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S K Smith

Publications and source records attributed to S K Smith.

At least 37 records · Page 2Linked to original sources
PubMed2002

Streamlined approach to functional analysis of promoter-region polymorphisms.

We have developed a rapid method for identifying functional promoter-region polymorphisms. Using a modified pGL3 luciferase expression T-vector, we can amplify by PCR, clone, identify allelic pairs of a polymorphic gene promoter region, and prepare plasmids for cell culture 10 promoters (20 allele pairs) per week per researcher. By utilizing 96-well plate technology and an internal control plasmid expressing secreted alkaline phosphatase, each of these allele pairs can be tested for relative promoter activity in each of three cell lines (HEK293t, TE671, and JEG3) with similar resources.

Alleles↗
PubMed2001

Physical properties of erythrocyte ghosts that determine susceptibility to secretory phospholipase A2.

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A(2) (sPLA(2)) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA(2) activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA(2) was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

Erythrocyte Membrane↗
PubMed2001

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Biological Transport↗
PubMed2001

Cross-protection studies with gut membrane glycoprotein antigens from Haemonchus contortus and Teladorsagia circumcincta.

Gel profiles of the peanut and ConA lectin binding integral membrane glycoproteins of Teladorsagia circumcincta and Haemonchus contortus were compared and found to be considerably different. However, some of the Teladorsagia polypeptides were recognized by antisera specific for Haemonchus amino-, metallo- or aspartyl peptidases, enzymes which are known to be protective antigens for that parasite. As expected, an experimental vaccine containing these Haemonchus proteases was extremely effective against homologous challenge, reducing egg and worm counts by more than 99% and 92%, respectively, but it did not provide any useful cross-protection against either T. circumcincta, Trichostrongylus axei or Cooperiaoncophora. A reciprocal experiment, where sheep were immunized with the equivalent glycoproteins from T. circumcincta, showed that, while they were not protected against homologous challenge, there was some cross-protection against Haemonchus as measured by a significant reduction in worm egg output.

Animals↗
PubMed2001

Fetal endocrine signals and preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal axis. Cortisol, derived from the fetal adrenal in cases of intrauterine compromise or from the maternal adrenal in response to stress, or generated locally from cortisone in choriodecidual trophoblasts, provides a crucial link to uterine stimulation. Cortisol contributes to the increased production of prostaglandins (PGs) by fetal membranes and the decidua through the upregulation of PG synthase and the downregulation of PG dehydrogenase enzymes. Cortisol also stimulates placental corticotropin-releasing hormone (CRH) output, although CRH may both relax and stimulate uterine activity depending on the distribution and affinity of its receptor subtypes. Other agents such as cytokines may intercede in this sequence to stimulate PGs and/or CRH, giving rise to a cascade phenomenon that results in preterm birth.

Corticotropin-Releasing Hormone↗
PubMed2001

Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells.

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common disorders of women, including menorrhagia and endometriosis. In pregnancy, failure of the endometrial spiral arterioles to undergo remodeling leads to preeclampsia. Here we report that in addition to vascular endothelial growth factor A (VEGF-A), human endometrium expresses messenger ribonucleic acids (mRNAs) encoding VEGF-C, placenta growth factor (PlGF), the angiopoietins, angiopoietin 1 (Ang1) and Ang2, and the receptors VEGFR-3 (Flt-4), Tie 1, and Tie 2. Levels of VEGF-C, PlGF, and Tie 2 changed during the menstrual cycle. Intense hybridization for VEGF-C and PlGF mRNAs was found in uterine nature killer cells in secretory phase endometrium and for Ang2 mRNA in the same cells in the late secretory phase. Interleukin-2 (IL-2) and IL-15 up-regulated VEGF-C, but not PlGF or Ang2, mRNA levels in isolated NK cells. Conditioned medium from decidual NK cells did not induce human umbilical vein endothelial cell apoptosis. These results indicate that human endometrium expresses a wide range of angiogenic growth factors and that uterine nature killer cells may play an important role in the abnormal endometrial angiogenesis that underlies a range of disorders affecting women.

Angiogenesis Inducing Agents↗
PubMed2001

Distribution of vascular endothelial growth factor (VEGF) and its binding sites at the maternal-fetal interface during gestation in pigs.

