kDa Heat Shock Research Articles

Effect of lentiviral vector-mediated KSR1 gene silencing on the proliferation of renal tubular epithelial cells and expression of inflammatory factors in a rat model of ischemia/reperfusion injury

Renal ischemia/reperfusion (I/R) is a common cause of acute renal failure in many clinical settings. Our study aimed to elucidate the role of lentiviral vector-mediated KSR1 gene silencing in inflammatory factor expression and proliferation of renal tubular epithelial cells (RTECs) in a rat model of I/R injury. Male Sprague-Dawley (SD) rats were used for I/R model establishment and subject to different treatments, followed by the measurement of neurological severity score (NSS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, 47-kDa heat-shock protein (HSP47), KSR1, and factors related to the Ras/MAPK pathway, as well as cell apoptosis. As compared with the blank group, the neurologic impairment induced by I/R in the siKSR1, U0126, and siKSR1 + U0126 groups was alleviated. Compared with the control group, the other five groups showed increased levels of TNF-α, IL-6, IL-1β, HSP47, N-ras, Raf-1, c-fos, TNF-α, IL-6, p38 MAPK, and cell apoptosis, accompanied by a declined mRNA and protein level of Bcl-2. As compared with the blank and NC groups, the siKSR1, U0126, and siKSR1 + U0126 groups showed decreased levels of TNF-α, IL-6, IL-1β, HSP47, N-ras, Raf-1, c-fos, TNF-α, IL-6, p38 MAPK, cleaved caspase-3, cleaved caspase-9, p53, and cell apoptosis, accompanied by an increased mRNA and protein level of Bcl-2. Our findings demonstrated that KSR1 gene silencing might inhibit the expression of inflammatory factors in RTECs and promote their proliferation by inactivating the Ras/MAPK pathway in the rat model of I/R injury.

Subacute exposure to residual oil fly ash (ROFA) increases eHSP70 content and extracellular-to-intracellular HSP70 ratio: a relation with oxidative stress markers.

The purpose of this study was to evaluate whether exposure to particles induces an imbalance in 70-kDa heat shock proteins (HSP70). Since intracellularly (iHSP70) it has anti-inflammatory roles whereas extracellularly (eHSP70) it has pro-inflammatory roles, we evaluate the effect of residual oil fly ash (ROFA) exposure on eHSP70-to-iHSP70 ratio (H index), a biomarker of inflammatory status that is related to oxidative stress in plasma and lymphoid tissue. Wistar rats that received ROFA suspension for three consecutive days (750μg) showed an increase in plasma eHSP70 levels (mainly the 72-kDa inducible form). Also, ROFA promoted alterations on plasma oxidative stress (increased protein carbonyl groups and superoxide dismutase activity, and decrease sulfhydryl groups). There was an increase in H index of the plasma/thymus with no changes in circulating leukocyte level, iHSP70, or oxidative stress markers in lymphoid tissues. Our results support the hypothesis that eHSP70 content and H index represent inflammatory and oxidative biomarkers.

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.

A Rationally Designed Hsp70 Variant Rescues the Aggregation-Associated Toxicity of Human IAPP in Cultured Pancreatic Islet β-Cells.

Molecular chaperones are key components of the protein homeostasis system against protein misfolding and aggregation. It has been recently shown that these molecules can be rationally modified to have an enhanced activity against specific amyloidogenic substrates. The resulting molecular chaperone variants can be effective inhibitors of protein aggregation in vitro, thus suggesting that they may provide novel opportunities in biomedical and biotechnological applications. Before such opportunities can be exploited, however, their effects on cell viability should be better characterised. Here, we employ a rational design method to specifically enhance the activity of the 70-kDa heat shock protein (Hsp70) against the aggregation of the human islet amyloid polypeptide (hIAPP, also known as amylin). We then show that the Hsp70 variant that we designed (grafted heat shock protein 70 kDa-human islet amyloid polypeptide, GHsp70-hIAPP) is significantly more effective than the wild type in recovering the viability of cultured pancreatic islet β-cells RIN-m5F upon hIAPP aggregation. These results indicate that a full recovery of the toxic effects of hIAPP aggregates on cultured pancreatic cells can be achieved by increasing the specificity and activity of Hsp70 towards hIAPP, thus providing evidence that the strategy presented here provides a possible route for rationally tailoring molecular chaperones for enhancing their effects in a target-dependent manner.

