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Biomedical subjects

T S Nowak

Publications and source records attributed to T S Nowak.

At least 55 records · Page 3Linked to original sources
PubMed1990

Protein synthesis and the heart shock/stress response after ischemia.

It has been appreciated for many years that the recovery of brain protein synthesis activity following a transient ischemic insult lags considerably behind the normalization of brain energy metabolism. More recently, selective increases or decreases in the synthesis of specific proteins have been documented to occur during postischemic recirculation, the best characterized of such changes being the induction of proteins characteristic of the "heat shock" or "stress" response. This review will summarize these developments in the study of changes in gene expression following ischemia, with an emphasis on regional differences in the vulnerability of overall translational activity as well in the expression of stress proteins and their mRNAs. The neuronal localization of the 70 kDa heat shock protein, hsp70, after ischemia is contrasted with its largely glial and vascular induction following a hyperthermic stress. The lasting depression of protein synthesis and sustained expression of hsp70 mRNA in vulnerable hippocampal CA1 neurons appear to be mechanistically related and may constitute markers for cellular pathophysiology leading to neuronal cell loss. Elucidating the mechanisms responsible for cell-specific regulation of stress proteins and other gene products may eventually contribute to a more precise understanding of the evolution of brain injury at the molecular level following diverse insults.

Animals↗
PubMed1990

Cumulative effect of repeated ischemia on brain edema in the gerbil. Biochemical and physiological correlates of repeated ischemic insults.

Using a bilateral carotid artery occlusion model in the gerbil, we evaluated the cumulative effect of repeated ischemic insults on various physiological and biochemical parameters in brain. The most striking consequence of repeated occlusions is a profound, delayed increase in brain edema between 6- and 24-hr recirculation, after a series of three 5-min occlusions carried out at 1-hr intervals. This increment in brain water is accompanied by morphological evidence of compressed capillaries with increased filling of larger vessels, consistent with impaired microcirculation even though total blood flow and total vascular volume return toward control levels. The effect on edema is most severe when occlusions are repeated during the period of postischemic hypoperfusion, although the mechanisms responsible for this effect remain to be determined. Histograms of cerebral cortical oxygen tension show a shift toward progressively lower values during the recirculation interval after each occlusion, suggesting that secondary hypoxia accompanying hypoperfusion may worsen the impact of successive ischemic intervals. Although levels of PCr and ATP indicate adequate recovery of energy metabolism between occlusions, slight elevations of brain lactate persist, consistent with continued hypoxia. Under the conditions employed in these studies, repeated occlusions give rise to progressively more prolonged deficits in brain protein synthesis activity, which may thus provide a useful index of the severity of the accumulated ischemic insult. Continued studies using this well-defined model should provide further insight into the pathophysiology of ischemic brain edema.

Animals↗
PubMed1989

Induction of stress protein HSP70 in nerve cells after status epilepticus in the rat.

The accumulation of the stress protein HSP70 was found to be an excellent marker for prolonged seizure related metabolic activity of neurons. After kainic acid (KA) induced status epilepticus we observed HSP70 immunoreactivity in the hippocampal CA4 and CA1 sectors, the subiculum, the basolateral and the lateral nuclei of the amygdala, the mediodorsal nucleus of the thalamus, the caudal part of the striatum, the claustrum and in neurons of certain neocortical areas. HSP70-positive nerve cells appeared normal in conventional histological stains. Conversely, degenerating neurons (e.g. in the hippocampal CA3 sector) remained unlabeled.

Animals↗
PubMed1989

Effect of acute electrode placement on regional CBF in the gerbil: a comparison of blood flow measured by hydrogen clearance, [3H]nicotine, and [14C]iodoantipyrine techniques.

