Search

The p53 tumor suppressor gene, an important regulator of the cell cycle, has been implicated in apoptotic cell death in vitro, and more recently in neuronal degeneration in vivo. The present study investigated the importance of p53 expression in the apoptotic death of hippocampal granule cells following adrenalectomy. Mice, either homozygous or heterozygous for the p53 null allele and wild-type controls were sacrificed 16 days after adrenalectomy. Hippocampal morphology was assessed in paraffin sections stained with hematoxylin and eosin. Cells exhibiting features characteristic of apoptosis were evident in hippocampi from wild-type mice. A significant decrease in the number of apoptotic cells was observed in both homozygous and heterozygous mice. These findings demonstrate that absence or attenuation of p53 expression protects granule cells from adrenalectomy-induced apoptosis and, combined with the results of other studies, suggest that p53 is required for certain types of neuronal degeneration.

Background and Purpose: Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA., Methods: Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture., Results: Both in situ hybridization and PCR studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however, astrocyte-endothelial cocultures had markedly decreased thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures., Conclusions: These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.

Recent evidence has implicated aberrant cell cycle regulation as a possible mechanism of apoptosis in non-dividing cells. We previously demonstrated increased expression of the p53 tumor suppressor gene, a prominent cell cycle regulator, in apoptotic neurons. Here we investigated the potential involvement of cyclin D1, a G1 phase cell cycle protein under p53 regulation, in kainic acid-mediated neuronal degeneration. Adult male Sprague-Dawley rats were treated systemically with kainic acid and sacrificed between 1 h and 5 days following seizure onset. Cyclin D1 expression was studied by Western blot analysis and immunohistochemistry using a rabbit polyclonal anti-cyclin D1 antibody. In untreated control rats low levels of cyclin D1 expression were detected in multiple brain regions. Between 8 and 16 h after the onset of kainic acid-induced seizures, increased cyclin D1 immunoreactivity was observed in vulnerable hippocampal pyramidal cells. Five days after seizure onset increased cyclin D1 expression was evident in reactive astrocytes. These results support a role for cyclin D1 in certain neuronal death pathways, and suggest that cyclin D1 has multiple and cell type-specific functions in the central nervous system.

Prior to 21 days of age, the immature rat brain is relatively resistant to excitotoxicity caused by the glutamate analogue, kainate. As stress-inducible proteins (GRP78, GRP94 and HSP70) have been proposed to possess molecular chaperone activity and protect cells from the deleterious effects of damaged proteins, we examined the pattern of expression of their respective messenger RNAs following systemic kainate at different postnatal ages. In untreated rats between seven and 21 days old, there was a higher basal level of grp78 and grp94 expression compared to hsp70. Unlike hsp70, which was inducible only in 21-day-old rats, kainate-mediated grp94 induction occurred in several regions of the brain as early as postnatal day 7. Grp78 messenger RNA expression was also increased by kainate treatment in 14-day-old rats, and the induction was most pronounced in the kainate-resistant dentate gyrus. With increasing age, longer lasting expression of both grp78 and grp94 messenger RNAs was observed in kainate-vulnerable regions, similar to observations in the adult rat brain. These results demonstrate non-overlapping expression patterns of glucose-regulated proteins and HSP70 in the immature central nervous system, suggesting that they serve different functions. While hsp70 induction could be a marker for potential cell injury and death, increased expression of grp78 and grp94 could play a neuroprotective role in the developing rat brain.

Several recent studies have demonstrated that expression of the tumour-suppressor gene p53 increases within the nervous system after injury. In various cell lines wild-type-p53, induced by DNA damage, has been shown to function to halt cell-cycle progression and under certain circumstances to induce programmed-cell death or apoptosis. Since wild type-p53 can act as a transcription factor to regulate the expression of p53-responsive genes it is possible that either, or both, functions of p53 are mediated by down-stream effector genes. However wild-type-p53 only weakly activates transcription and it remains to be determined whether p53-responsive genes are expressed in lesioned brain. Here we report that excitotoxic lesion of rat brain with the N-methyl-D-aspartate receptor agonist, quinolinic acid, induces expression of p53 messenger RNA and protein in brain regions showing delayed DNA fragmentation and that expression of p53 messenger RNA precedes DNA damage detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling. In addition, using in situ hybridization and immunocytochemistry we demonstrate increased expression of the p53-responsive gene Gadd-45 (preceding p53 expression) and re-expression of the p53-responsive gene Bax (following p53 expression), in these same areas. Bax has been shown to promote neuronal death by interacting with Bcl-2 family members while Gadd-45 expression has been associated with suppression of the cell-cycle and DNA repair. These results suggest that p53 protein may function as an active transcription factor in lesioned brain perhaps initiating the re-expression of Bax in injured brain regions. However, since Gadd-45 precedes p53 expression it appears unlikely that p53 is involved in regulating the early expression of Gadd-45. Taken together however, these results suggest that p53, Bax and Gadd-45 may play important roles in the response (damage/recovery) of the brain following excitotoxic injury.

