Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii

Heat shock proteins are ubiquitous and highly conserved. Recently they have become implicated in the import of proteins into organelles. All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast. In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome. Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P. lutherii chloroplast chromosome is 115 kb. We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803. The greatest identity is found with the cyanobacterium Synechocystis PCC6803.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum

It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.     

Inhibition of hsp70–1 and hsp70–3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic

Mouse 70-kDa heat shock proteins Hsp70–1 and Hsp70–3 (Hsp70–1/3) are stress-inducible protein chaperones thought to protect embryonic cells and tissues from the effects of a wide range of environmental exposures. Hsp70–1/3 are expressed constitutively, and at times are stress-inducible during various stages of preimplantation embryogenesis. In order to elucidate the functions of constitutive and stress-inducible Hsp70 expression in mouse preimplantation embryos, the consequences of inhibiting expression with antisense oligonucleotides complementary to the mRNAs of hsp70–1 and hsp70–3 (AO70–1/3) were evaluated. Transfection of preimplantation embryos (four-cell stage) with 2.5 μM AO70–1/3 had no effect on in vitro blastocoel formation. However, transfection with 5 or 10 μM AO70–1/3 reduced in vitro blastocyst development to 30% and 0%, respectively (approximately 90% control embryos developed to blastocyst). Thus constitutive expression of Hsp70–1/3 appears significant to preimplantation embryogenesis. Limiting expression of Hsp70–1/3 with 5 μM AO70–1/3 also heightened embryo sensitivity to arsenic, resulting in less than 5% in vitro development to blastocyst in the presence of the subtoxic dose of 0.4 μM sodium arsenite. Whether the combined effect of AO70–1/3 and arsenic is due to blocking inducible expression of the Hsp70s, or due to further reducing the amount of constitutively expressed Hsp70s available to the embryo is not known at this time. However, these results clearly indicate that some minimal amount of Hsp70–1 and/or Hsp70–3 is required for preimplantation embryogenesis, and that increasing the demand for Hsp70s by arsenic exposure heightens this requirement. Mol. Reprod. Dev. 51:373–380, 1998. © 1998 Wiley-Liss, Inc.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Antisense oligonucleotide to the 70-kDa heat shock cognate protein inhibits synthesis of myelin basic protein

Transfection of rat oligodendrocytes with an oligonucleotide sequence complementary to the mRNA encoding the initial ten amino acids of the rat 70-kDa heat shock cognate protein (HSC70) resulted in a rapid (within 24 h) and significant reduction in HSC70 synthesis (69% of control cells transfected with sense oligonucleotide). A further decrease to approximately 44% of controls was detected after 2 days. At that time, HSC70 protein content fell to approximately 49% of controls, and a significant reduction in the synthesis of myelin basic protein (MBP) was first detected (66% of controls). After 5 days, HSC70 synthesis returned to control levels. As HSC70 protein content recovered, so did the synthesis of MBP. Throughout the 5-day experimental period, only minor changes were detected in cell morphology, overall pattern of protein synthesis and the synthesis and content of proteolipid protein (PLP) and the pi isoenzyme of glutathione-S-transferase (pi). These data show that when HSC70 protein content is sufficiently reduced by antisense oligonucleotide, synthesis of MBP (but not PLP or pi) is correspondingly down-regulated, and provide evidence consistent with the role of HSC70 as a chaperone for MBP. Special issue dedicated to Dr. Marion E. Smith.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos

