Recent evidence indicates that different forms of stress, including hypoxia, can induce specific proteins called heat-shock or stress proteins in various types of mammalian cells. These studies examined whether myocardial ischemia can result in increased levels of proteins with molecular weight and isoelectric point characteristics similar to those described for heat-shock or stress proteins. The left anterior descending coronary artery of the dog heart was completely occluded; normal and ischemic myocardial samples were obtained 6 hours after occlusion; and total cardiac proteins and RNA were prepared. Ribonucleic acid was translated in vitro in a modified rabbit reticulocyte lysate system, and [35S]-methionine-labelled translational products as well as unlabelled cardiac proteins were separated by two-dimensional gel electrophoresis. Total proteins were visualized by silver staining and in vitro translation products quantified by fluorometry. A translatable mRNA coding for a 71,000 dalton peptide with an isoelectric point of 5.8 was markedly increased in the ischemic myocardium after 6 hours of ischemia. A protein with similar migration characteristics was detected in ischemic myocardium but not in normal myocardium. These results indicate that an mRNA coding for a translational product with similar migration characteristics of heat-shock protein 71 is induced by ischemia in the dog heart.
Hyperthermia, hypoxia, and other conditions induce the appearance of heat shock or stress proteins in cells. We have previously shown that in the ischemic dog myocardium the level of a messenger RNA (mRNA) coding for a protein with migration characteristics similar to heat shock/stress protein 71 increases. Using a human heat-shock protein (hHSP) 70 genomic clone and anti-HSP70 antibodies as probes, we demonstrate in this report that heart stress protein (SP) 71 mRNA and its translational products (71 kDa polypeptides) are members of the stress protein family. In rabbit hearts, the ischemia-induced mRNAs translate into three isoforms with different isoelectric points (6.0, 6.1, and 6.15), in contrast to dog heart mRNA that translates into a protein with a pI of 5.8. The levels of SP71 mRNA in the dog and rabbit ischemic myocardium increased by sixfold and 18-fold, respectively. In the same samples, the levels of creatine kinase M mRNA decreased by about 40%, whereas those of myosin heavy chain mRNA remain unaltered. Our comparative analysis of three different mRNAs indicates that ischemia manifests its effects by differentially changing the levels of specific mRNAs coding for proteins with separate and distinct roles in the cell.
Parafibromin, a tumor suppressor protein encoded by HRPT2/CDC73 and implicated in parathyroid cancer and the hyperparathyroidism-jaw tumor familial cancer syndrome, is part of the PAF1 transcriptional regulatory complex. Parafibromin has been implicated in apoptosis and growth arrest, but the mechanism by which its loss of function promotes neoplasia is poorly understood. We report here that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of HRPT2/CDC73, rescues the loss-of-ventral-eye phenotype of lobe (Akt1s1). Such rescue is consistent with previous reports that hyx/ parafibromin is required for the nuclear transduction of Wingless/Wnt signals and that Wingless signaling antagonizes lobe function. A screen employing double hyx/lobe heterozygotes identified an additional interaction with orb and orb2, homologs of mammalian cytoplasmic polyadenylation element binding protein (CPEB), a translational regulatory protein. Hyx and orb2 heterozygotes lived longer and were more resistant to starvation than controls. In mammalian cells knockdown of parafibromin expression reduced levels of CPEB1. Chromatin immunoprecipitation demonstrated occupancy of CPEB1 by endogenous parafibromin. Bioinformatic analysis revealed a significant overlap between human transcripts potentially regulated by parafibromin and CPEB. These results show that parafibromin may exert both transcriptional and, through CPEB, translational control over a subset of target genes and that loss of parafibromin (and CPEB) function may promote tumorigenesis in part by conferring resistance to nutritional stress.
Our results suggest that the non-genetic sporadic AD (sAD) rat model developed by single-time STZ-ICV infusion exhibits protein aggregation and dementia probably resulting from increased mitochondrial fragmentation and functional aberrations. The present study reinforces the validity of this model for studying pathogenesis and potential therapies of sAD.
Replication-competent forms of herpes simplex virus 1 (HSV-1) defective in the viral neurovirulence factor infected cell protein 34.5 (ICP34.5) are under investigation for use in the therapeutic treatment of cancer. In mouse models, intratumoral injection of ICP34.5-defective oncolytic HSVs (oHSVs) has resulted in the infection and lysis of tumor cells, an associated decrease in tumor size, and increased survival times. The ability of these oHSVs to infect and lyse cells is frequently characterized as exclusive to or selective for tumor cells. However, the extent to which ICP34.5-deficient HSV-1 replicates in and may be neurotoxic to normal brain cell types in vivo is poorly understood. Here we report that HSV-1 defective in ICP34.5 expression is capable of establishing a productive infection in at least one normal mouse brain cell type. We show that ␥34.5 deletion viruses replicate productively in and induce cellular damage in infected ependymal cells. Further evaluation of the effects of oHSVs on normal brain cells in animal models is needed to enhance our understanding of the risks associated with the use of current and future oHSVs in the brains of clinical trial subjects and to provide information that can be used to create improved oHSVs for future use.Several types of replication-competent neuroattenuated herpes simplex viruses (HSVs) are currently being evaluated in clinical cancer trials for safety and therapeutic activity (32), as well as for vaccine development (20). A critical safety concern associated with the clinical use of these oncolytic HSVs (oHSVs) is their ability to enter, replicate in, and spread to a wide range of cell types in different regions of the nervous system. One potential complication resulting from invasion of the central nervous system by HSV is herpes simplex encephalitis (HSE), an infection that causes lifelong neurological damage or death. A limited number of genes have been demonstrated to contribute to the virus's ability to trigger HSE. The viral gene ␥34.5 encodes the neurovirulence protein infected cell protein 34.5 (ICP34.5) (29). Viruses lacking the ␥34.5 gene (e.g., R3616 and 1716) were found to be 5 logs less neurovirulent than wild-type strains of 19,36), as quantified by the intracranial LD 50 , i.e., the lethal dose in 50% of mice inoculated intracerebroventricularly with the virus. The basis for this neuroattenuation was initially reported to be the inability of the ␥34.5 deletion viruses to infect or replicate in brain cells (4). Subsequent immunohistochemical studies on infected brain tissue of intracerebroventricularly inoculated mice suggested that ␥34.5 deletion viruses retained the ability to infect a wide range of brain cell types and to replicate in and, by day 7, destroy ependymal cells (ECs) (16,21).To create a more neuroattenuated and thus safer virus, the virus G207 was constructed from the ␥34.5 deletion virus R3616 by insertional mutagenesis of the U L 39 gene (25). The U L 39 gene encodes the large subunit of the viral ribonucleotide reductase (vR...
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