Background and Purpose: Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury., Methods: Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion., Results: All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively., Conclusions: Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.

The p53 tumor suppressor gene has been implicated in apoptotic cell death. The present study was conducted to investigate whether expression of p53 protein is increased in association with kainic acid-induced neuronal apoptosis. Adult male Sprague-Dawley rats were treated systemically with the glutamate analog kainic acid, and sacrificed either 4 or 30 h after the onset of seizure activity. Immunohistochemistry was performed on paraffin-embedded sections using an anti-p53 polyclonal antibody. At both time points, increased p53 immuno-reactivity was observed predominantly in the nucleus of apoptotic neurons. These findings lend additional support to the hypothesis that p53 is a marker of neuronal apoptosis in the CNS, and suggest that nuclear accumulation of p53 protein may be an important mediator of neuronal death.

Previous work has shown that tissue plasminogen activator (tPA) is a key enzyme in the control of fibrinolysis within the vascular system. The sources of brain tPA and the mechanisms by which tPA secretion and production occur within cerebral microcirculation are not well established. In this study, expression of tPA was investigated in cerebral capillaries and capillary-depleted brain isolated from cortices of 4- to 5-week-old rats and guinea pigs. In both species, a single tPA band of M(r) 67,000 was detected in cerebral capillaries by Western blot analysis. The tPA signal was absent from capillary-depleted brain. These results were corroborated at the messenger ribonucleic acid level. Reverse transcription-polymerase chain reaction analysis revealed the presence of tPA complementary deoxyribonucleic acid in samples derived from cerebral microvessels and demonstrated very low or undetectable tPA expression in capillary-depleted brain. Immunohistochemical analysis confirmed tPA localization in endothelial cells of brain capillaries. We conclude that microvascular endothelium, i.e., the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation. Delineation of the molecular mechanisms of blood-brain barrier-mediated fibrinolysis will likely contribute to future stroke prevention efforts.

Proposed mechanisms of neurodegeneration focus generally on the triggering of toxic biochemical pathways by an increased intracellular concentration of Ca2+. Recent evidence also suggests that Ca2+ causes transcriptional activation of so-called 'cell-death genes'. Efforts to elucidate the basis of selective vulnerability have relied on animal models of delayed neuronal death in the hippocampus. Biochemical and morphological data indicate that delayed neuronal death is a form of programmed cell death, or apoptosis. Observations that specific genes are activated transcriptionally for prolonged times in neuronal populations that are undergoing delayed death suggest that active gene expression is part of the neuronal-death cascade. Although a direct causal role remains to be proven, evidence implicates certain genes in neuronal-death pathways.

The excitotoxic effects of N-methyl-D-aspartic acid (NMDA) and kainic acid (KA) were studied in organotypic hippocampal slices maintained in vitro for various periods of time. Cultures aged to equivalent Postnatal Day (EPD) 10-12, 15-17, and 23-26 were exposed to 50 microM KA or 50 microM NMDA and were analyzed at 0, 3, 6, 9, 12, 24, 48 h, or 5 days after the initiation of the excitotoxin exposure. Neuronal injury was determined by: (1) propidium iodide (PI) uptake; (2) lactate dehydrogenase (LDH) release; (3) morphological damage in hematoxylin and eosin (H/E) stained sections; (4) loss of Nissl stain. Changes in PI uptake and LDH release after KA or NMDA treatment indicated that there was a developmental shift towards increasing sensitivity to KA toxicity during in vitro development, whereas cultures of all ages were equally sensitive to NMDA toxicity. The profile of damage in H/E-stained sections after treatment with KA or NMDA indicated a transient phase of damaged morphology at 12 and 24 h that was not evident after 5 days. To determine whether the disappearance of morphological manifestations of neuronal damage 5 days after treatment was due to recovery of morphology or to neuronal death, neuronal loss in Nissl-stained sections was also quantified. KA treatment did not cause significant neuronal loss in any hippocampal region in EPD 10-12 cultures, indicating that the neurons were able to successfully recover from the damage demonstrated in H/E sections at 12 and 24 h in these cultures. KA treatment in mature cultures (EPD 23-26) and NMDA treatment in all cultures produced a marked loss of identifiable Nissl-stained neurons at 5 days, indicating neuronal death and disintegration. The results provide further support for the similarities between the organotypic hippocampal culture model and in vivo excitotoxic models and also confirm that excitotoxic neuronal injury can be reversible under some conditions.