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.

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.

Genome-Wide Characterization of Heat-Shock Protein 70s from Chenopodium quinoa and Expression Analyses of Cqhsp70s in Response to Drought Stress.

Heat-shock proteins (HSPs) are ubiquitous proteins with important roles in response to biotic and abiotic stress. The 70-kDa heat-shock genes (Hsp70s) encode a group of conserved chaperone proteins that play central roles in cellular networks of molecular chaperones and folding catalysts across all the studied organisms including bacteria, plants and animals. Several Hsp70s involved in drought tolerance have been well characterized in various plants, whereas no research on Chenopodium quinoa HSPs has been completed. Here, we analyzed the genome of C. quinoa and identified sixteen Hsp70 members in quinoa genome. Phylogenetic analysis revealed the independent origination of those Hsp70 members, with eight paralogous pairs comprising the Hsp70 family in quinoa. While the gene structure and motif analysis showed high conservation of those paralogous pairs, the synteny analysis of those paralogous pairs provided evidence for expansion coming from the polyploidy event. With several subcellular localization signals detected in CqHSP70 protein paralogous pairs, some of the paralogous proteins lost the localization information, indicating the diversity of both subcellular localizations and potential functionalities of those HSP70s. Further gene expression analyses revealed by quantitative polymerase chain reaction (qPCR) analysis illustrated the significant variations of Cqhsp70s in response to drought stress. In conclusion, the sixteen Cqhsp70s undergo lineage-specific expansions and might play important and varied roles in response to drought stress.

The 70-kDa Heat-shock Protein as a Potential Biomarker of Quality of the Parapenaeus longirostris Shrimp Flesh

ABSTRACTThe deep-water rose shrimp, Parapenaeus longirostris, is a target species of the Mediterranean fisheries mostly caught by trawlers offshore, processed, and frozen on board. The effects of thawing on shrimp muscle exudate collected at 0, 1, 2, 3 days after thawing were investigated. In total, 70-kDa heat-shock protein (Hsp70), alpha (α)-enolase, and manganese superoxide dismutase (Mn-SOD) were selected as metabolic and stress-related proteins and analyzed by immunoblotting on exudates. Data were compared for the amount of exudates collected and the pH values. Among the investigated proteins, only the Hsp70 levels showed a decrease related to the post-thawing period and correlated with both the significant increase of the exudate amount and the pH values. These data strongly suggest the potential use of Hsp70 as an early predictive biomarker for quality of the P. longirostris shrimp after thawing.

DNAJC12 deficiency: A new strategy in the diagnosis of hyperphenylalaninemias.

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4). Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH4 by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in CSF and responded to supplementation with BH4, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism. Late diagnosis result in permanent neurological disability, while early diagnosed and treated patients develop normally. Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH4 are genetically excluded.

Non-protease native allergens partially purified from bodies of eight domestic mites using p-aminobenzamidine ligand

BackgroundOptimised purification steps for concentrating trace target native antigens are needed. Combining the p-aminobenzamidine ligand with protease inactivation enables partial purification of mite non-protease allergens lacking proteases. ObjectiveWe sought to analyse in detail proteins obtained using this method from eight species of synanthropic acaridid mites and tested IgE reactivity using pooled human sera. Materials and methodsProteins affinity bound to p-aminobenzamidine as a ligand were identified by MALDI TOF/TOF. After electroblotting, the proteins were visualised using the fluorescent SYPRO-Ruby protein blot stain, and IgE reactivity was further analysed using pooled human sera collected from patients allergic to house dust mites. ResultsMS/MS identification confirmed previous results that no proteases were purified. Protein patterns corresponding to the allergens Der f 7, Der f 30 and actins indicated that these proteins are purified using p-aminobenzamidine and are present across a wide spectrum of acaridid mites. When using Dermatophagoides farinae, apolipophorins (Der f 14), chitinase-like Der f 15 and 18, 70-kDa heat shock protein, and a Der f Alt a10 allergen homolog (gi|37958173) were also detected. The target antigens tropomyosins and paramyosins showed similar IgE binding among the mite species tested. IgE reactivity with miscellaneous D. farinae antigen was also observed. ConclusionsPartial purification of mite non-protease antigens using a strategy combining p-aminobenzamidine with protease inactivation was verified by 1D-E and 2D-E analyses. IgE binding to p-aminobenzamidine-purified native non-protease mite antigens was tested using pooled sera. This preliminary study allows for further work on individual serum samples, allowing confirmation of immunoreactivity.