Although sensitive to various disrupters, pre-implantation embryos possess some cellular cytoprotective mechanisms that allow continued survival in the face of a deleterious environment. For stresses such as heat shock, embryonic resistance increases as development proceeds. Present objectives were to determine whether (1) arsenic compromises development of pre-implantation bovine embryos, (2) developmental changes in embryonic resistance to arsenic mimic those seen for resistance to heat shock, and (3) developmental patterns in induction of apoptosis by arsenic are correlated with similar changes in resistance of embryos to inhibitory effects of arsenic on development. Bovine embryos produced by in vitro fertilization were exposed at the two-cell stage or at day 5 after insemination (embryos > or = 16-cells in number) to either sodium arsenite (0, 1, 5, or 10 microM) or heat shock (exposure to 41 degrees C for 0, 3, 4.5, 6, or 9 hr). Arsenic induced apoptosis and increased group 2 caspase activity for embryos at the > or = 16-cell stage, but not for embryos at the two-cell stage. In contrast to these developmental changes in apoptosis responses, exposure to arsenic reduced cell number 24 hr after exposure for both two-cell embryos and embryos > or = 16-cells. Similarly, the percentage of embryos that developed to the blastocyst stage at day 8 after fertilization was reduced by arsenic exposure at both stages of development. Heat shock, conversely, reduced development to the blastocyst stage when applied at the two-cell stage, but not when applied to embryos > or = 16-cells at day 5 after insemination. In conclusion, arsenic can compromise development of bovine pre-implantation embryos, the temporal window of sensitivity of embryos to arsenic is wider than for heat shock, and cellular cytoprotective responses that embryos acquire for thermal resistance are not sufficient to cause increased embryonic resistance to arsenic exposure. It is likely that despite common cellular pathologies caused by arsenic and heat shock, arsenic acts to reduce development in part through biochemical pathways not activated by heat shock. Moreover, the embryo does not acquire significant resistance to these perturbations within the time frame in development examined.     

The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions

The human heat shock protein 70 (Hsp70) family contains at least eight homologous chaperone proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.     

Effect of heat shock,pretreatment and hsp70 copy number on wing development in Drosophila melanogaster

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.     

Heat shock response and mammal adaptation to high elevation (hypoxia)

The mammal’s high elevation (hypoxia) adaptation was studied by using the immunological and the molecular biological methods to understand the significance of Hsp (hypoxia) adaptation in the organic high elevation, through the mammal heat shock response. (1) From high elevation to low elevation (natural hypoxia): Westem blot and conventional RT-PCR and real-time fluorescence quota PCR were adopted. Expression difference of heat shock protein of 70 (Hsp70) and natural expression of brain tissue of Hsp70 gene was determined in the cardiac muscle tissue among the different elevation mammals (yak). (2)From low elevation to high elevation (hypoxia induction): The mammals (domestic rabbits) from the low elevation were sent directly to the areas with different high elevations like 2300, 3300 and 5000 m above sea level to be raised for a period of 3 weeks before being slaughtered and the genetic inductive expression of the brain tissue of Hsp70 was determined with RT-PCR. The result indicated that all of the mammals at different elevations possessed their heat shock response gene. Hsp70 of the high elevation mammal rose abruptly under stress and might be induced to come into being by high elevation (hypoxia). The speedy synthesis of Hsp70 in the process of heat shock response is suitable to maintain the cells’ normal physiological functions under stress. The Hsp70 has its threshold value. The altitude of 5000 m above sea level is the best condition for the heat shock response, and it starts to reduce when the altitude is over 6000 m above sea level. The Hsp70 production quantity and the cell hypoxia bearing capacity have their direct ratio.     

Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre-treatment against 3,4-methylenedioxymethamphetamine toxicity in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.     

Heat shock response and mammal adaptation to high elevation (hypoxia)

The mammal's high elevation(hypoxia) adaptation was studied by using the immu-nological and the molecular biological methods to understand the significance of Hsp(hypoxia) ad-aptation in the organic high elevation,through the mammal heat shock response.(1) From high ele-vation to low elevation(natural hypoxia) :Western blot and conventional RT-PCR and real-time fluo-rescence quota PCR were adopted.Expression difference of heat shock protein of 70(Hsp70) and natural expression of brain tissue of Hsp70 gene was determined in the cardiac muscle tissue among the different elevation mammals(yak) .(2) From low elevation to high elevation(hypoxia induction) :The mammals(domestic rabbits) from the low elevation were sent directly to the areas with different high elevations like 2300,3300 and 5000 m above sea level to be raised for a period of 3 weeks be-fore being slaughtered and the genetic inductive expression of the brain tissue of Hsp70 was deter-mined with RT-PCR.The result indicated that all of the mammals at different elevations possessed their heat shock response gene.Hsp70 of the high elevation mammal rose abruptly under stress and might be induced to come into being by high elevation(hypoxia) .The speedy synthesis of Hsp70 in the process of heat shock response is suitable to maintain the cells' normal physiological functions under stress.The Hsp70 has its threshold value.The altitude of 5000 m above sea level is the best condition for the heat shock response,and it starts to reduce when the altitude is over 6000 m above sea level.The Hsp70 production quantity and the cell hypoxia bearing capacity have their direct ratio.