Tumor suppressor p53 encodes a protein that is an important regulator of the cell cycle. However, under certain conditions increased p53 expression results in programmed cell death or apoptosis. We used in situ hybridization histochemistry to investigate the role of p53 in the adrenalectomy-induced degeneration of hippocampal granule cells. Three days after adrenalectomy, a subpopulation of granule cells exhibiting morphological features of apoptosis expressed increased amounts of p53 mRNA. Both adrenalectomy-induced p53 expression and granule cell degeneration were prevented by daily administration of corticosterone. In situ end-labeling of nuclei containing fragmented DNA revealed a distribution similar to that of cells with increased p53 expression. These results demonstrate an association between p53 induction and apoptosis in the central nervous system and support the idea that cell cycle-related genes play a role in neuronal death pathways.

MR scanning is used in the clinical evaluation of patients with dementia but lacks a reliable method of visual inspection. Two neurologists conducted multiple pilot trials of alternate methods for visual inspection of MRIs, including methods that produced at least 75% interrater agreement in repeat trials, and selected a final method for rating ventricular:brain ratio (VBR), cortical atrophy, and white matter changes. Two other neurologists, new to the method, tested interrater reliability for each component of the method after a brief training session. The correlation of VBR measurement was 0.884 (p = 0.0001). The weighted kappa scores were 0.68 for overall frontal lobe atrophy, 0.38 for right temporal lobe atrophy, 0.20 for left temporal lobe atrophy, and 0.54 for parietal lobe atrophy. The weighted kappa scores were 0.77 for overall periventricular white matter hyperintensities and 0.72 for centrum semiovale hyperintensities. The proposed method may provide a rapid and reliable way to assess VBR, frontal lobe atrophy, parietal lobe atrophy, and white matter changes on brain MRIs in the evaluation of dementia, but it was less reliable for the assessment of temporal lobe atrophy.

The p53 tumor-suppressor gene encodes a growth-regulatory protein that has been implicated in programmed cell death. To investigate the possible role of p53 in neuronal death, we studied p53 expression associated with excitotoxicity in the adult rat brain. Within hours of systemic administration of the glutamate analogue kainic acid, p53 mRNA levels were increased in neurons exhibiting morphological features of damage within kainate-vulnerable brain regions. A similar distribution was found for neurons exhibiting DNA damage as evidenced by in situ end-labeling of fragmented DNA. Pretreatment with the protein synthesis inhibitor cycloheximide prevented both kainate-mediated p53 induction and neuronal damage. The distinctive pattern of excitotoxin-mediated p53 expression suggests that p53 induction is a marker of irreversible injury in postmitotic cells of the central nervous system and could have functional significance in determining selective neuronal vulnerability.

Tropomodulin is a 40.6 kDa tropomyosin-binding protein associated with actin filaments in muscle and the membrane cytoskeleton in erythrocytes. We have detected tropomodulin mRNA and protein in brains of rats by northern and western blot analyses. In situ hybridization of rat brain and spinal cord sections shows tropomodulin expression in the cerebellum, neocortex, hippocampus, and anterior horn of the spinal cord. Tropomodulin expression is first observed around day 15 after birth and increases through day 24. The temporal and spatial changes in tropomodulin expression during cerebellar development parallel those for brain tropomyosin. Tropomodulin mRNA increases in the dentate gyrus of the hippocampus following prolonged seizure activity induced by kainic acid administration; the increase is clearly evident 8 h after initiation of seizures and is still present 1 week later. However, Western blot analysis of tropomodulin protein level in the dentate gyrus before and after seizure induction show only slight increases in tropomodulin protein concentration, suggesting tight regulation of tropomodulin expression at the translational level. The developmental expression of tropomodulin, together with the induction of tropomodulin mRNA production in the dentate gyrus after kainic acid treatment, suggests a role for tropomodulin in neuronal organization and plasticity.