Role of the brain in adaptation of the common and golden carps to adverse factors of chemical origin

Using an indirect solid-phase immunoassay (ELISA test), cytochrome P-450, serotoninmodulated anticonsolidation protein (SMAP) and 70-kDa heat shock protein (HSP70) levels were analyzed in the brain and liver of the common carp Cyprinus carpio L. and golden carp Carassius auratus caught in reservoirs of the Arpachay river (clean zone) and Araks river (polluted zone) in Nakhchivan Autonomous Republic of Azerbaijan. Upregulation of cytochrome P-450 (p < 0.05) and SMAP (p < 0.01) in the common carp brain and downregulation of cytochrome P-450 (p < 0.05) and HSP70 (p < 0.05) in the golden carp liver were detected in fish caught in the Araks river reservoir as compared to levels of the same substances in fish from the Arpachay river reservoir. It is concluded that the brain plays a regulatory role in adaptation of animals to adverse environmental conditions and in protection of tissues from negative effects of pollutants.

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.

Proteomic identification of protein markers of stages of heart formation in humans

On the basis of the results of proteomic analysis and mass spectrometric identification of human myocardium proteins exhibiting pronounced quantitative changes in the dynamics of prenatal cardiogenesis, changes in the expression level of proteins of three families (mitochondrial, contractile, and heat shock) have been identified. The complex of human myocardium mitochondrial proteins (for example, α and β isoforms of ATP synthase, aconitase 2, creatine phosphokinase M-subunit, and 60-kDa heat shock protein) largely finishes its development according to the adult type by developmental week 24. The formation of the protein composition of human myocardium contractile structures (for example, desmin, myosin regulatory light chain 2, fetal ventricular essential isoform 1, canonical α-tropomyosin, and fetal isoform 6) reflects the initial stage of myofibril development until developmental week 8 (replacement of fetal isoforms of contractile proteins with adult ones with the involvement of the phosphorylated isoform of 27-kDa heat shock protein), the stage of their qualitative and quantitative structuring by developmental weeks 20–24, and the final formation of the adult phenotype of contractile structures by 2 years of life.

The Role of BiP Retrieval by the KDEL Receptor in the Early Secretory Pathway and its Effect on Protein Quality Control and Neurodegeneration.

Protein quality control in the early secretory pathway is a ubiquitous eukaryotic mechanism for adaptation to endoplasmic reticulum (ER) stress. An ER molecular chaperone, immunoglobulin heavy chain-binding protein (BiP), is one of the essential components in this process. BiP interacts with nascent proteins to facilitate their folding. BiP also plays an important role in preventing aggregation of misfolded proteins and regulating the ER stress response when cells suffer various injuries. BiP is a member of the 70-kDa heat shock protein (HSP70) family of molecular chaperones that resides in the ER. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity, leading to protein folding and maturation. BiP also possesses a retrieval motif in its carboxyl terminal. When BiP is secreted from the ER, the Lys-Asp-Glu-Leu (KDEL) receptor in the post-ER compartments binds with the carboxyl terminal KDEL sequence of BiP and returns BiP to the ER via coat protein complex I (COPI) vesicular transport. Although yeast studies showed that BiP retrieval by the KDEL receptor is not essential in single cells, it is crucial for multicellular organisms, where some essential proteins require retrieval to facilitate folding and maturation. Experiments in knock-in mice expressing mutant BiP with the retrieval motif deleted revealed a unique role of BiP retrieval by the KDEL receptor in neuronal development and age-related neurodegeneration.

A mild cold stress that increases resistance to heat lowers FOXO translocation in Drosophila melanogaster.

