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Biomedical subjects

S M Smith

Publications and source records attributed to S M Smith.

At least 109 records · Page 6Linked to original sources
PubMed1998

Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory.

Activation of cAMP synthesis by intracellular Ca2+ is thought to be the main mode of cAMP generation in the brain. Accordingly, the Ca2+-activated adenylyl cyclases I and VIII are expressed prominently in forebrain neurons. The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca2+ and that this effect is blocked selectively by inhibitors of calcineurin such as FK506 and cyclosporin A. Moreover, adenylyl cyclase IX is inhibited by the same range of intracellular free Ca2+ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca2+/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Circadian changes of type II adenylyl cyclase mRNA in the rat suprachiasmatic nuclei.

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN.

Adenylyl Cyclases↗
PubMed1998

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons.

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.

Animals↗
PubMed1998

A bidirectional regulation between the TR2/TR4 orphan receptors (TR2/TR4) and the ciliary neurotrophic factor (CNTF) signaling pathway.

Previously, we reported that the nuclear orphan receptor TR4 could induce transcriptional activity via the 5th intron of the ciliary neurotrophic factor (CNTF) alpha receptor gene (CNTFR-I5). Here we show CNTF could increase TR4 expression and enhance the DNA-binding capacity of TR4. Interestingly, the expression of TR2, a close family member of TR4, could also be induced by CNTF. In return, TR2 induced CNTFRalpha transcriptional activity through binding to a direct repeat response element of AGGTCA within CNTFR-I5. The possibility of this mutual influence between TR2 and the CNTF signaling was further strengthened by in situ hybridization. Similar expression patterns of TR2 and CNTFRalpha were observed in most of the developing neural structures such as the ganglia, neural epithelia, spinal cord, and the periventricular areas of brain. Together, our data suggest that an interaction between TR2/TR4 and the CNTF signaling pathway may occur, supporting the hypothesis that TR2/TR4 may play important roles in neurogenesis.

Amino Acid Sequence↗
PubMed1998

Cyclic glucans produced by the intramolecular transglycosylation activity of potato D-enzyme on amylopectin.

Potato D-enzyme catalyses an intramolecular transglycosylation reaction on amylose to produce cycloamylose, a novel cyclic alpha-1, 4 glucan. To determine if a similar activity could be observed with a high molecular weight branched substrate, recombinant potato D-enzyme was incubated with amylopectin. The substrate was converted into two fractions of lower molecular mass. Fraction I comprised 15% cyclic molecules of which the majority contained both alpha-1,4 and alpha-1,6 links. These were shown to be branched molecules with branches shorter than those in amylopectin. Fraction II comprised 80% cyclic molecules of which the majority contained only alpha-1,4 links (cycloamylose). Since fraction II appeared before fraction I, we propose that D-enzyme first catalysed the cyclisation of the outer side chains of amylopectin and then the cyclisation of inner chains to produce branched clusters. These results demonstrate that D-enzyme can catalyse the transfer of branched glucans, and suggest novel ways in which it may participate in starch metabolism in plants.

Amylopectin↗
PubMed1998

Expression of retinol binding protein and transthyretin during early embryogenesis.

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardiac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough [1994] Dev. Dyn. 200:155-162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell explants. Fluorography of proteins from endoderm-conditioned medium revealed 1-2 dozen bands, the most prominent of which migrated at approximately 17 and 25 kD. The bulk of the 17-kD band, which migrates near FGFs and subunits of the transforming growth factor-beta family, was identified by N-terminal sequencing as transthyretin (TTR). A component of the 25-kD band was identified by Western blotting as retinol binding protein (RBP). RT/PCR analysis revealed that mRNAs for both proteins are in the embryo as early as stage 3. In situ hybridization localized these mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, RBP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of RBP by stage 7, followed by sequential increases to stage 25; by contrast, content of RBP in isolated hearts peaked at stage 14, then declined. Immunohistochemistry revealed the presence of RBP protein in the extracellular matrix subjacent to lateral plate endoderm beginning at stage 8; upon formation of the definitive heart, intense staining was observed in the cardiac "jelly." By contrast TTR was intracellular, first detected as subtle deposits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanchnic mesoderm. At stages 11-14, TTR was detected only in myocardial cells. Such localization of RBP and TTR may indicate a role in the transport and distribution of retinol and thyroid hormone, respectively, from yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development.

Amino Acid Sequence↗
PubMed1998

Nutrition, endocrinology, and body composition during space flight.

Space flight induces endocrine changes that perturb metabolism. This altered metabolism affects both the astronauts' body composition and the nutritional requirements necessary to maintain their health. During the last 25 years, a combination of studies conducted on Skylab (the first U.S. space laboratory), U.S. Shuttle flights, and Soviet and Russian flights provides a range of data from which general conclusions about energy and protein requirements can be drawn. We have reviewed the endocrine data from those studies and related it to changes in body composition. From these data it appears that protein and energy intake of astronauts are similar to those on Earth. However, a combination of measures, including exercise, appropriate diet, and, potentially, drugs, is required to provide the muscle health needed for long duration space flight.

