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.     

A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR‐like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast‐localized and highly expressed in actively photosynthesizing tissue ( Sadanandom A et al., 2000 ). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast‐localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR‐catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time‐scale, was ultimately DTT‐dependent and led to recovery of Hsp21 conformation and chaperone‐like activity, both of which are lost upon methionine sulfoxidation ( Härndahl et al., 2001 ). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.     

Expression of HSP70 and CYP1A protein in ovary and liver of juvenile rainbow trout exposed to beta-naphthoflavone

Cytochrome P4501A (CYP1A) and the 70-kDa stress protein (HSP70) were determined using Western blotting in the ovary and liver of juvenile female rainbow trout (Oncorhynchus mykiss) exposed for 4 days to beta-naphthoflavone (betaNF) following a single intraperitoneal injection. Ovarian CYP1A protein was observed in both control and betaNF-exposed fish, indicating constitutive and inducible expression of CYP1A in immature trout ovaries. CYP1A protein levels determined using densitometry were 14- and 46-fold greater in betaNF-exposed trout compared to controls in the liver and ovary, respectively. Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity, a specific catalytic marker of CYP1A, was also induced 38-fold above controls following betaNF exposure. Hepatic HSP70 protein expression was significantly higher in whole cell homogenates, but not in cytosolic fractions, collected from betaNF-exposed fish in comparison to control fish. There was no difference in ovarian HSP70 levels determined in whole cell homogenates between control and betaNF-exposed fish. The observation that unlike liver, ovarian HSP70 expression remained unchanged following induction of CYP1A protein may be related to the sensitivity of the teleost ovary to environmental toxicants that act as aryl hydrocarbon receptor agonists.     

Cryptic Expression of the 70-kDa Heat Shock Protein,hsp72, in Gerbil Hippocampus after Transient Ischemia

The 70 kDa heat shock protein, hsp72, is known to be induced following transient global ischemia in brain, as detected by immunocytochemistry and in situ hybridization techniques. However, while hsp72 mRNA is expressed rapidly following postischemic recirculation, immunocytochemistry fails to detect hsp72 protein for many hours after such insults, even in cell populations that readily express Fos and other proteins encoded by ischemia-induced mRNAs. In the present study, hsp72 expression in gerbil hippocampus was compared by immunocytochemistry and immunoblot methods at several intervals following 10 min ischemia. As established in previous studies, hsp72 immunoreactivity remained undetectable in postischemic neurons at 6 h following such insults. In contrast, immunoblots of dissected gerbil hippocampus demonstrated nearly maximal accumulation of hsp72 at this time point. These results indicate that the protein is present, but cryptic to detection in perfusion-fixed sections, during early recirculation. The constitutively expressed heat shock cognate protein, hsc70, did not show significant changes in level or distribution by either method, except for a decrease in CA1 staining at 48 h. These results confirm that hsp72 rapidly accumulates to high levels in postischemic hippocampus, and suggest that further studies of its subcellular localization during this interval may offer insight into its functional role as a component of the stress response in neurons after such insults.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.