Previous studies have shown that subjecting Drosophila melanogaster flies to a mild stress at young or middle age can increase lifespan and resistance to severe stresses throughout life and that the NF-κB-like transcription factor DIF, the 70kDa heat-shock proteins, and the Drosophila Forkhead box class O (dFOXO) transcription factor could explain some of these effects. The present study showed that two dFOXO mutants do not survive longer heat if previously subjected to a mild cold stress, contrarily to wild-type flies. This cold pretreatment had nearly no effect on dFOXO nuclear translocation in wild-type males. Heat stress strongly increased dFOXO translocation, but this effect was lowered in cold-pretreated males. Because cold-pretreated wild-type males survived longer heat and had nevertheless a lower dFOXO translocation after this heat stress, one can conclude that dFOXO is required to resist heat but that the cold pretreatment makes that other mechanisms partly substitute to dFOXO translocation.

Abstract 2121: Deguelin-derivative, L80 suppresses tumor growth and metastasis via inhibition of STAT3 activation in triple-negative breast cancer

Abstract Triple-negative breast cancer TNBC is associated with aggressive metastasis and poor clinical outcomes due to a lack of effective treatment options. Therefore, new drugs that effectively target both cancer cell proliferation and metastasis are needed to improve clinical outcomes. The 90-kDa molecular chaperone heat-shock protein, HSP90 has been implicated in cancer progression and metastasis by modulating the stabilization and maturation of many oncogenic proteins. Our objective was to investigate the mechanism of action of C-terminal HSP90 inhibitor, deguelin-derivative L80 on TNBC proliferation and metastasis in vitro and in vivo. L80 induced apoptosis and suppression of cell viability in TNBC cell lines via inhibition of Akt activation with concomitant nuclear accumulation of p27. L80 treatment also caused a marked suppression of cell migration and invasion. These responses were associated with inhibition of STAT3 phosphorylation (Tyr705) as evidenced by downregulation of STAT3 downstream target genes including cyclin D1 and survivin. The syngeneic orthotopic mouse model with 4T1 cells, L80 administration resulted in significant reduction in tumor growth together with decreased number of Ki-67-positive cells and downregulation of phospho-STAT3. Double-label immunofluorescence analysis showed that individuals receiving L80 exhibited a marked reduction in co-localization and expression of HIF-1α and HSP90 compared to their control groups in vivo. Finally, we observed that L80 administration significantly suppressed lung metastasis, as determined by in vivo bioluminescent imaging system. Our findings suggest that L80 may be potentially effective for the treatment for metastatic TNBC patients. Note: This abstract was not presented at the meeting. Citation Format: Tae-Min Cho, Eunhye Oh, Daeil Sung, Yoon-Jae Kim, Ji Young Kim, Jeewoo Lee, Jae Hong Seo. Deguelin-derivative, L80 suppresses tumor growth and metastasis via inhibition of STAT3 activation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2121. doi:10.1158/1538-7445.AM2017-2121

BAG-1L Protects SH-SY5Y Neuroblastoma Cells Against Hypoxia/Re-oxygenation Through Up-Regulating HSP70 and Activating PI3K/AKT Signaling Pathway.

BCL-2-associated athanogene-1(BAG-1) is a multifunctional and anti-apoptotic protein that was first identified as a binding partner of BCL-2. But the effects and mechanisms for BAG-1 against hypoxic damage is unclear up to now. Whether BAG-1 could protect the human brain against hypoxic damage through up-regulating 70kDa heat shock proteins (HSP70) and PI3K/AKT pathway activation? In present study, we examined the changes of HSP70 and AKT and p-AKT protein level in SH-SY5Y cells with BAG-1L gene over-expression subjected to hypoxia/re-oxygenation injury. BAG-1L over-expression increased neuronal viability, and it reduced apoptosis of neurons after hypoxia/re-oxygenation for 8h. BAG-1L over-expression enhanced the HSP70 protein levels and increased p-AKT/total AKT ratio after hypoxia/re-oxygenation for 8h. These results suggest that BAG-1L over-expression protects against hypoxia/re-oxygenation injury, at least in part, by interacting with HSP70, and by accelerating the activation of PI3K/AKT pathways.

