Biomedical subjects
Roger Smith
Publications and source records attributed to Roger Smith.
Corticotrophin releasing hormone and the timing of birth.
Corticotrophin-releasing hormone (CRH) is the hypothalamic peptide that controls the function of the pituitary-adrenal axis in response to stress. CRH is also expressed abundantly in the human placenta and is present in high concentrations in maternal and fetal plasma during late pregnancy. During pregnancy, CRH derived from the placenta is thought to play a crucial role in the regulation of fetal maturation and the timing of delivery, and CRH has also been implicated in the control of fetal-placental blood flow. Elevated CRH concentrations, as compared with gestational age matched controls, occur in patients in preterm labour. The exponential curve depicting the CRH increase is shifted to the left in women who will subsequently deliver preterm and to the right in women who will deliver post dates. This has led to the suggestion that CRH production is linked to a placental clock which determines the length of gestation. Clinically, maternal plasma CRH concentrations may be useful in identifying women at high risk of preterm delivery and CRH antagonists may be useful in preventing preterm labour. As significant CRH production by the placenta is restricted to primates, future research must take into account the species specificity of the mechanisms regulating parturition. A number of significant gaps remain in our knowledge of the function of this peptide in pregnancy. This review examines the current evidence regarding the role of CRH in human parturition.
Differential enrichment of high- and low-molecular weight proteins and concurrent RNA extraction.
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Neonatal neurodevelopmental outcomes following tocolysis with glycerol trinitrate patches.
OBJECTIVE: The object of this study was to determine the effects of maternal tocolysis with glycerol trinitrate (GTN) patches on the neurodevelopment of infants. STUDY DESIGN: This was a randomized, multicenter, controlled trial comparing the efficacy of GTN patches with standard beta2 agonist as tocolytic therapy. The previously reported outcomes of this study indicated no difference in neonatal mortality or morbidity to hospital discharge. One hundred fifty-six surviving infants from 2 Australian centers were psychometrically assessed using the Griffiths Mental development Scales (revised) at 18 months of age. RESULTS: There was no difference in psychometric performance between those infants enrolled in either the GTN (81 infants) or beta2 agonist (75 infants) arm of the study. CONCLUSION: This randomized trial supports no significant difference between GTN patches in comparison with standard beta2 agonist for tocolytic therapy. The results underscore the association between premature labor and adverse infant outcomes.
A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva.
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G --> A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.
Endocrine regulation of human fetal growth: the role of the mother, placenta, and fetus.
The environment in which the fetus develops is critical for its survival and long-term health. The regulation of normal human fetal growth involves many multidirectional interactions between the mother, placenta, and fetus. The mother supplies nutrients and oxygen to the fetus via the placenta. The fetus influences the provision of maternal nutrients via the placental production of hormones that regulate maternal metabolism. The placenta is the site of exchange between mother and fetus and regulates fetal growth via the production and metabolism of growth-regulating hormones such as IGFs and glucocorticoids. Adequate trophoblast invasion in early pregnancy and increased uteroplacental blood flow ensure sufficient growth of the uterus, placenta, and fetus. The placenta may respond to fetal endocrine signals to increase transport of maternal nutrients by growth of the placenta, by activation of transport systems, and by production of placental hormones to influence maternal physiology and even behavior. There are consequences of poor fetal growth both in the short term and long term, in the form of increased mortality and morbidity. Endocrine regulation of fetal growth involves interactions between the mother, placenta, and fetus, and these effects may program long-term physiology.
Pattern of maternal serum corticotropin-releasing hormone concentration during pregnancy in the common marmoset (Callithrix jacchus).
Corticotropin-releasing hormone (CRH), a potent neuropeptide, is produced by the placenta of anthropoid primates. No other mammals, including prosimian primates, are known to produce placental CRH. In humans, placental CRH appears to play an important role in the progression of pregnancy to parturition. Maternal circulating CRH begins to rise early in pregnancy and increases until parturition. Gorillas and chimpanzees share this pattern of increasing maternal CRH during pregnancy with humans. In humans, chimpanzees, and gorillas, maternal CRH and estradiol concentrations are correlated, consistent with the hypothesis that CRH is involved in the biosynthetic pathway for placental estrogen production. In contrast, in baboons, maternal circulating CRH rises precipitously early in pregnancy and then declines, though CRH is detectable until birth. This research was designed to investigate the pattern of maternal circulating CRH in the common marmoset during pregnancy. Blood samples were taken across gestation from nine subjects over 11 pregnancies, and the plasma was assayed for CRH. The pattern of maternal circulating CRH in the common marmoset was similar to that of the baboon, with a rapid rise starting at about 50 days postconception and a peak at approximately 70 days postconception. By 110 days postconception, CRH concentration had plateaued at a significantly lower value. The peak and mean values for CRH were associated with fetal number (e.g., females gestating triplets had higher values than females gestating twins). Urinary estradiol showed no association with plasma CRH concentration. Marmosets appear to differ from the great apes in this regard, and to share a pattern of maternal CRH during pregnancy with the baboon, indicating that the baboon and marmoset pattern may be ancestral. The function of the early rapid rise of CRH in baboons and marmosets, and the significance of this difference between monkeys and apes, are not known.
