Search PubMed⌕ Search

Biomedical subjects

M L Smith

Publications and source records attributed to M L Smith.

At least 181 records · Page 10Linked to original sources

Hyperthermia nullifies the ameliorating effect of dizocilpine maleate (MK-801) in focal cerebral ischemia.

The present study was inspired by two previous findings from the laboratory. The first was that dizocilpine maleate (MK-801) fails to reduce infarct size when the middle cerebral artery (MCA) is permanently occluded by an intraluminal filament technique in rats. In seeking the reasons for this we measured temperature and found that the body temperature of occluded animals increases to 39.0-39.5 degrees C during the first 2-3 h. In order to explore whether the rise in temperature was responsible for the lack of effect of MK-801, two groups of animals were studied, both containing animals which were subjected to 2 h of transient MCA occlusion and given MK-801 15 min before, as well as 6 and 24 h after ischemia. In one group, temperature was allowed to rise spontaneously during ischemia (39.0-39.5 degrees C). In the other, body temperature was maintained close to normal during ischemia, and for the first 6 h postischemically, by cooling of the ambient air. Infarct volume was assessed by triphenyltetrazolium chloride staining after 48 h of recovery. The results showed that MK-801 failed to reduce infarct size in animals whose body temperature rose during ischemia. In contrast, the drug markedly reduced infarct volume in temperature-controlled animals; in fact, 5/8 animals had no infarcts but selective neuronal damage only. The results suggest that amelioration of focal ischemic damage cannot be expected if body and brain temperature is allowed to rise above normal.

Animals↗

Stimulus and sex differences in performance of mental rotation: evidence from event-related potentials.

We examined stimulus and sex differences in reaction time (RT) and event-related potentials (ERPs) during mental rotation of letters and abstract designs (PMA figures). RTs replicated stimulus and angle effects found in previous studies, but no sex differences were found for either set of stimuli. ERP latency data showed women began stimulus evaluation earlier, and PMA rotations began later over smaller angles, whereas letter rotations began later over larger angles. ERP amplitude data replicated hemisphere, electrode, and angle effects found in earlier studies. Amplitude measures also showed greater involvement of anterior cortical areas for evaluation of letter figures and posterior right temporal lobe for PMA figures, and greater positivity of women's waveforms than men's over late evaluation and early rotation components.

Electric Stimulation↗

Induction of p53-, MDM2-, and WAF1/CIP1-like molecules in insect cells by DNA-damaging agents.

Cellular responses following DNA damage are ubiquitous in the biological world. In response to DNA damage, cell cycle checkpoints are activated, which delay cell cycle progression and most likely serve to allow time for repair. One important checkpoint in mammalian cells, activated in the G1 phase of the cell cycle, is dependent on the p53 tumor suppressor gene product. While p53 is responsible for inducing G1 arrest, the product of the MDM2 gene is believed to alleviate the arrest, allowing continuation of the cell cycle after a transient delay. Inasmuch as MDM2 and WAF1/CIP1 are transactivated by p53, while MDM2 binds to and modulates the activity of p53, a "feedback loop" is thus created. This pathway has been highly conserved in mammalian cells, but its presence outside of vertebrates is unknown. By using human MDM2 and WAF1/CIP1 cDNA probes, and monoclonal antibodies to p53 and Mdm2, we demonstrate in insect cell lines evidence for the existence of p53-, MDM2-, and WAF1/CIP1-like molecules and a p53-regulated pathway following treatment by DNA-damaging agents.

Animals↗

Regulation of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage.