Production performance, immunity, and heat stress resistance in Jersey cattle fed a concentrate fermented with probiotics in the presence of a Chinese herbal combination

The goals of this study were to investigate the production performance, immunity, and heat stress resistance of Jersey cattle fed a concentrate fermented with compound probiotics in the presence of a Chinese herbal combination (CHC). First, the appropriate concentrations for the six Chinese herbs required to stimulate probiotic proliferation were investigated in vitro. Next, the optimal time and moisture content were determined for fermentation. We then tested the feeding effect of our fermented product on 36 growing cattle and 36 milking cows. The animals were randomly assigned to receive the product or not, resulting in half of each group receiving the treatment or serving as the control group. For 60days, the experimental group was fed with the fermented product and the control group was fed with a basic concentrate that did not contain the probiotics or the Chinese herbs. The results showed that the cattle that received the fermented concentrate showed improved weight gain and milking cows that received the fermented concentrate showed increased average milk yield, milk fat content, and solid-not-fat content (P<0.05). In addition, the serum levels of growth hormone, the 70kDa heat shock protein (HPS70), and immunoglobulin G (IgG) of the cattle in the experimental group were significantly higher than the levels of cattle in the control group. A similar increase in the serum levels of HPS70, IgG, immunoglobulin A, and interferon γ was observed in milking cows that received the treatment (P<0.05). No significant differences (P>0.05) were observed in the serum levels of interleukin-2. Moreover, milking cows that received the fermented concentrate showed increased apparent digestibility of crude protein, ether extract, and crude fiber (P<0.05). For the growing cattle, only crude protein digestibility was significantly increased in the experimental group (P<0.05). Finally, we found that the ammonia content in the barn was decreased after the animals received the fermented concentrate. These results demonstrated that our fermented concentrate improved production performance, immunity, and heat stress resistance in Jersey cattle. We also demonstrated that this CHC and a mixture of probiotic strains acted as an ideal leavening agent for cattle’s concentrate.

Functional characterization of natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible 70kDa heat shock gene in humans

The cellular stress response system is responsible for maintaining cellular homeostasis during periods of stress caused by environmental insults including pathogens and disease. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. Therefore, we hypothesized that natural occurring variations in the sequence of Hsp70s will affect cellular and subsequently species adaptation. To investigate the relationship between adaptation, disease susceptibility, and Hsp70s' variation we tested whether natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter its function. Specifically, the wild‐type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site‐directed mutagenesis. We then determined whether any of these mutations affected the intracellular localization of HSPA1A. These experiments were performed by using GFP‐tagged HSPA1A and fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, and visualized using confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Furthermore, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly‐glutamine carrying huntingtin proteins. This assay determined that one of the mutations caused increased cell death as compared to the WT, suggesting that it may alter HSPA1A's ability to prevent protein aggregation. We also determined whether these mutations affect the ability of HSPA1A to refold heat‐denatured luciferase. These assays revealed that only one mutation resulted in significantly lower levels of refolded enzyme, suggesting a putative loss‐of‐chaperone function. Lastly, live‐dead assays revealed that human cell lines carrying two of these mutations had significantly less resistance to heat and ethanol stress than cells expressing the WT protein. Given that these natural variants are found at a very low frequency in humans we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress.Support or Funding InformationThis project was supported by funds from NIH and CSUF to NN

Physiological plasticity related to zonation affects hsp70 expression in the reef-building coral Pocillopora verrucosa.

This study investigates for the first time the transcriptional regulation of a stress-inducible 70-kDa heat shock protein (hsp70) in the scleractinian coral Pocillopora verrucosa sampled at three locations and two depths (3 m and 12 m) in Bangka Island waters (North Sulawesi, Indonesia). Percentage of coral cover indicated reduced habitat suitability with depth and at the Tanjung Husi (TA) site, which also displayed relatively higher seawater temperatures. Expression of the P. verrucosa hsp70 transcript evaluated under field conditions followed a depth-related profile, with relatively higher expression levels in 3-m collected nubbins compared to the 12-m ones. Expression levels of metabolism-related transcripts ATP synthase and NADH dehydrogenase indicated metabolic activation of nubbins to cope with habitat conditions of the TA site at 3 m. After a 14-day acclimatization to common and fixed temperature conditions in the laboratory, corals were subjected for 7 days to an altered thermal regime, where temperature was elevated at 31°C during the light phase and returned to 28°C during the dark phase. Nubbins collected at 12 m were relatively more sensitive to thermal stress, as they significantly over-expressed the selected transcripts. Corals collected at 3 m appeared more resilient, as they showed unaffected mRNA expressions. The results indicated that local habitat conditions may influence transcription of stress-related genes in P. verrucosa. Corals exhibiting higher basal hsp70 levels may display enhanced tolerance towards environmental stressors.