The relationship between heat shock protein 72 (HSP72) and c-fos gene expression following systemic administration of kainic acid was investigated by combining immunocytochemistry for HSP72 with in situ hybridization for c-fos. Increased HSP72 expression was detected in adult rat hippocampus 4 h after seizure-onset. Transient co-expression of c-fos and HSP72 occurred in neurons that are resistant to kainic acid, whereas prolonged co-expression was observed in vulnerable neurons. The spatial distribution and developmental time course of kainic acid-induced HSP72 expression were similar to those of kainic acid-induced neurodegeneration. The results demonstrate a relationship between c-fos and HSP72 gene expression and suggest that prolonged co-expression of these genes plays a role in kainic acid-induced neuronal death.

In this report, we demonstrate that the brain-specific ras nucleotide-exchange factor, cdc25NEF-B, is expressed in specific neuronal populations in the juvenile rat brain. Because cdc25NEF-B likely regulates one or more of the vertebrate ras proteins, H-, K- and N-ras, we also examined their levels of expression and pattern of expression in the juvenile rat brain. We find cdc25NEF-B to be highly expressed in the hippocampus, some deep nuclei, neocortex, and the granule cell layer of the anterior lobules of the cerebellum. Our observations suggest a functional link between cdc25NEF-B and H-ras in a neuronal signal transduction pathway.

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed by in situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainate-vulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen idea that prolonged c-fos expression is a marker of neuronal death.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine synthesis and is regulated by both transcription-dependent and transcription-independent mechanisms. We compared the effects of asparagine, an amino acid previously shown to increase ODC activity in adult hippocampal slices, on ODC mRNA and activity in adult and neonatal hippocampal slices. In addition, we evaluated the effects of asparagine on ODC activity following seizure activity elicited by systemic administration of kainic acid (KA) in both adult and neonatal rats. Asparagine produced an increase in ODC gene expression and activity in both adult and neonatal hippocampal slices. The increase in ODC activity elicited by asparagine in hippocampal slices was the same in control animals as in animals sacrificed 16 h after KA-induced seizure activity. The asparagine-elicited increase in ODC activity in neonatal and adult hippocampal slices was blocked by the RNA synthesis inhibitor, actinomycin D. Finally, polyamines produced an inhibition of ODC activity in neonatal hippocampal slices. The results indicate that the regulation of the expression and activity of ODC is similar in neonatal and adult hippocampus.

The present study investigated the changes in sulfated glycoprotein-2 (SGP-2) messenger RNA at various times following kainic acid-induced seizure onset in adult and neonatal rat brain. Double labelling using immunostaining of the astrocyte-specific glial fibrillary acidic protein indicated that SGP-2 expression was rapidly and transiently increased in granule cells of the dentate gyrus up to 8 hours after seizure onset. Thereafter, and up to 7 days following seizure onset, the majority of cells exhibiting increased SGP-2 expression were astrocytes located in the molecular layer of the dentate gyrus and in the alveus, as well as in regions adjacent to CA3 and CA1 pyramidal cells. No increase in SGP-2 mRNA was detected in pyramidal neurons selectively damaged by KA. In addition, increased expression of SGP-2 following KA administration was not observed in neonatal rat hippocampus prior to postnatal day 21. The results argue against a role for SGP-2 in KA-induced neuronal death and demonstrate a surprisingly rapid increase in astroglial gene expression following seizure activity, thus supporting a role for SGP-2 in synaptic plasticity.

Induction of the proto-oncogene c-fos is often considered to be a marker of increased neuronal activity. We have used in situ hybridization to study the pattern of c-fos expression in limbic structures following kainic acid-induced seizures during the postnatal period in the rat. Prior to postnatal day 13 (P13), seizure activity did not result in c-fos induction in any limbic structure. Between P13 and P25, a gradual increase in c-fos expression was observed in hippocampus and cortical structures. These results were corroborated by nuclear run-off transcription assay. Thus, alterations in c-fos transcription that may facilitate stimulus-transcription coupling occur during postnatal development. The possible relationship between the postnatal maturation of c-fos expression and the increase in susceptibility of specific neuronal populations to seizure-induced cell damage is discussed.