Levels of mRNA for c-fos, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), TrkB, and TrkC were studied using in situ hybridization in the rat brain at different reperfusion times after unilateral middle cerebral artery occlusion (MCAO). Short-term (15 min) MCAO, which does not cause neuronal death, induced elevated BDNF mRNA expression confined to ipsilateral frontal and cingulate cortices outside the ischemic area. With a longer duration of MCAO (2 h), which leads to cortical infarction, the increase was more marked and elevated BDNF mRNA levels were also detected bilaterally in dentate granule cells and CA1 and CA3 pyramidal neurons. Maximum expression was found after 2 h of reperfusion. At 24 h BDNF mRNA expression had returned to control values. In the ischemic core of the parietal cortex only scattered neurons were expressing high levels of BDNF mRNA after 15 min and 2 h of MCAO. Analysis of different BDNF transcripts showed that MCAO induced a marked increase of exon III mRNA but only small increases of exon I and II mRNAs in cortex and hippocampus. In contrast to BDNF mRNA, elevated expression of c-fos mRNA was observed in the entire ipsilateral cerebral cortex, including the ischemic core, after both 15 min and 2 h of MCAO. Two hours of MCAO also induced transient, bilateral increases of NGF and TrkB mRNA levels and a decrease of NT-3 mRNA expression, confined to dentate granule cells. The upregulation of BDNF mRNA expression in cortical neurons after MCAO is probably triggered by glutamate through a spreading depression-like mechanism. The lack of response of the BDNF gene in the ischemic core may be due to suppression of signal transduction or transcription factor synthesis caused by the ischemia. The observed pattern of gene expression after MCAO agrees well with a neuroprotective role of BDNF in cortical neurons. However, elevated levels of NGF and BDNF protein could also increase synaptic efficacy in the postischemic phase, which may promote epileptogenesis.

Animals↗

Critical values for plasma glucose in aggravating ischaemic brain damage: correlation to extracellular pH.

The objective of the present experiments was to characterize conditions under which pre-ischaemic hyperglycaemia aggravates brain damage following transient forebrain ischaemia. Specifically, we wished to explore whether accentuated damage is a threshold function of plasma glucose concentration or pH, as assessed by measurements of extracellular pH (pHe). Forebrain ischaemia of 10 min duration was induced in rats at varying degrees of hyperglycaemia, with continuous measurements of pHe, and the animals were allowed to survive for 7 days before histopathological evaluation of the density and distribution of brain damage. Ischaemic brain damage appeared as a threshold function of plasma glucose concentration. At values of 4-6 mM virtually no damage was observed in any other structure than the CA1 sector of the hippocampus and, even in that structure, damage was variable. At glucose concentrations of 8-12 mM moderate damage was observed infrequently in caudoputamen, parietal cortex, and thalamus. At values above 12 mM, damage increased dramatically in these areas, and additional structures were recruited in the damage process (cingulate cortex, the CA3 sector of the hippocampus, and substantia nigra). Measurements of pHe in parietal cortex showed a threshold for seizure induction at values of 6.4-6.5, probably corresponding to intracellular pH values of 6.2-6.3. The threshold for aggravation of histopathological damage was similar. It is concluded that a moderate increase in plasma glucose in the threshold range predisposes the tissue to aggravated damage, probably by activating biochemical reactions or pathophysiological events with a steep pH dependence.

Animals↗

Psychosocial correlates of immune responsiveness and illness episodes in US Air Force Academy cadets undergoing basic cadet training.

This study examined psychosocial correlates of immune function and illness in 89 male first-year US Air Force Academy cadets. A psychosocial questionnaire was administered to cadets prior to their arrival at the academy and was readministered during cadet orientation and during the stressful environment of Basic Cadet Training (BCT). Immune responsiveness was analyzed by PHA-, PMA-, or anti-CD3-stimulated thymidine uptake in mononuclear leucocytes. Illness episodes were assessed via medical chart review and self-reported symptoms. There were significant increases in distress levels as cadets entered BCT. No psychosocial measure assessed prior to arrival at the academy predicted level of PHA-, PMA-, and anti-CD3-stimulated thymidine uptake or risk of illness. However, hostility levels reported during BCT predicted risk of illness in the four weeks following psychosocial assessment (odds ratio = 7.1; 95% confidence interval: 1.4-36.1). Elevated response to environmental stressors and lower well-being levels also predicted impending illness, but only in the cohort of cadets who had not contracted food poisoning prior to assessment during BCT (OR = 9.3, CI = 1.9-46.7; OR = 0.09, CI = 0.02-0.53). These results suggest that self-report measures of hostility, response to environmental stressors and well-being may be useful predictors of impending illness episodes in males encountering high stress environments.

Adaptation, Psychological↗

The effects of frontal- or temporal-lobe lesions on susceptibility to interference in spatial memory.

