Anti-60-kDa heat shock protein antibodies in fetal serum: a biomarker for unexplained small for gestational age fetuses.

Introduction: Small for gestational age (SGA) is an important problem affecting 10% of pregnancies and is associated with significant perinatal morbidity. In about 80% of cases, a probable etiology or a major risk factor can be identified. But almost 20% of SGA cases are considered unexplained. The 60-kDa heat shock protein (HSP60) is a highly immunogenic protein whose synthesis is greatly upregulated under nonphysiological conditions. Bacterial and human HSP60 share a high degree of sequence homology, and immunity to conserved epitopes may result in development of autoimmunity following a bacterial infection. We hypothesized that unexplained SGA could be the consequence of immune sensitization to human HSP60. Methods: Unexplained SGA fetuses were identified by ultrasound biometry with normal Doppler velocimetry and with no detectable maternal or fetal abnormalities. Fetal sera were obtained by cordocentesis performed for a karyotype analysis in cases of unexplained SGA (study group) or for screening of Rhesus incompatibility (control group). Fetal sera were tested for HSP60 antigen and for IgG and IgM anti-HSP60 by ELISA as well as for other immune and hematological parameters. Results: Maternal parameters were similar between the 12 study cases and the 23 control cases. The mean gestational age at cordocentesis was 29 weeks. IgM anti-HSP60 was detected in 12 cases (100%) and in no controls (p < 0.00017), while IgG anti-HSP60 was detected in 7 cases (58%) and only 1 control (p < 0.001). Three of the 4 cases with the highest IgM antibody levels died. There were no differences in fetal serum levels of HSP60 antigen or other immune and hematological markers between the two groups. Conclusion: Fetuses with unexplained SGA are positive for IgM and IgG antibody to human HSP60 and the specific IgM antibody level is predictive of fetal mortality. Detection of these antibodies indicates that a placental perturbation and a fetal autoimmune reaction to HSP60 are associated with this developmental delay.

How do plants feel the heat?

In plants, the heat stress response (HSR) is highly conserved and involves multiple pathways, regulatory networks and cellular compartments. At least four putative sensors have recently been proposed to trigger the HSR. They include a plasma membrane channel that initiates an inward calcium flux, a histone sensor in the nucleus, and two unfolded protein sensors in the endoplasmic reticulum and the cytosol. Each of these putative sensors is thought to activate a similar set of HSR genes leading to enhanced thermotolerance, but the relationship between the different pathways and their hierarchical order is unclear. In this review, we explore the possible involvement of different thermosensors in the plant response to warming and heat stress.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia.

We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of "mitochondrial chaperonopathies" and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Gentamicin-induced preconditioning of proximal tubular LLC-PK1 cells stimulates nitric oxide production but not the synthesis of heat shock protein

Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 µg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared to G/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed after preconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.

Amputation and heat induced protein synthesis in the regenerating forelimb of Notophthalmus viridescens

This thesis compares the responses of regenerating forelimb tissues of the newt Notophthalmu..f vlridescens to the stresses of hyperthermia and ID.echanical injury of amputation. In particular, both quantitative and qualitative changes in the synthesis of soluble proteins in stump tissues, including those of the heat shock protein family (HSP70-1ike) were examined. Results from SDS-PAGEfluorography indicate that the trauma of amputation mimics the heat shock response both quantitatively and temporally in its transient repression of the synthesis of most normal cellular proteins, and qualitatively. in the locaJized expression of two unique proteins (hsp30 and hsp70). Fluorography of proteins separated by twodimensional gets revealed that thelCl4:alizedt amputation induced 70kDa protein (amp70) was distinct from the more basic newt hsp/hsc70 isoforms. Although limb amputation resulted in an increase in the synthesis of HSP70 mRNA analogous to that induced by heat 3.b.OCKf amp70 did not cross-react with murine monoclonal antibodies directed against both the inducible and cognate HSP70 proteins of the human. Thus, the possible relationship of amp70 to other members of the HSP70-1ike protein family remains unclear. Western analyses indicated that the levels of the constitutive form of HSP70 (hsc70) were found to be regulated in a stage-dependent manner in the distal stump tissues of the regen,erating forelimb of the newt. The highest levels were found in the mid-late bud stage, a period during which rapidly dividing blastema cells begin to redifferentiate in a proximodistal direction. Immediately after amputation) hsc70 synthesis and accumulation was depressed below steady-state levels measured in the unamputated limb~ The results are discussed in light of a possible role for HSPs and amputatio~ induced proteins in the epimorphic regeneration of the amphibian limb.