Regional cerebral blood flow (rCBF) was compared in the gerbil by means of [3H]nicotine, [14C]-iodoantipyrine, and hydrogen clearance techniques. In agreement with other studies, nicotine and iodoantipyrine methods gave virtually identical results. With these methods, it was observed that a reduction in blood flow occurred shortly after insertion of an electrode into the striatum for hydrogen clearance measurement, affecting rCBF throughout the impaled hemisphere. The reduction was moderate (30%) in the striatum and hippocampus, but much greater (70%) in cortical regions. Identical deficits were observed following brief penetrations involving only cortex. Following chronic electrode placement in the striatum, regional blood flow values obtained with [3H]nicotine returned to the control range within 6 h. Blood flow estimates obtained in the striatum with the implanted electrode increased with a similar time course, so that by 6-24 h, hydrogen clearance gave values indistinguishable from control values obtained with [3H]nicotine. These results clearly demonstrate that reduction of CBF subsequent to electrode placement can account for the low values frequently obtained with the hydrogen clearance method in small animals. The distribution of the deficit and the time course of its recovery are similar to blood flow changes associated with spreading depression. While mechanisms responsible for this effect remain to be fully identified, chronic implantation is a practical solution that allows the continued use of hydrogen clearance as a convenient method for repeated measurement of blood flow in the same animal.

Animals↗
PubMed1989

Cerebral vascular volume after repeated ischemic insults in the gerbil: comparison with changes in CBF and brain edema.

The time course of changes in cerebral intravascular volume was evaluated during 24 h following a series of three 5-min carotid artery occlusions spaced at 1-h intervals and compared with the changes occurring after single 5- or 15-min occlusions. Quantitative estimates of cerebral red cell volume, plasma volume, and total blood volume were obtained from the distribution spaces of 51Cr-labeled erythrocytes and 125I-albumin infused prior to killing at varied recirculation intervals. Significant reductions in vascular volume occurred in all ischemic brain regions within 1 h following a single 5-min occlusion, which recovered to control values within 6 h. A similar time course was seen after repeated occlusions. The reductions in volume remained significant at 6 h after a single 15-min occlusion, but there was no difference from control by 24 h. Thus, the time course of total vascular volume correlates well with that of CBF changes previously described, and both blood flow and blood volume are at normal levels during the time of severe edema 24 h after repeated occlusions. Calculated cerebral hematocrit was 60-70% of that obtained from the femoral artery, but was identical in all brain regions and was constant throughout the postischemic recirculation period, with the exception of a transient reduction in both peripheral and cerebral hematocrit observed at 6-h recirculation following single 15-min occlusions. These results suggest that changes in CBF and blood volume reflect primarily the status of larger vessels and that values in the normal range may be observed even under conditions of severe edema and impaired perfusion at the capillary level.

Animals↗
PubMed1988

Localization of 70-kDa stress protein induction in gerbil brain after ischemia.

Induction of the 70-kDa heat shock protein, hsp70, has been demonstrated in brain following experimental stroke. In the present study, hsp70 was localized in gerbil brain at intervals after transient ischemia using a monoclonal antibody specific for stress-inducible forms of hsp70-related proteins. Induced immunoreactivity was found only in neurons, primarily in hippocampus, striatum, entorhinal cortex and some neocortical regions. Notably hsp70 accumulation was minimal in hippocampal CA1 neurons which die after brief ischemic episodes, but was most pronounced in dentate granule cells and CA3 neurons which are spared. The peak of CA3 immunoreactivity occurred at 48-h recirculation, at the onset of CA1 neuron loss at 2-4 days, demonstrating that hsp70 induction is also a component of this delayed hippocampal pathophysiology rather than a direct response to the metabolic disruption of the initial ischemic episode. These results suggest that hsp70 immunocytochemistry may serve as a marker for neuronal circuitry involved in proposed excitotoxic mechanisms after ischemia and other stresses. Control animals showed immunoreactivity in ependymal cells lining the ventricles, indicating a role for hsp70 in normal functioning of these specialized cells.

Animals↗
PubMed1988

Microvascular disturbances and edema formation after repetitive ischemia of gerbil brain.