Pigs show epitheliochorial placentation, in which the maternal uterine epithelium and the fetal trophectoderm become closely apposed. There is no invasion of trophoblast into the maternal tissue, and nutrient and waste exchange take place across two epithelial layers beneath which a complex network of capillaries forms. Later in gestation, the epithelial cells become indented by blood vessels, which greatly reduces the distance for diffusion between the two circulatory systems. Vascular endothelial growth factor is a secreted homodimeric angiogenic growth factor that is involved in physiological and pathological angiogenesis. Its receptors are generally restricted to endothelial cells. Ligand binding, in situ hybridization and immunohistochemistry were carried out in pig placenta throughout gestation to investigate the possible role of vascular endothelial growth factor and its receptors in non-invasive placentation. In situ hybridization and immunohistochemistry revealed that mRNA and immunoreactivity for vascular endothelial growth factor were localized in both maternal and fetal epithelial cells at the maternal-fetal interface and over the maternal glands, although the signal was generally weaker in the maternal glands. Ligand binding was used to localize for vascular endothelial growth factor receptors; no binding was observed over the maternal glands, but very strong binding was localized to the endometrial blood vessels. At the interface between maternal and fetal tissue, a similar pattern was observed whereby the numerous small capillaries at the bases of the two apposed epithelia bound vascular endothelial growth factor specifically. It is concluded that vascular endothelial growth factor produced by the maternal and fetal epithelial layers promotes the growth of capillaries locally, which would facilitate the development of two vascular networks for the efficient transfer of nutrients and waste products.

Animals↗
PubMed2001

The regulation and localization of angiopoietin-1, -2, and their receptor Tie2 in normal and pathologic human placentae.

BACKGROUND: Angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) act on the endothelial cell Tie-2 receptor to regulate vascular integrity and remodeling. The local balance of these factors and the level of other angiogenic factors determine whether blood vessels grow, are maintained or regress. Profound angiogenesis and vascular remodeling occur in the placenta and this is altered in preeclampsia, a major cause of maternal and fetal morbidity and mortality. MATERIALS AND METHODS: The mRNAs encoding Ang-1, Ang-2, and Tie-2 were detected and localized in human placentae throughout gestation. The mechanism of regulation angiopoietin mRNAs level was determined by explant culture in ambient and reduced oxygen, and in the presence of actinomycin D. RESULTS: In situ hybridization showed that Ang-2 mRNA was abundant in the syncytiotrophoblast in the first trimester of human pregnancy. Ang-1 mRNA could not be detected by in situ hybridization, but was by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting. Placental vascular structure is altered in preeclampsia and intrauterine growth restriction, conditions where feto-placental oxygenation is perturbed. In villous explant cultures, a reduction in oxygen tension significantly raised the levels of Ang-2 mRNA, and this was dependent on transcription. However, similar experiments showed that the stability of the Ang-1 message was greatly reduced under these conditions. Thus, hypoxia has opposite effects on Ang-1 and Ang-2 mRNA levels. Placentae obtained from women with preeclampsia had reduced levels of Ang-2 mRNA compared to gestationally matched controls. There was no difference in the levels of Ang-1 mRNAs. CONCLUSIONS: These data show that the relative levels of Ang-1 and Ang-2 mRNA are regulated by local oxygen tension by different mechanisms and that this may be important during normal human placentation.

Actins↗
PubMed2000

Identification of syntaxin 1A as a novel binding protein for presenilin-1.

Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

Alzheimer Disease↗
PubMed2000

Gonadotrophin-releasing hormone analogues for pain associated with endometriosis.

BACKGROUND: Endometriosis is a common gynaecological condition that frequently presents with the symptom of pain. The precise pathogenesis (mode of development) of endometriosis is unclear but it is evident that endometriosis arises by the dissemination of endometrium to ectopic sites and the subsequent establishment of deposits of ectopic endometrium. The observation that endometriosis is rarely seen in the hypo-oestrogenic (low levels of oestrogen) post-menopausal woman led to the concept of medical treatment by induction of a pseudo-menopause using Gonadotrophin Releasing Hormone Analogues (GnRHas). When administered in a non-pulsatile manner (the pituitary is normally stimulated by pulses of natural GnRH and all analogues act on the pituitary at a constant level) their use results in down regulation (switching off) of the pituitary and a hypogonadotrophic hypogonadal state (low levels of female hormones due to non stimulation of the ovary). OBJECTIVES: To determine the effectiveness of Gonadotrophin Releasing Hormone analogues (GnRHas) in the treatment of the painful symptoms of endometriosis by comparing them with no treatment, placebo, other recognised medical treatments, and surgical interventions. SEARCH STRATEGY: The search strategy of the Menstrual Disorders and Subfertility review group (please see Review Group details) was used to identify all randomised trials of the use of GnRHas for the treatment of the painful symptoms of endometriosis. SELECTION CRITERIA: Trials were included if they were randomised, and considered the effectiveness of GnRHas in the treatment of the painful symptoms of endometriosis. DATA COLLECTION AND ANALYSIS: Twenty-six studies had data appropriate for inclusion in the review. The largest group (15 studies) compared GnRHas with danazol. There are 5 studies comparing GnRHas with GnRHas plus add-back therapy, 3 comparing GnRHa with GnRHa in a different form or dose, one compares them with gestrinone, one with the combined oral contraceptive pill, and one with placebo. Data was extracted independently by two reviewers. The authors of 11 studies have been contacted to clarify missing or unclear data. Only 4 have replied to date. Data on relief of pain, change in revised American Fertility Society (rAFS) scores, and side effects was collected. MAIN RESULTS: No difference was found between GnRHas and any of the other active comparators with respect to pain relief or reduction in endometriotic deposits. The side effect profiles of the different treatments were different, with danazol and gestrinone having more androgenic side effects, while GnRHas tend to produce more hypo-oestrogenic symptoms. REVIEWER'S CONCLUSIONS: There is little or no difference in the effectiveness of GnRHas in comparison with other medical treatments for endometriosis. GnRHas do appear to be an effective treatment. Differences that do exist relate to side effect profiles. Side effects of GnRHas can be ameliorated by the addition of addback therapy.