Heterotopically (infrarenal) transplanted cardiac isografts are histologically normal, spontaneously beating, hemodynamically unloaded hearts that undergo rapid and predictable atrophy. To study the factors that regulate cardiac growth we examined the effect of left ventricular (LV) isovolumic balloon placement on the weight, in vitro protein synthesis rate, and LV RNA content of the transplanted heart. At day 0 the donor LV weight was 367 +/- 8 mg, which decreased to 226 +/- 14 mg (p less than 0.001) after transplantation. Isovolumic loading with a latex balloon (volume, 111 +/- 10 microliters) significantly increased the LV weight of the transplanted heart to 397 +/- 13 mg when compared with that of the unloaded heart 14 days after surgery and prevented the atrophy when compared with the LV weight at day 0. Sham-loaded transplants had LV weights of 256 +/- 17 mg, which did not differ from the LV weight of unloaded transplanted hearts. Protein synthesis rates (nmol lysine/LV/hr) and total LV RNA and LV 18S RNA content were significantly greater in the balloon-loaded hearts when compared with the unloaded isografts and returned to levels similar to those measured in the in situ hearts. The ability of isovolumic loading of the LV to prevent atrophy via increased protein synthesis and the maintenance of LV RNA in the heterotopically transplanted heart further supports the important role of cardiac work in the regulation of cardiac growth.

In the present study we examined the relationship between the induction of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats and activation of immediate early genes (IEGs; c-fos and zif/268) using several different high-frequency stimulation paradigms. Stimulation parameters that effectively induced LTP were not associated with IEG activation. Conversely, stimulation parameters that failed to induce LTP consistently resulted in IEG activation. These results suggest that there is a negative correlation between IEG activation and LTP, and that activation of IEGs is neither necessary nor sufficient for the induction of LTP.

We used in-situ hybridization to study the effect of acute stress on induction of the immediate early genes (IEGs), c-fos and zif/268, in the rat brain. After one hour of restraint plus intermittent tail shock, messenger RNA (mRNA) levels for both genes were significantly increased bilaterally in the neocortex, particularly in layers IV, V and VI, and in the CA1 region of the hippocampus. This regionally-specific response suggests that IEGs may have a role in the mediation of acute stress responses in the central nervous system.

This study concerns effects of the testes on two macromolecules in the rat hippocampus that were previously not known to be responsive to this endocrine axis. Castration for 3 weeks elevated the expression of glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) in male rat hippocampus, as shown by Northern blots and immunocytochemistry. SGP-2 mRNA was colocalized with GFAP, implying increased prevalence in astrocytes after castration. During hippocampal responses to deafferentation by entorhinal cortex lesions that damage the perforant path and induce synaptic reorganization, both mRNA and protein for SGP-2 and GFAP increase. Moreover, prior castration had an additive effect with entorhinal cortex lesions in the increase in GFAP and SGP-2 mRNA. These data suggest that testicular hormones regulate hippocampal astrocyte activity in intact adult rats as well as during synaptic reorganization in response to deafferenting lesions.

Heterotopic cardiac isografts are vasculary perfused organs that maintain structural and functional integrity. We have used this model to study the time course of change in total heart and left ventricular size which results from mechanical unloading of the myocardium. When compared to the in situ (working) heart there is a 19% decrease in the size of the heterotopic (non-working) heart (P less than 0.01) as early as 3 days post-transplantation. By 14 days there is 50% decrease in the size of the transplanted heart which is maintained at this atrophic size when measured after 4 weeks. Left ventricular protein synthesis was assayed by the simultaneous in vitro perfusion of the host and transplanted hearts under identical hemodynamic conditions. The hourly incorporation of 14C-lysine into total left ventricular protein was 21 nmol in the transplant compared to 50 nmol in the in situ heart (P less than 0.01). This incorporation remained significantly lower throughout the period of study. In contrast, both the total (mumol lysine/g protein nitrogen/h) and fractional rates of protein synthesis which were lower in the transplanted left ventricle at days 3 and 7 returned to control values by day 28. The present studies demonstrate that heterotropic cardiac transplantation leads to a prompt and reproducible decline in cardiac mass and in total protein synthesis. These studies further support the role of cardiac work as an important determinant in the regulation of cardiac protein synthesis.

Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.

Albumin synthesis was studied in the isolated perfused rabbit liver under the influence of the stresses of fasting and acute alcohol and acetaldehyde exposure. Fasting clearly depressed albumin production and disaggregated the endoplasmic membrane-bound polysomes. Acute exposure to alcohol produced the same results. Acetaldehyde 2 mg% resulted in a depression of albumin synthesis but the polysomes were not disaggregated. The metabolism of alcohol was necessary for polysome disaggregation. The acute effects of ethanol and fasting were quite similar and it might be considered that the alcohol was acting like a pharmacologic fast. Employing the liver from a fasted donor specific amino acids infused into the liver at levels of 10 mM reversed the acute effects of fasting and the acute effects of exposure to ethanol. However when the two stresses of fasting and alcohol were combined the same amino acids were not effective. In studying albumin synthesis and/or secretion it is necessary to carefully define the nutritional status of the experimental model.