Three transient episodes of 5 min ischemia spaced at 1-h intervals were produced in Mongolian gerbils by bilateral carotid artery occlusion with an implanted vascular occlusion device. The interval of 1 h was chosen to allow for the development of post-ischemic hypoperfusion between the ischemic episodes. Three minutes and 1 h after each ischemic episode, and 6 and 24 h after the third occlusion, Evan's blue (EB) was injected intravenously to trace circulating blood, and the number of perfused capillaries was determined in various brain regions by fluorescence microscopy. Brain edema was evaluated by measuring specific gravity in tissue samples taken from adjacent areas. Repetitive ischemia caused progressively increasing brain edema and a progressive reduction of the number of perfused capillaries. Immediately after each ischemic episode, transient recruitment of capillaries occurred, thus excluding no-reflow as a main pathogenetic factor of microcirculatory disturbances. The pattern of microcirculation 6 and 24 h after the last occlusion revealed a redistribution of circulating blood, characterized by a reduction in the number of EB-filled capillaries associated with a noticeable dilatation of the larger vascular channels. Our studies suggest a close interrelationship between post-ischemic microcirculatory hypoperfusion and the development of brain edema, the degree and extent of which progresses with the repetition of ischemic episodes when they are carried out during the periods of hypoperfusion.

Animals↗
PubMed1988

Effects of amphetamine on protein synthesis and energy metabolism in mouse brain: role of drug-induced hyperthermia.

Changes in brain protein synthesis activity, and in brain levels of glucose, glycogen, and several high-energy phosphate metabolites, were evaluated under conditions of amphetamine-induced hyperthermia in mice. Protein synthesis showed a striking dependence on rectal temperature (TR), falling abruptly at TR above 40 degrees C. A similar result was obtained following direct heating of the animals. Protein synthesis activity in liver showed the same temperature dependence observed for brain. Increased synthesis of a protein with characteristics of the major mammalian stress protein, hsp 70, was demonstrated in both brain and liver following amphetamine administration. Brain protein synthesis showed significant recovery within 2 h after amphetamine administration whereas that of liver remained below 30% of control activity, suggesting significant temporal and quantitative differences in the response of individual tissues to elevated temperatures. Brain glycogen levels after amphetamine administration were significantly lower under conditions of ambient temperature which resulted in more severe drug-induced hyperthermia but did not correlate as strikingly as protein synthesis with the temperatures of individual animals. Brain glycogen also fell in animals whose temperatures were increased by brief exposure at high ambient temperature. Brain glucose levels did not consistently change with hyperthermia. Slight decreases in high-energy phosphates with increasing TR were likely the result of fixation artifact. These results demonstrate the fundamental role of hyperthermia in the reduction of protein synthesis in brain and other tissues by amphetamine, and suggest that temperature also constitutes a significant source of variability in the effects of this drug on brain energy metabolism, in particular glycogenolysis.

Adenosine Triphosphate↗
PubMed1987

Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults.

An experimental model for repeated ischemic attacks, which allows easy induction of cerebral ischemia of any desired duration and frequency, has been developed in the gerbil. With this procedure, a pronounced cumulative effect on development of edema and tissue injury was observed using 3 separate, 5-min bilateral occlusions of the common carotid arteries spaced at various time intervals. This effect was most evident when the occlusions were carried out at 1-h intervals, i.e., during the period of marked postischemic hypoperfusion. Such animals, killed after 24 h of recirculation, showed significantly more severe edema and brain tissue injury in the areas exposed to ischemia than was observed in animals killed 24 h after single 5- or 15-min occlusions. The changes of regional CBF, assayed with a [3H]nicotine method, indicated a relatively rapid onset of hypoperfusion of similar degree after each release of arterial occlusion. The hypoperfusion recovered significantly within 6 h of recirculation following either single or multiple occlusions, and no residual hypoperfusion was observed in animals which, when killed at 24 h, showed severe edema and brain tissue injury. This model should prove useful in elucidating the pathophysiological mechanisms operative in repetitive cerebral ischemia.