Body Composition↗
PubMed1998

Cerebral blood flow is proportional to cardiac index in patients with septic shock.

PURPOSE: In patients with septic shock, the cardiac index is often increased. Maldistribution of blood flow and regional hypoperfusion has been implicated as a key factor in the pathogenesis of organ dysfunction in these patients. We have investigated the relationship between cerebral blood flow and cardiac index in patients with septic shock. MATERIALS AND METHODS: We used Doppler ultrasound techniques to investigate limb and carotid blood flow in 15 patients with septic shock and 9 nonseptic controls. RESULTS: In the nonseptic control patients, common femoral and brachial blood flow were proportional to cardiac index (r=0.73 and 0.76; P=.038 and .017, respectively) reflecting a protective redistribution of flow to more vital organs. However, this relationship was absent in patients with septic shock (r=0.23 and 0.21). Furthermore, in the septic patients but not the nonseptic controls, cerebral blood flow was correlated with the cardiac index (r=0.66, P < .05 vs r=-0.36, NS in nonseptic controls). Carotid flow was independent of mean arterial pressure, PaCO2 and PaO2 in patients with septic shock. CONCLUSIONS: These data are consistent with a loss of autoregulation of cerebral blood flow and a change in the control of limb blood flow in humans with septic shock.

Aged↗
PubMed1998

Esophageal irritation due to alendronate sodium tablets: possible mechanisms.

Animal studies were done using an in vivo dog model to examine the possible mechanism for the esophageal adverse events reported with alendronate sodium tablets. These studies showed that under low pH conditions alendronate sodium can cause esophageal irritation. No esophageal irritation occurred at pH 3.5 or higher where the drug exists primarily as the sodium salt. The animal studies also showed that alendronate sodium can exacerbate preexisting esophageal damage. Exposure of the esophageal mucosa for a prolonged period to alendronate sodium tablet can also cause mild esophageal irritation. These findings suggest that the esophageal irritation in patients taking Fosamax can be from prolonged contact with the tablet, reflux of acidic gastric contents with alendronate sodium, and exacerbation of preexisting esophageal damage. The findings also suggest that other bisphosphonates can cause esophageal injury under similar conditions.

Alendronate↗
PubMed1998

Retinoids and their receptors in vertebrate embryogenesis.

Vitamin A and its derivatives, the retinoids, participate in formation of diverse embryonic structures, including face, heart, eye, limb and nervous system. Studies of retinoid-deficient and -treated embryos, and of receptor null mutants, provide evidence that this participation involves interactions between retinoids and their receptors. Targeted retinoid application and retinoid deficiency, using in ovo avian embryos, has identified early cardiogenic contributions, including cardiocyte gene expression and differentiation, heart tube fusion and laterality, and segmental identity. Also useful is a mammalian model, which targets retinoid deficiency to distinct gestational windows, circumventing limitations of traditional deficiency studies and current null mutant technologies. Rat embryos made deficient in retinoids during gestational d 11.5-13.5 exhibit specific cardiac, limb, ocular and nervous system deficits. That many of the anomalies previously reported in retinoid receptor null mutants are observed in deficiency confirms that ligand-receptor interactions are essential for embryonic development. Other defects are novel, reemphasizing the functional redundancy of retinoid receptors and that retinoid receptors have multiple and overlapping contributions to morphogenesis.

Animals↗
PubMed1998

Natural infection of a household pet red-capped mangabey (Cercocebus torquatus torquatus) with a new simian immunodeficiency virus.

A seroprevalence survey was conducted for simian immunodeficiency virus (SIV) antibody in household pet monkeys in Gabon. Twenty-nine monkeys representing seven species were analyzed. By using human immunodeficiency virus type 2 (HIV-2)/SIVsm, SIVmnd, and SIVagm antigens, one red-capped mangabey (RCM) (Cercocebus torquatus torquatus) was identified as harboring SIV-cross-reactive antibodies. A virus isolate, termed SIVrcm, was subsequently established from this seropositive RCM by cocultivation of its peripheral blood mononuclear cells (PBMC) with PBMC from seronegative humans or RCMs. SIVrcm was also isolated by cocultivation of CD8-depleted RCM PBMC with Molt 4 clone 8 cells but not with CEMx174 cells. The lack of growth in CEMx174 cells distinguished this new SIV from all previously reported sooty mangabey-derived viruses (SIVsm), which grow well in this cell line. SIVrcm was also successfully transmitted (cell free) to human and rhesus PBMC as well as to Molt 4 clone 8 cells. To determine the evolutionary origins of this newly identified virus, subgenomic pol (475 bp) and gag (954 bp) gene fragments were amplified from infected cell culture DNA and sequenced. The position of SIVrcm relative to those of members of the other primate lentivirus lineages was then examined in evolutionary trees constructed from deduced protein sequences. This analysis revealed significantly discordant phylogenetic positions of SIVrcm in the two genomic regions. In trees derived from partial gag sequences, SIVrcm clustered independently from all other HIV and SIV strains, consistent with a new primate lentivirus lineage. However, in trees derived from pol sequences, SIVrcm grouped with the HIV-1/SIVcpz lineage. These findings suggest that the SIVrcm genome is mosaic and possibly is the result of a recombination event involving divergent lentiviruses in the distant past. Further analysis of this and other SIVrcm isolates may shed new light on the origin of HIV-1.