Although acute perfusion of guinea pig hearts with ethanol does not affect cardiac protein synthesis, the latter is inhibited after prolonged ingestion of ethanol when tested in an in vitro system with the working right ventricle. This study reports on the added stress of ischemia on such hearts. Hearts were removed from maturing guinea pigs after 13-16 weeks of ingesting 10% ethanol and were perfused in vitro under conditions of relative ischemia (one-sixth of normal coronary flow) with maintenance of right ventricular load and outflow resistance identical to normal pre-ischemic levels. With this degree of ischemia, there was a 4-6 fold increase in lactate production, an 80% drop in ATP, and a 90% decrease in creatine phosphate after 150 min of the ischemia. Incorporation of both labeled lysine and phenylalanine into cardiac protein was also diminished to 35% of control in the left ventricle and 55% of control in the right. This diminution of protein synthesis was the same in hearts from ethanol-drinking and matched control animals. Thus, prior prolonged ingestion of ethanol did not worsen the inhibition of protein synthesis by oxygen deprivation. There were, however, two significant differences in hemodynamic response to the ischemia by the right ventricles of hearts from ethanol-drinking guinea pigs compared to their matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

CCl4, 2.5 ml/kg body weight, was administered via a gastric tube to fed rabbits 2 hr before the livers were removed and perfused. Electron microscope studies of the liver showed that CCl4 caused a decrease in the rough endoplasmic reticulum and an increase in the smooth reticulum. The reticulum was dilated and vesiculated with few attached ribosomes. Sucrose gradient analysis of the endoplasmic reticulum bound polysomes showed them to be considerably disaggreated, and albumin synthesis and 14C-incorporation into proteins secreted into the perfusate were decreased. These effects were partially reversed when these livers were perfused with either 1 mM spermine of 10 mM arginine. Perfusion with both arginine and spermine increased endoplasmic reticulum bound polysome aggregation to about 92% of control and albumin synthesis increased from a low of 3.3 mg/100 g wet liver weight to 8.2 mg/hr or 37% of control. 14C-Incorporation into total hepatic protein and secretory proteins increased as well. The combination of spermine and arginine was more effective in stimulating albumin synthesis than either agent alone. The ability of spermine to partially reverse a specific toxic effect of carbon tetracholoride, namely polysome disaggregation, and to stimulate protein production is noted.

Acetaldehyde infusions inhibit albumin synthesis in the liver from fed donors but not in the livers from fasted donors. The inhibition of acetaldehyde metabolism with 4-MP and disulfiram reverses this finding, suggesting that acetaldehyde per se is not the toxic agent. Disulfiram stimulates albumin synthesis in livers from fasted donors, and the presence of acetaldehyde does not prevent this process. The effects of ethanol infusions cannot be explained as due to the presence of acetaldehyde; some intermediate metabolic step may be the basis of the inhibition of albumin production and polysome disaggregation in the presence of ethanol.

Cardiac stress produced by hypertension or excess volume loading results in different types of hypertrophy. Elevated left ventricular pressure rapidly results in increased myocardial protein synthesis in vivo and in vitro, but such rapid alterations are not consistently seen in volume loading. The difference in response is difficult to clarify since it is not possible to effect alterations in left ventricular pressure or perfusion without profoundly affecting coronary perfusion. The present study describes cardiac protein synthesis in the right ventricle of the young guinea pig heart in vitro utilizing a perfusion model in which the right ventricle could be stressed by elevations of pressure or volume loading in the presence of constant and restricted coronary perfusion. With coronary flow maintained at 25 ml/min/g dry wt, an increase in right ventricular pressure from normal levels of 3 mm Hg to 11 mm Hg resulted in a 60% increase of myocardial incorporation of lysine-14 C into protein. However, with further increases of right ventricular pressure to 22 mm Hg, protein synthesis dropped back to normal levels. The fall-off in protein synthesis was not due to decreased contractility, alterations in intracellular lysine pool specific activity, or alterations in total coronary flow or pressure. A 60% increase in coronary perfusion was associated with a similar response of protein synthesis to progressive elevations of pressure. Since the ATP levels rose and lactate production fell, a deficiency of O2 did not entirely explain the decline of protein synthesis with maximal pressures. At all levels of coronary perfusion, volume loading for 3 hr did not result in increased protein incorporation of lysine-14 C. The studies indicate a relationship between ventricular pressure and protein synthesis unrelated to coronary flow per se and suggest a pressure receptor triggering protein synthesis within the ventricular wall. Such a relationship is not apparent in short term volume loading in vitro.