Cellular effects induced by the antitumor agent azinomycin B.
Studies on the mechanism of action of the antitumor agent azinomycin B in vitro suggest that the drug elicits its lethal effects by the formation of interstrand crosslinks within the major groove of DNA. Here, we demonstrate the biological effects of the drug in vivo. Fluorescence imaging revealed localization of azinomycin B in the nuclear region of yeast. Moreover, experiments with oligonucleotide microarrays examined the effects of the drug across the yeast transcriptome. The results demonstrated a robust DNA damage response that supports the proposed role of the drug as a covalent DNA modifying agent. RT-PCR analysis validated the gene changes, and flow cytometry of azinomycin-treated yeast cells demonstrated a phenotypic S phase shift consistent with transcriptional effects.
Proteomic study of plasma proteins in pregnant women with asthma.
OBJECTIVE AND BACKGROUND: The course of asthma may be altered during pregnancy with at least one-third of women experiencing a worsening of asthma and 20% having an exacerbation during pregnancy. This study used the novel proteomic technique, surface-enhanced laser desorption ionization-time of flight mass spectrometry to determine if the presence of asthma during pregnancy was associated with alterations in plasma proteins. METHODS: Plasma collected from healthy (n = 23) and asthmatic (n = 27) pregnant women at 18 and 30 weeks gestation was applied to strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using Ciphergen Protein Biology System IIc and significant differences in individual peak intensities between groups determined. RESULTS: At 18 weeks gestation, 91 peaks were significantly different between pregnant women with and without asthma, representing 28% of the total peaks identified. At 30 weeks gestation, 51 peaks were significantly different. There were two peaks that were significantly different between groups at both 18 and 30 weeks gestation and expressed at a similar level at both time points. One was increased in asthmatics (MW = 6444 Da) whereas the other decreased in asthmatics compared with non-asthmatic women (MW = 1846 Da). CONCLUSIONS: This study demonstrated that there are differences in protein patterns between pregnant women with and without asthma. Other techniques are needed to define the molecular species and classify pathophysiological significance. Surface-enhanced laser desorption ionization-time of flight mass spectrometry has potential as a tool to monitor disease progression in situations such as pregnancy.
Identification of a family of DNA-binding proteins with homology to RNA splicing factors.
We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.
A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide.
A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (C(T)) shift 0.1 cycles greater than PCR tube controls. Experiments with HSV-1-positive cerebrospinal fluid (CSF) examined the extraction, amplification, and detection properties of the AOM tubes compared to the Qiagen DNA blood mini kit. The AOM extraction, amplification, and detection of HSV-1 in CSF displayed differences of less than one C(T) when compared to Qiagen-extracted samples. Experiments testing the AOM method using clinical CSF samples displayed 100% concordance with reported results. AOM tubes have no adverse effects on amplification or fluorescence acquisition by real-time PCR and can be effectively used for the extraction, amplification, and detection of HSV from CSF. The AOM single tube method is a fast, reliable, and reproducible technique for the extraction, amplification, and detection of HSV in CSF.
Determination of steroids in human plasma using temperature-dependent inclusion chromatography for metabolomic investigations.