Animals↗
PubMed1985

Changes in brain energy metabolism and protein synthesis following transient bilateral ischemia in the gerbil.

The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.

Adenine Nucleotides↗
PubMed1985

Synthesis of a stress protein following transient ischemia in the gerbil.

In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70-kilodalton stress protein, identified by two-dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70-kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.

Animals↗
PubMed1985

Role of hyperthermia in effects of electroconvulsive shock on protein synthesis in the rabbit.

The effects of electroconvulsive shock (ECS) on rectal temperature (TR) and on protein synthesis in brain and liver were compared in rabbit, rat, and mouse. Protein synthesis status was assessed using an in vitro amino acid incorporation method which provides information equivalent to polyribosome profiles. In the rabbit, TR rose from 39.5 +/- 0.4 degrees C to 40.4 +/- 0.2 degrees C within 10 min following a single ECS, and significant hyperthermia persisted for at least 60 min. This effect was markedly attenuated in animals housed at 4 degrees C. In vitro protein synthesis activities of rabbit brain and liver preparations were significantly reduced following ECS only in those animals whose TR exceeded 40 degrees C. In the rat, ECS gave rise to a significant hyperthermia, but in no case did TR exceed 40 degrees C, and protein synthesis activity of brain supernatants was not affected. In the mouse, ECS reduced TR and had no effect on in vitro protein synthesis activity. These results demonstrate that the unique sensitivity of protein synthesis in rabbit tissues to electroconvulsive shock is a direct consequence of the hyperthermia that arises following ECS in this species.

Amino Acids↗
PubMed1985

Nimodipine levels in gerbil brain following parenteral drug administration.

Nimodipine binding to the particulate fraction of gerbil brain homogenate was characterized using tritiated (3H)-nimodipine as the radioactive ligand. Binding was monophasic and saturable, with the apparent affinity constant (KD) = 0.4 nM and the maximum number of binding sites (Bmax) = 12 nmol/kg wet wt. A competitive binding assay was validated for the measurement of nimodipine using gerbil brain as the source of receptors for the drug. Binding characteristics were sufficiently similar in specimens from different animals to allow the use of homogenates from individual animals as the source of both membrane-binding sites and competing ligand. Nimodipine could be detected in the brains of animals sacrificed soon after drug injection, and reached a peak level within 15 minutes. Brain drug level at a given time was a linear function of dose administered. One hour after a 1-mg/kg dose, the level of drug measured in brain was approximately 100 nmol/kg wet wt, more that 200 times the KD. Sufficient drug to mediate a maximal pharmacological effect accumulated in brain even after a dose of only 0.25 mg/kg. Thus, in this species, effective tissue nimodipine levels may be achieved at doses which minimize the risk of systemic hypotension.

Animals↗
PubMed1984

An in vitro amino acid incorporation method for assessing the status of in vivo protein synthesis.

A quantitative in vitro amino acid incorporation assay is described which can be used to assess the status of in vivo protein synthesis. The preparation and incubation conditions employed result in constant precursor specific activity and limit amino acid incorporation to completion of nascent peptide chains. Results obtained with this method correlate well with measurements of polyribosome profiles using sucrose gradient centrifugation. The assay is easily applied to a large number of samples, and requires only a fraction of the time and tissue necessary for conventional measures of polysome aggregation. The method has been found suitable for studies of protein synthesis in mouse brain and liver, and in gerbil brain, but not in mouse kidney. Products of in vitro protein synthesis can be separated by standard electrophoretic techniques, allowing a characterization of proteins whose mRNAs are actively translated in vivo.

Amino Acids↗
PubMed1983

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition.

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/or elongation reactions may predominate, depending on the construction of the system under study.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays.

A several-fold greater counting efficiency is observed for protein labeled with [3H]leucine than for free [3H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for 14C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.

Carbon Radioisotopes↗