Patients with unilateral frontal- or temporal-lobe lesions and normal control subjects studied multiple arrays of pictures and were tested for recall of the locations of the pictures. One condition consisted of three trials of the same pictures in different spatial arrangements, recall being tested immediately after each presentation. In a second condition (using different stimuli), the subject was given two trials with one set of pictures, but a new set of pictures was viewed on the third trial. All groups showed a build-up of proactive interference across trials using the same pictures, and a release of proactive interference when they studied new pictures. Patients with frontal-lobe lesions were more susceptible to proactive interference than were the other groups.

Adolescent↗

Cholinergic and GABAergic neurons in the nucleus basalis region of young and aged rats.

Antibodies directed against choline acetyltransferase and glutamic acid decarboxylase were used in combination with recently developed stereological techniques to quantify changes in cholinergic, GABAergic, and total neuron number (Nissl-stain) within adjacent tissue sections through the horizontal limb/nucleus basalis in young (3 months, n = 6) and aged (27 months, n = 6) Fischer-344 male rats. Unbiased estimates of total neuron number within these regions were produced using a three-dimensional optical probe, the optical disector, in combination with a systematic random sampling scheme. Estimates of cell counts in immunostained tissue sections were conducted throughout the entire horizontal limb/nucleus basalis region. A significant 30% decrease in both cholinergic and total neuron number was detected in the aged animals; GABAergic neuron number remained unchanged. Total neuron number was significantly correlated with both cholinergic (r = 0.94) and glial cell number (r = 0.63), but not with GABAergic cell number. Based on neuron counts within an individual thick tissue section, the cholinergic neurons comprised only 11-15% of all neurons in the nucleus basalis of young and aged animals. Cholinergic neuron loss accounted for only 20% of the total age-related neuron loss within the horizontal limb/nucleus basalis in Fischer-344 male rats. These results indicate that age-related cholinergic neuron loss within the basal forebrain is reflected in reductions in total neuron number; however, GABAergic neurons, many of which project to the cortex, are unaffected by age. The magnitude of the age-related total neuron loss cannot be entirely accounted for by cholinergic cell loss. Therefore, an unidentified non-cholinergic, non-GABAergic component within the basal forebrain is also lost during aging and may contribute to the cognitive deficits previously ascribed to cholinergic dysfunction.

Aging↗

The production and characterization of murine monoclonal antibodies to human Gadd45 raised against a recombinant protein.

The production of two different murine monoclonal antibodies to human Gadd45, a protein that is induced in response to DNA damage, is reported. Antibodies were generated in a SJL mouse using a recombinant form of the human Gadd45 protein. Monoclonal antibody 4TCYA1, which recognizes the denatured form of human Gadd45 in Western blots, was selected based upon the recognition of Gadd45 induced by functional p53 in the human myeloid leukemia cell line, ML-1. A second monoclonal antibody, designated 30T.14, immunoprecipitates native human Gadd45 in lysates produced from RKO cells, a colorectal carcinoma cell line that expresses relatively high basal levels of Gadd45, as well as from cell lysates made from ML-1 cells after exposure to ionizing irradiation (IR). Since 4TCYA1 fails to immunoprecipitate Gadd45, and 30T.14 fails to bind to IR-induced Gadd45 in immunoblotting, these two monoclonal antibodies probably recognize different epitopes.

Animals↗

The neurochemical and metabolic cascade following brain injury: moving from animal models to man.

Experimental traumatic brain injury produces a series of cellular events contributing to a neurochemical and neurometabolic cascade. This cascade is defined by the release of neurotransmitters resulting in a massive ionic flux, which, consequently, produces an increase in glycolysis. This increase in glycolysis is followed by a metabolic diaschisis, which is related to the degree and extent of behavioral deficits. Clinical efforts have now determined that a similar cascade occurs in human head injury, validating the animal model as well as providing new assessment strategies for the management and treatment of brain injury.

Animals↗

Mechanisms of secondary brain damage in global and focal ischemia: a speculative synthesis.

