15 HEAT SHOCK COGNATE 70 PROTEIN IMPROVES THE LONG-TERM SURVIVAL OF RAM SPERMATOZOA DURING STORAGE AT 17°C IN A COMMERCIAL EXTENDER

Heat shock proteins (HSPs) are highly represented among oviductal epithelial cell (OEC) plasma membrane proteins (Sostaric et al. 2006 J. Proteome Res. 5, 3029–3027); their abundance increases in oviductal fluid in response to the presence of spermatozoa (Georgiou et al. 2005 Mol. Cell. Proteomics 4, 1785–1796), and they are capable of binding directly to spermatozoa (Boilard et al. 2004 Biol. Reprod. 71, 1879–1889). As a result of these observations, a role for HSPs in prolonging the functional life of spermatozoa in the oviduct prior to fertilization has been proposed. Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical AI (Salamon and Maxwell 2000 Anim. Reprod. Sci. 62, 77–111). This is due, in part, to extenders being unable to maintain the functional life of ram spermatozoa to the same extent as the oviduct. Therefore, in this study, we aimed to determine the effect of supplementing extenders with the HSPs, HSC70 and HSP70, on the maintenance of ram sperm function during storage. Eight freshly collected ram semen ejaculates were split and diluted in the extenders, INRA96 and RSD-1, to 25 � 106 spermatozoa mL–1, either alone (control) or supplemented with HSC70, HSP70, or α-tubulin (protein with a molecular weight similar to that of the two HSPs). The final concentration of protein in each case was 4 µg mL–1, and the samples were stored at 17�C for 48 h. At 1 h, 6 h, 24 h, and 48 h, the viability and DNA integrity of the stored spermatozoa was determined using the live and dead stains, SYBR�-14 and ethidium homodimer, and the Sperm-Ovis-Halomax kit, respectively. In each case, the mean percentage (%) of viable and DNA-fragmented (DF) spermatozoa was determined and log-transformed prior to analysis using factorial ANOVA. Statistical significance was defined as P < 0.05. In INRA96, but not RSD-1, the beneficial effect of protein supplementation on the % of viable spermatozoa observed during storage was significant (P < 0.001). Most noteworthy was INRA96 supplemented with HSC70, which maintained the % of viable spermatozoa observed during storage significantly (P < 0.001) better than INRA96 alone. Protein supplementation had no significant effect on the % of DF spermatozoa observed during storage. However, the % of DF spermatozoa observed increased significantly (P < 0.001) during storage, irrespective of the presence or absence of protein. On the basis of these findings, it is not implausible to suggest that supplementing the commercially available INRA96 extender with HSC70 would improve the fertility rates observed following cervical AI using stored ram semen. Furthermore, the increase in the incidence of DNA-fragmented spermatozoa observed during storage offers a potential explanation as to why poor fertility rates are often observed when ram semen stored in conventional extenders is used for cervical AI.

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The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender

Reproduction Fertility and Development ◽

10.1071/rd11173 ◽

2012 ◽

Vol 24 (4) ◽

pp. 543 ◽

Cited By ~ 19

Author(s):

R. E. Lloyd ◽

A. Fazeli ◽

P. F. Watson ◽

W. V. Holt

Keyword(s):

Heat Shock ◽

Nuclear Dna ◽

Sperm Concentration ◽

Dna Integrity ◽

Sperm Viability ◽

Fertility Rates ◽

Term Survival ◽

Nuclear Dna Fragmentation ◽

Ram Sperm

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL–1) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48 h and this experiment was repeated using 25 × 106 and 800 × 106 spermatozoa mL–1. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

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HSPA2 overexpression protects V79 fibroblasts against bortezomib-induced apoptosis

Biochemistry and Cell Biology ◽

10.1139/o11-083 ◽

2012 ◽

Vol 90 (2) ◽

pp. 224-231 ◽

Cited By ~ 10

Author(s):

Piotr Teodor Filipczak ◽

Wojciech Piglowski ◽

Magdalena Glowala-Kosinska ◽

Zdzislaw Krawczyk ◽

Dorota Scieglinska

Keyword(s):