The occurrence of cardiomy opathy in chronic alcoholics is well known, but the causes are as yet unclear (Mitchell and Cohen, 1970). Metabolic effects of ethanol, such as accumulation of triglycerides despite a decrease in fatty acid extraction (Regan et al., 1966; 1969), have been suggested as a cause of ultimate impairment of myofibrillar function. The suggestion has also been made that the detrimental effects of ethanol may actually be an acetaldehyde effect, mediated through the release of norepinephrine causing chronic chronotropic and inotropic effects which may often play a role in the development of the myopathy (James and Bear, 1967). It has been reported that acute exposure to alcohol decreases to one-third that of the control the capacity of the liver to synthesize albumin (Rothschild et al., 1971). In view of the rapid inhibitory effect, it was felt to be of interest to study the effect of alcohol in the perfused heart to see whether myocardial protein synthesis was similarly inhibited. In addition, since alcohol is apparently not metabolized by the heart (Gailis and Verdy, 1971; Lochner, Cowley, and Brink, 1969). The effect of a primary metabolite, acetaldehyde (James and Bear, 1967), synthesized in liver was also studied. The results indicated that acute exposure to levels of alcohol which decreased albumin synthesis in the perfused liver had no effect on protein synthesis in the perfused heart. However, acetaldehyde, at levels that produce a marked chronotropic and inotropic effect, markedly inhibited protein synthesis of total cardiac protein. To further define the inhibition of protein synthesis by acetaldehyde, the effects of ethanol and acetaldehyde on cardiac micorsomes were also studied in cell-free systems. Some of these data were reported previously (Schreiber et al., 1972; 1974).

One-sided lateral funiculotomy at the C-2 segment induced axon reaction in the contralateral red nucleus of adult cats. Two to 60 days postoperatively the animals were sacrificed and the mesencephalon was fixed in ethanol-acetic acid, 3:1. Ten micrometer paraffin sections including both red nuclei were stained for RNA with azure B after incubation in DNAse. Cytophotometric measurements of RNA content of neurons from the caudal 600-1000 micrometer of each red nucleus were made with a Zeiss Cytoscan system using an automatic scanning stage. In contrast to the heightened RNA synthesis that has been reported for axotomized peripheral (extrinsic) neurons, the axotomized central (intrinsic) neurons of the red nucleus showed no evidence of accumulation of cytoplasmic or nucleolar RNA. Rather depletion of cellular RNA occurred. Further indication of the regressive nature of rubral axon reaction derived from morphometric measurements that showed cytoplasmic, nuclear and nucleolar atrophy of the neurons of the red nucleus contralateral to operation with the exception of a possible transient cytoplasmic enlargement 9 days postoperatively. From the cytophotometric and morphometric data here reported we are led to suggest that the frequently observed failure of axonal repair in mammalian CNS results from the innately regressive nature of the axon reaction of many mammalian central neurons.

Human coronaviruses are important human pathogens and have also been implicated in multiple sclerosis. To further understand the molecular biology of human coronavirus 229E (HCV-229E), molecular cloning and sequence analysis of the viral RNA have been initiated. Following established protocols, the 3'-terminal 1732 nucleotides of the genome were sequenced. A large open reading frame encodes a 389 amino acid protein of 43,366 Da, which is presumably the nucleocapsid protein. The predicted protein is similar in size, chemical properties, and amino acid sequence to the nucleocapsid proteins of other coronaviruses. This is especially evident when the sequence is compared with that of the antigenically related porcine transmissible gastroenteritis virus (TGEV), with which a region of 46% amino acid sequence homology was found. Hydropathy profiles revealed the existence of several conserved domains which could have functional significance. An intergenic consensus sequence precedes the 5'-end of the proposed nucleocapsid protein gene. The consensus sequence is present in other coronaviruses and has been proposed as the site of binding of the leader sequence for mRNA transcriptional start. This region was also examined by primer extension analysis of mRNAs, which identified a 60-nucleotide leader sequence. The 3'-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the Coronavirus family and lends support to the theory that this region is important for the replication of negative-strand RNA.

