginate, but the lower toxicity of P. aeruginosa and/or differential activation of proteine kinase C (PKC) by the two bacteria, might explain their differences in host HSP induction. While O2- is insufficient to induce HSP synthesis in m phi, hydroxyl radicals, generated in the presence of exogenous iron, is a likely additional signal, along with PKC activation, for HSP induction during bacterial phagocytosis.     

On the cardioprotective effect of adenosine

In isovolumically perfused Langendorff heart preparations of guinea pigs adenosine-depending on the experimental protocol-more or less could prevent the hypoxia-induced decrease in myocardial adenosine triphosphate [ATP], creatine phosphate [CP], glycogen and increase in lactate, i.e. showed cardioprotection.     

Whole body heat shock fails to protect mouse heart against ischemia/reperfusion injury: role of 72 kDa heat shock protein and antioxidant enzymes

The transgenic mice overexpressing heat shock protein 72 (HSP72) or antioxidants have been reported to be more resistant to myocardial ischemia/reperfusion injury. However, it remains unknown whether whole body heat stress (HS) which may induce HSP72 or endogenous antioxidants affords similar protection in the mouse heart. Adult male mice were treated with either HS (42 degrees C for 15 min) or anesthesia only (SC) against a group of non-stressed controls (NC). At 6 or 24 h later, the hearts were excised and perfused at a constant pressure of 55 mmHg in Langendorff mode. Following 30 min equilibration, hearts were subjected to 20 min of global ischemia and 30 min reperfusion (37 degrees C). Ventricular force was measured by a force-displacement transducer attached to the apex. Leakage of intracellular enzymes (CK, LDH) was measured in coronary efflux. Infarct size was determined by tetrazolium staining. The results showed that no significant differences between HS, SC, and NC groups in ventricular contractile function, CK and LDH release, or infarct size were observed at either time window. HS enhanced the expression of HSP72 in mouse hearts by two- to three-fold, whereas antioxidant enzyme activities (catalase and MnSOD) did not change significantly. We conclude that HS does not precondition the isolated perfused mice hearts against ischemia/reperfusion injury, despite induction of HSP72.