Heat stress response for physiological traits in dairy and dual purpose cattle populations on phenotypic and genetic scales

The main objective of this thesis was to determine the potential impact of heat stress (HS) on physiological traits of lactating cows and semen quality of bulls kept in a temperate climate. The thesis is comprised of three studies. An innovative statistical modeling aspect common to all three studies was the application of random regression methodology (RRM) to study the phenotypic and genetic trajectory of traits in dependency of a continuous temperature humidity index (THI). In the first study, semen quality and quantity traits of 562 Holstein sires kept on an AI station in northwestern Germany were analyzed in the course of THI calculated from data obtained from the nearest weather station. Heat stress was identified based on a decline in semen quality and quantity parameters. The identified general HS threshold (THI = 60) and the thermoneutal zone (THI in the range from 50 to 60) for semen production were lower than detected in studies conducted in tropical and subtropical climates. Even though adult bulls were characterized by higher semen productivity compared to younger bulls, they responded with a stronger semen production loss during harsh environments. Heritabilities (low to moderate range) and additive genetic variances of semen characteristics varied with different levels of THI. Also, based on genetic correlations genotype, by environment interactions were detected. Taken together, these findings suggest the application of specific selection strategies for specific climate conditions. In the second study, the effect of the continuous environmental descriptor THI as measured inside the barns on rectal temperatures (RT), skin temperatures (ST), vaginal temperatures (VT), respiration rates (RR), and pulse rate (PR) of lactating Holstein Friesian (HF) and dual-purpose German black pied cattle (DSN) was analyzed. Increasing HS from THI 65 (threshold) to THI 86 (maximal THI) resulted in an increase of RT by 0.6 °C (DSN) and 1 °C (HF), ST by 3.5 °C (HF) and 8 °C (DSN), VT by 0.3 °C (DSN), and RR by 47 breaths / minute (DSN), and decreased PR by 7 beats / minute (DSN). The undesired effects of rising THI on physiological traits were most pronounced for cows with high levels of milk yield and milk constituents, cows in early days in milk and later parities, and during summer seasons in the year 2014. In the third study of this dissertation, the genetic components of the cow’s physiological responses to HS were investigated. Heat stress was deduced from indoor THI measurements, and physiological traits were recorded on native DSN cows and their genetically upgraded crosses with Holstein Friesian sires in two experimental herds from pasture-based production systems reflecting a harsh environment of the northern part of Germany. Although heritabilities were in a low range (from 0.018 to 0.072), alterations of heritabilities, repeatabilities, and genetic components in the course of THI justify the implementation of genetic evaluations including heat stress components. However, low repeatabilities indicate the necessity of using repeated records for measuring physiological traits in German cattle. Moderate EBV correlations between different trait combinations indicate the potential of selection for one trait to simultaneously improve the other physiological attributes. In conclusion, bulls of AI centers and lactating cows suffer from HS during more extreme weather conditions also in the temperate climate of Northern Germany. Monitoring physiological traits during warm and humid conditions could provide precious information for detection of appropriate times for implementation of cooling systems and changes in feeding and management strategies. Subsequently, the inclusion of these physiological traits with THI specific breeding values into overall breeding goals could contribute to improving cattle adaptability by selecting the optimal animal for extreme hot and humid conditions. Furthermore, the recording of meteorological data in close distance to the cow and visualizing the surface body temperature by infrared thermography techniques might be helpful for recognizing heat tolerance and adaptability in cattle.

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alpha A-, alpha beta- and R120G alpha B-Crystallins

alpha B-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alpha B-crystallin in detail, and also that of alpha A-crystallin and the disease-related mutant R120G (alpha B-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alpha B-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. H-1 NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alpha B-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils. (C) 2007 Elsevier Ltd. All rights reserved.

Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.