Acute exposure of the heart to ethanol does not appear to alter the rate of young guinea pig cardiac protein synthesis when assayed in vitro. In contrast, the primary metabolite of ethanol, acetaldehyde, markedly diminishes synthesis despite its chronotropic and inotropic effects. On the other hand, after 11-13 weeks of ethanol-drinking during growth and maturation, the synthetic capacity of the working right ventricle was decreased when measured in vitro with normal perfusate. Assay of synthesis of the contractile proteins myosin heavy and light chains, actin and tropomyosin suggests a change in synthesis or pool size of actin reflected in an alteration of relative synthesis of this protein compared to that of heavy chains. The relative synthesis of the other proteins remained at control levels. When hearts from ethanol-drinking and matched control animals were perfused under conditions of severe ischemia, there was a profound fall in protein synthesis in all hearts, and ethanol did not enhance the inhibition of synthesis. However, the hearts from ethanol-drinking animals showed a more marked and significant impairment of maintaining ejection pressure with a marked increase in coronary resistance as the perfusion progressed. It is postulated that some impairment of protein metabolism may occur during prolonged ethanol exposure, which may influence the cardiac response of another induced stress, e.g., ischemia.

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.

1. Reperfusion after ischemia and perfusion with total anoxia were studied in the isolated guinea pig heart model which permits right ventricular loading and constant coronary perfusion. Deprivation of oxygen in both situations resulted in a marked shift of circulation from the left to the right ventricle with markedly increased spaces of distribution of 99mTc radionuclides and albumin in the latter. 2. In view of the complexities of measuring protein synthesis during ischemia, continuous anoxic perfusion was used to evaluate this parameter in anoxic induced arrest. There was a profound fall in protein synthesis associated with this arrest, accompanied by a fall in ATP, creating phosphate, glycogen, potassium, and a rise in lactate production. The fall in protein synthesis was more marked in the left ventricle, despite the absence of work while it was still beating. 3. The changes in synthesis were almost completely prevented by initiating cardiac arrest with high K+ (16 meq/l) at the same time as anoxia; energy metabolism remained near normal, and recovery of contractility was nearly complete. 4. The studies demonstrated the differences in vascular distribution between the ventricles after ischemia or with perfusion anoxia, the possible difference in availability of substrate to the two ventricles under these conditions, as well as the difference in protein synthetic response, and further support the protective effect of potassium induced arrest on the hypoxic heart.

The occurrence of cardiomyopathy in chronic alcoholism has been well documented, but the cause of the myopathy is still unclear. Some of the mechanisms described have included accumulation of triglycerides, altered fatty acid extraction, membrane alterations with decreased response to Ca2+ and to catecholamines and alterations in cardiac protein synthesis. The present article briefly reviews the effects of ethanol or its primary metabolite, acetaldehyde, on cardiac protein synthesis and possible relation to cardiomyopathy.

Newly weaned guinea pigs weighing approximately 300 g were fed normal laboratory diets with drinking water containing 5.5% ethanol as the sole source of liquid for periods of 8-11 weeks. Growth was continuous with this diet. After this period, hearts were removed from anesthetized animals and perfused for 3 hr in a perfusion system in which pressure may be induced in the right ventricle in the face of constant coronary flow. Protein synthesis, assayed from the incorporation of labeled lysine and phenylalanine, was compared to that in hearts from identically treated weight-matched control animals who had been drinking water without ethanol. Protein synthesis in hearts from ethanol-drinking animals was decreased in the right ventricles exposed to normal pulmonary pressure, but was unchanged in the contracting but not working left ventricles. The data suggest that prolonged exposure even to low levels of ethanol in the growing animal may interfere with the cardiac protein synthetic response to the normal work stress.

99mTc colloid scans, hepatobiliary scans with IDA derivatives, 67Ga scans, and labeled red blood cells or indium-labeled white blood cells are the major imaging procedures that are currently widely available to visualize the liver. The use of labeled antibodies to a specific tumor is being explored as an investigative procedure but is complicated by the high circulating background activity. In this overview of planar liver radionuclide imaging, it was emphasized that these procedures are noninvasive, may be performed at the bedside, are inexpensive, and provide important data for formulating the further investigation of intrahepatic masses, gallbladder disease, vascular and inflammatory diseases. The functional status of the liver is the basis of the radionuclide imaging procedures, and the more accurate anatomic technique do not give these data.