Adaptation, Physiological↗
PubMed1998

The Shc 66 and 46 kD isoforms are differentially downregulated at parturition in the fetal mouse lung.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

3T3 Cells↗
PubMed1998

Collagen cross-link excretion during space flight and bed rest.

Extended exposure to weightlessness results in bone loss. However, little information exists as to the precise nature or time course of this bone loss. Bone resorption results in the release of collagen breakdown products, including N-telopeptide and the pyridinium (PYD) cross-links, pyridinoline and deoxypyridinoline. Urinary pyridinoline and deoxypyridinoline are known to increase during bed rest. We assessed excretion of PYD cross-links and N-telopeptide before, during, and after long (28-day, 59-day, and 84-day) Skylab missions, as well as during short (14-day) and long (119-day) bed-rest studies. During space flight, the urinary cross-link excretion level was twice those observed before flight. Urinary excretion levels of the collagen breakdown products were also 40-50% higher, during short and long bed rest, than before. These results clearly show that the changes in bone metabolism associated with space flight involve increased resorption. The rate of response (i.e. within days to weeks) suggests that alterations in bone metabolism are an early effect of weightlessness. These studies are important for a better understanding of bone metabolism in space crews and in those who are bedridden.

Adult↗
PubMed1998

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos.

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.

Acyl-CoA Dehydrogenase↗
PubMed1998

Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct.

The ability of both acute and chronic ethanol exposures to elicit cell death within specific embryonic and adult tissues is believed to partly underlie ethanol's pathogenicity; however, the mechanism underlying this cell death is unknown. This study partially characterized the mechanism of ethanol-induced neural crest cell death in a chick embryo model of fetal alcohol syndrome. In situ DNA end-labeling demonstrated this cell death was apoptotic and occurred at embryonic ethanol levels as low as 42 mM. Regardless of the initial exposure time, this apoptosis always appeared at a distinct developmental time point simultaneous with the normal deletion of a cranial neural crest subset. This suggested that ethanol might act through aberrant activation of the endogenous death pathway; however, ethanol exposure failed to induce two components of this pathway, the homeotic transcription factor msx-2 and the growth factor bone morphogenetic protein 4. Both endogenous and ethanol-induced death were blocked by local application of an interleukin-1beta converting enzyme/CED-3 protease (caspase) inhibitor, showing that the two paths converge mechanistically and suggesting the potential to prevent this aspect of ethanol's teratogenicity. Ethanol exposure did not significantly alter cell proliferation within neural crest-populated regions, suggesting that susceptibility to ethanol-induced death did not involve exit from the cell cycle. Apoptotic deletion of cranial neural crest could partially explain the craniofacial deficits characteristic of the fetal alcohol syndrome.

Adult↗
PubMed1998

Child fatality review in Georgia: a young system demonstrates its potential for identifying preventable childhood deaths.

BACKGROUND: Child fatality review (CFR) by interagency teams can contribute to the prevention of childhood deaths. We investigated the potential usefulness of Georgia's CFR, legislated in 1990 primarily to prevent death from child maltreatment, for identifying preventable deaths from injury and sudden infant death syndrome (SIDS). METHODS: Using CFR report data and death certificate data, we examined reviewed and nonreviewed childhood deaths in Georgia in 1991 and examined data by etiology, county, risk factors, and preventability. RESULTS: Injury or SIDS caused 33.2% of childhood deaths in Georgia in 1991; CFR reviewed 29.4% of these. Child fatality review was most sensitive for investigating death from intentional injury (40.5%) and SIDS (35.3%). Review teams reassigned the cause of five deaths (2.0%) to child abuse or neglect. County participation was low (31.4%). Overall, 29.0% of deaths were judged preventable. CONCLUSIONS: Georgia's CFR has potential for identifying preventable childhood deaths. Refinements in the system can increase the number and accuracy of death investigations. By participating in the system, physicians may make meaningful contributions to preventing childhood death in their own communities.

Adolescent↗
PubMed1997

An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

Amino Acid Sequence↗