The objective of this article is to amalgamate previous results into a speculative synthesis that sheds light on the causes of secondary brain damage following either global/forebrain or focal ischemia. The hypothesis is based on the well-founded assumption that the pathophysiology of the brain damage incurred by global or forebrain ischemia is different from that of focal ischemia. In the former, the ischemia is usually dense and of brief duration and, provided that reperfusion is adequate, cell damage is conspicuously delayed, mostly affecting selectively vulnerable neurons. In contrast, focal ischemia is either long-lasting or permanent, and it is usually less severe, particularly in the perifocal penumbral regions. The lesion is typically pan-necrotic ("infarction"), initially affecting the focus supplied by the occluded artery, later invading the penumbra zone. Available results allow a restatement of the calcium hypothesis of cell death. In global or forebrain ischemia, calcium influx through channels gated by voltage or glutamate receptors is envisaged to trigger reactions that limit the survival of neurons during reperfusion, leading to secondary neuronal death after hours or days of survival. It can be hypothesized that the initial insult leads to a sustained alteration of membrane calcium handling, resulting in slow, gradual calcium overload of mitochondria. Alternatively, a sustained perturbation of the intracellular signal transduction pathway leads to changes in transcription or translation, bereaving the cells of heat shock and stress proteins, of trophic factors, or of enzymes required for survival. However, with the possible exception of the gerbil, neither microvascular failure nor primary mitochondrial dysfunction is believed to be involved. In focal ischemia, similar reactions are probably triggered by calcium influx, whether this is sustained (the focus) or intermittent (the penumbra). However, these play a minor role in cell death since they are overridden by reactions producing mediators of rapidly developing secondary damage, affecting either microvessels or mitochondria. Very probably, some of these mediators are free radicals, or nitric oxide, or other reactive metabolites, emanating from lipid hydrolysis and arachidonic acid metabolism. During continuous ischemia, or during recirculation following 1-3 h of ischemia, these mediators activate adhesion molecules in endothelial cells or polymorphonuclear leucocytes, or oxidize key proteins. The result is either failure of microcirculation ("capillary plugging"), or sustained mitochondrial failure. Since calcium influx is an initial event, agents reducing presynaptic depolarization and calcium entry through glutamate receptor-gated and other calcium channels have predictably a narrow therapeutic window; however, since spin trapping agents of the nitrone class act many hours after the induction of focal ischemia, their therapeutic window is potentially very wide. This may be because expression of mRNAs for adhesion molecules and their synthesis are relatively slow processes, and because the nitrones act on events that involve adhesion of leukocytes to the endothelial cells, with plugging of capillaries and postcapillary venules, and on the ensuing inflammatory response.

Animals↗

Simultaneous measurement of one-carbon and polyglutamate derivatives of folic acid in rat liver using enzymatic interconversions of folates followed by ternary complex formation with thymidylate synthetase and 5-fluorodeoxyuridylic acid: standardization of the method.

A sensitive assay is needed for the measurement of individual folate derivatives in samples that contain low concentrations of folate. The ternary complex method for the determination of folylpolyglutamates has been combined with procedures for interconverting folate derivatives to provide a method capable of measuring 28 different folate derivatives in biological samples. The method takes advantage of the properties of the ternary complex formed with thymidylate synthetase, fluorodeoxyuridylic acid and 5,10-methylenetetrahydrofolic acid. In the presence of excess purified thymidylate synthetase and excess [3H]fluorodeoxyuridylic acid, limiting concentrations of folates were converted to 5,10-methylenetetrahydrofolate using purified folate interconverting enzymes. This process separated folate derivatives into four groups: Tetrahydrofolate + 5,10-methylenetetrahydrofolate; dihydrofolate; 5,10-methenyl-, 5-formyl-, and 10-formyltetrahydrofolates and 5-methyltetrahydrofolate. Within groups specificity was good, showing little overlap between folates. Sensitivities to 100 fmol of folate were achievable and to 1 pmol were standard. Recoveries were linear for each of the groups in this system to 50 pmol of folate. Ternary complexes containing different polyglutamates were separated by isoelectric focusing, visualized by fluorography and measured by densitometry. The densitometry was linear with folate concentration in the range 20-200 fmol for each of the polyglutamates. Primary and secondary coefficients of variation were determined. This method provides the sensitivity to measure individual folates in the femtomole range and the flexibility to determine the concentrations of 28 separate pools of folate derivatives, differentiating between derivatives of the pteridine moiety and glutamate chain length simultaneously.