Clinical and metabolomic investigations of complex human fluids require cost-effective methodologies that can rapidly assess the steroid hormone milieu of individual samples. The efficiency of quantification of many steroids is limited using immunoassays as these methods can only measure a single component of biological samples and are dependent upon the specificity of the antiserum used in the protocol. In this study, we optimised the solid-phase extraction protocol for the extraction of a range of steroids of varied polarity from estetrol to progesterone from human plasma. The final SPE procedure for efficient extraction of steroids was a washing mixture of 5 ml of 30% methanol and an elution solvent of 2 ml of 100% methanol using 0.5 g C-18 cartridges. This protocol resulted in a high recovery rate, ranging from 85.2 to 99.9% for both the internal standard (7,8-dimethoxyflavone) and steroids of interest. We also improved the separation methodology of our previous work using temperature dependent inclusion chromatography with a mobile phase composition of 35% acetonitrile and 12 mM of beta-cyclodextrin at 29 degrees C. Under these conditions most of the fluid components including estetrol were detected in the first 10 min with progesterone appearing at 43 min. This method is simplistic, inexpensive and reproducible with the capabilities of accurate quantification of steroids. Therefore it could have numerous clinical and metabolomic applications.
The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes.
Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4+ and CD8+ T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4+ and CD8+ T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.
Dietary eicosapentaenoic acid modulates CTLA-4 expression in murine CD4+ T-cells.
We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.
Quantifiable change in functional brain response to empathic and forgivability judgments with resolution of posttraumatic stress disorder.
Previous functional neuroimaging studies of posttraumatic stress disorder (PTSD) have mainly focused on symptom-provocation paradigms in combat-related PTSD. We sought to elucidate the effect of non-combat-related PTSD on the physiology of social cognition. Thirteen patients with PTSD underwent functional magnetic resonance imaging (fMRI) while they engaged in tasks that (i) involve speculation on another's intention, (ii) invoke empathy and (iii) involve making judgments of the forgivability of others' actions; each versus 'baseline' social reasoning judgments. A post-therapy fMRI scan followed a course of modified cognitive behavioural therapy. Post-therapy, we found increased activation in brain regions predicted on the basis of foregoing work in healthy subjects. These included significant left middle temporal gyrus activation in post-therapy response to empathy judgments and posterior cingulate gyrus activation in post-therapy response to forgivability judgments. The specific regions of the human brain activated by empathy and forgivability judgments changed with symptom resolution in PTSD. Time and therapy are likely contributory factors that lead to a degree of 'normalisation' of the neural response to these social cognition tasks.
Molecular dynamics simulations of nanoindentation and nanotribology.
We present results of parallel molecular dynamics simulations of nanoindentation and nanotribology experiments. The models we have developed describe both the sample and the indenter atomistically and model the effect of the cantilevers in an atomic force microscope through the use of springs. We show that the simulations are in good qualitative agreement with experiment and help to elucidate many of the mechanisms that take place during these processes. In particular, we illustrate the role that dislocations play both in nanoindentation and also in stick-slip. Further to this we show how real-time visualization and computational steering have been employed in these simulations to capture the dynamical events that take place.
Inflammatory aetiology of human myometrial activation tested using directed graphs.
There are three main hypotheses for the activation of the human uterus at labour: functional progesterone withdrawal, inflammatory stimulation, and oxytocin receptor activation. To test these alternatives we have taken information and data from the literature to develop causal pathway models for the activation of human myometrium. The data provided quantitative RT-PCR results on key genes from samples taken before and during labour. Principal component analysis showed that pre-labour samples form a homogenous group compared to those during labour. We therefore modelled the alternative causal pathways in non-laboring samples using directed graphs and statistically compared the likelihood of the different models using structural equations and D-separation approaches. Using the computer program LISREL, inflammatory activation as a primary event was highly consistent with the data (p = 0.925), progesterone withdrawal, as a primary event, is plausible (p = 0.499), yet comparatively unlikely, oxytocin receptor mediated initiation is less compatible with the data (p = 0.091). DGraph, a software program that creates directed graphs, produced similar results (p= 0.684, p= 0.280, and p = 0.04, respectively). This outcome supports an inflammatory aetiology for human labour. Our results demonstrate the value of directed graphs in determining the likelihood of causal relationships in biology in situations where experiments are not possible.
Abrupt and gradual extinction among Late Permian land vertebrates in the Karoo basin, South Africa.
The Karoo basin of South Africa exposes a succession of Upper Permian to Lower Triassic terrestrial strata containing abundant terrestrial vertebrate fossils. Paleomagnetic/magnetostratigraphic and carbon-isotope data allow sections to be correlated across the basin. With this stratigraphy, the vertebrate fossil data show a gradual extinction in the Upper Permian punctuated by an enhanced extinction pulse at the Permian-Triassic boundary interval, particularly among the dicynodont therapsids, coinciding with negative carbon-isotope anomalies.