Heat Shock ◽

Cell Line ◽

Somatic Cells ◽

Proteasome Inhibitors ◽

Chinese Hamster ◽

Culture Conditions ◽

Term Survival ◽

Hsp70 Family ◽

Induced Apoptosis ◽

Shock Proteins

Human HSPA2 is a member of the HSPA (HSP70) family of heat-shock proteins, encoded by the gene originally described as testis-specific. Recently, it has been reported that HSPA2 can be also expressed in human somatic tissues in a cell-type specific manner. The aim of the present study was to find out whether HSPA2 can increase the resistance of somatic cells to the toxic effect of heat shock, proteasome inhibitors, and several anticancer cytostatics. We used a Chinese hamster fibroblast V79 cell line because these cells do not express the HSPA2 and cytoprotective HSPA1 proteins under normal culture conditions and show limited ability to express HSPA1 in response to heat shock and proteasome inhibitors. We established, by retroviral gene transfer, a stable V79/HSPA2 cell line, which constitutively overexpressed HSPA2 protein. The major observation of our study was that HSPA2 increased long-term survival of cells subjected to heat shock and proteasome inhibitors. We found, that HSPA2 confers resistance to bortezomib-induced apoptosis. Thus, we showed for the first time that in somatic cells HSPA2 can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress.

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Small Heat Shock Proteins Can Confer Tolerance to Nanomaterial-induced Toxicity

HortScience ◽

10.21273/hortsci.49.8.1116 ◽

2014 ◽

Vol 49 (8) ◽

pp. 1116-1121 ◽

Cited By ~ 6

Author(s):

Hanseul Park ◽

Eunhye Ko ◽

Yeh-Jin Ahn

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Small Heat Shock Protein ◽

Multiwalled Carbon ◽

E Coli ◽

Transgenic Cell ◽

Heat Shock Cognate 70 ◽

Leaf Tissues ◽

Shock Proteins ◽

Transgenic Cell Line

The expression of a small heat shock protein (sHSP) in plants and its possible function in conditions related to nanomaterial exposure were examined. Multiwalled carbon nanotubes (MWCNTs) and silver nanoparticles (AgNPs) induced toxicity that was indicated by the bending and curling of carrot leaf tissues. Both nanomaterials induced the expression of a small heat shock protein in carrot, DcHsp17.7, but reduced the level of a constitutive heat shock cognate 70. To examine the possible function of DcHsp17.7, the coding gene was heterologously expressed in Escherichia coli. Both nanomaterials reduced the viability of E. coli cell lines. However, the transgenic cell line heterologously expressing DcHsp17.7 showed higher levels of cell viability, compared with vector controls, when exposed to MWCNTs and, more notably, to AgNPs. To the best of our knowledge, this is the first study reporting the influence of nanomaterials on the expression of a plant sHSP and its possible function in conferring tolerance to nanomaterial stress.

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Dodecin as carrier protein for immunizations and bioengineering applications

10.1101/2020.03.19.990861 ◽

2020 ◽

Cited By ~ 1

Author(s):

Florian Bourdeaux ◽

Yannick Kopp ◽

Julia Lautenschläger ◽

Ines Gößner ◽

Hüseyin Besir ◽

...

Keyword(s):

Heat Shock ◽

Fluorescent Protein ◽

Spherical Shape ◽

Interacting Protein ◽

C Terminus ◽

Heat Shock Cognate 70 ◽

A Cell ◽

Green Fluorescent ◽

Shock Proteins

AbstractIn bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signaling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).For Table of Contents Only

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The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum.

The Journal of Cell Biology ◽

10.1083/jcb.100.2.642 ◽

1985 ◽

Vol 100 (2) ◽

pp. 642-647 ◽

Cited By ~ 9

Author(s):

J J Carrino ◽

T G Laffler

Keyword(s):

Cell Cycle ◽

Heat Shock ◽

Physarum Polycephalum ◽

Cell Cycle Regulation ◽

Heat Shocks ◽

Tubulin Mrna ◽

Shock Proteins ◽

Tubulin Protein ◽

Cycle Regulation ◽

Second Peak

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.

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