Animals↗

Baseline arterial pressure affects sympathoexcitatory responses to ventricular premature beats.

The seconds to minutes before sudden cardiac death are characterized by fluctuations of arterial pressure, cardiac rhythm, and probably sympathetic nerve activity. We explored the interrelations among these factors in seven patients undergoing clinical electrophysiological testing. We measured muscle sympathetic nerve activity (SNA) and arterial pressure responses to ventricular premature beats induced throughout the cardiac cycle under three conditions: 1) lowered arterial pressure and elevated SNA produced by intravenous nitroprusside, 2) baseline arterial pressure and SNA during saline infusion, and 3) elevated arterial pressure and decreased SNA activity produced by intravenous phenylephrine. Sympathetic responses to premature beats were inversely related to diastolic pressure. The magnitude of the sympathetic response was directly related to the prevailing arterial pressure and inversely related to baseline SNA. These data demonstrate that sympathoexcitation evoked by ventricular dysrhythmias is determined importantly by the prevailing arterial pressure and possibly by the background R-R interval and level of sympathetic activity. This effect may influence hemodynamic and electrophysiological stability during dysrhythmias.

Adult↗

Modulation of human sympathetic periodicity by mild, brief hypoxia and hypercapnia.

We determined the influence of brief mild normocapnic hyperoxia, hypoxia, and hyperoxic hypercapnia on human muscle sympathetic nerve activity and R-R intervals, as quantified by both time- and frequency-domain analyses. We obtained measurements in nine healthy young adult men and women during uncontrolled and frequency (but not tidal volume) controlled breathing. Responses were evaluated with forward selection and backward elimination statistical models, with muscle sympathetic nerve activity as the dependent variable, and power spectral techniques. Hyperoxia and hypoxia did not alter arterial pressure; hypercapnia increased diastolic pressure modestly. Average R-R intervals tended to increase during hyperoxia, and decrease during hypoxia and hypercapnia. During uncontrolled breathing, changes of inspiratory gases exerted only minor effects on muscle sympathetic nerve activity; during controlled breathing, both hypoxia and hypercapnia tended to increase muscle sympathetic nerve activity. Statistical modeling suggested that chemoreceptor stimulation increased muscle sympathetic neural outflows, but that increases of sympathetic traffic were opposed by secondary increases of ventilation. Inspiratory gases modulated the frequency distribution of muscle sympathetic nerve activity strikingly: hypoxia increased sympathetic power at respiratory frequencies and hypercapnia increased sympathetic power at both respiratory and (primarily in one subject) cardiac frequencies. Our data suggest that mild brief hypoxia and hypercapnia increase human muscle sympathetic nerve activity, but that this tendency is opposed by chemoreflex-induced increases of ventilation. Our results suggest also that chemoreceptor activity exerts important influences on the frequency content, as well as the quantity of sympathetic neural outflow.

Adult↗

Genomic instability and the role of p53 mutations in cancer cells.

The p53 tumor-suppressor gene encodes a cell-cycle checkpoint protein that functions in the G1 phase of the cell cycle. When DNA damage is incurred, p53 transactivates a number of downstream genes whose products, with diverse biologic activities, each make a contribution to the cellular response to DNA damage. One major p53-mediated stress response is the G1 cell-cycle arrest, or delay, which probably allows the cell time to repair DNA damage prior to S-phase entry. In cells lacking p53 function, which include most cancer cells, a condition of genomic instability results from checkpoint loss that culminates in gene amplifications, aneuploidy, and other chromosomal aberrations. These abnormalities contribute to the clonal evolution of cancer cells and tumor progression. The role of p53 in radioresistance and chemoresistance is discussed.

Animals↗

Characterization of human Gadd45, a p53-regulated protein.

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.

Animals↗

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen.

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.

Cell Division↗

Structure and function of a mating-type gene from the homothallic species Neurospora africana.

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.

Amino Acid Sequence↗