Danazol↗
PubMed2000

Angiogenic growth factor expression in placenta.

New blood vessel growth is generally a rare event in the healthy adult. However, a notable exception to this is the female reproductive tract where cyclic angiogenesis occurs. Striking new vessel growth and remodeling also occurs during placentation; thus angiogenesis is essential for reproductive success. Vascular endothelial growth factor is a potent stimulator of this process and its production and action is tightly regulated. Indeed the placenta is a rich source of a soluble variant of the flt-1 receptor which seems to protect the placenta from the effects of excess vascular endothelial growth factor. The balance between new vessel growth (in the placental villi for example) and endothelial cell loss in the spiral arteries within the decidua is a delicate one. This is influenced by the local production of promotors and inhibitors of endothelial cell activation. Perturbation of this may lead to maternal pathology during pregnancy.

Angiogenesis Inducing Agents↗
PubMed2000

Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus.

Jacalin lectin was used as a ligand to isolate a fraction containing two distinct protective antigens from detergent extracts of membranes from Haemonchus contortus. The first antigen was identified as a complex which appeared very similar to Haemonchus galactose-containing glycoprotein (H-gal-GP), which is a previously described protective protease complex, except that it was substantially depleted of one of the main H-gal-GP components, a 230 kDa metallopeptidase-containing band. The new complex was termed Haemonchus sialylated galactose-containing glycoprotein (H-sialgal-GP), because it bound to jacalin but not to peanut lectin and only jacalin will bind the sialylated form of galactosyl (beta-1, 3) N-acetylgalactosamine. Two protection trials with sheep showed that H-sialgal-GP and H-gal-GP were equally efficacious, reducing numbers of Haemonchus eggs by between 86% and 93% and worms by between 52% and 75%, respectively. The second jacalin-binding protective antigen fraction was separated from H-sialgal-GP by ion exchange and gel filtration chromatography. It was greatly enriched for two proteins termed p46 and p52 according to their apparent molecular weights. Immunization of sheep with these proteins gave protection values of 78% for eggs and 33% for worms, which are significantly lower than those obtained with either H-gal-GP or H-sialgal-GP. N-terminal amino acid sequence data from p46 and p52 showed that both proteins were closely related to a previously described 45 kDa Haemonchus membrane protein, which had conferred protection against Haemonchus in guinea-pigs.

Amino Acid Sequence↗
PubMed2000

Evaluation of immunization with gut membrane glycoproteins of Ostertagia ostertagi against homologous challenge in calves and against Haemonchus contortus in sheep.

Peanut and ConA lectins were used as ligands to isolate glycoproteins from detergent extracts of adult Ostertagia ostertagi membranes. As judged by their profiles following SDS-PAGE, these fractions closely resembled the equivalents from Haemonchus contortus which are derived from the nematode intestinal cell microvillar membranes and which are highly protective when used as antigens. Groups of calves were immunized with the peanut and ConA binding fractions of Ostertagia, either as separate or pooled antigens mixed with QuilA as adjuvant. All calves, including controls immunized with adjuvant only, were challenged with a single dose of infective Ostertagia larvae and faecal egg counts were monitored for 5 weeks. In two experiments where the antigen fractions were pooled, moderate (30-50%), but statistically significant reductions in egg output were observed, but the number of worms was not diminished. No significant protection was observed in a third trial where groups of calves were immunized with peanut or ConA binding proteins given separately. Two further trials were conducted in sheep immunized with the same Ostertagia fractions but challenged with Haemonchus. Irrespective of whether they were administered separately or together, the Ostertagia antigens cross protected efficiently against Haemonchus reducing egg counts by between 81% and 97% and worm numbers by between 57% and 84%.

Adjuvants, Immunologic↗
PubMed2000

Steroids and endometrial breakthrough bleeding: future directions for research.

Abnormal endometrial bleeding continues to be a significant problem for women using long-acting gestagens and not only reduces the use of these agents, thus restricting the options available to women, but also reduces the quality of their lives. This symposium addressed a range of basic and clinical studies that have tried over the past 3 years to define the problem. Two areas have been identified. The first is the structure of the blood vessels themselves and the second is the environment of the endometrium in relation to these vessels. This reflects a shift in research interest away from factors that simply control epithelial-mesenchyme interactions. The expression of angiogenic inhibitors and stimulators is being defined and the critical role of the matrix and the immunocompetent cells of the endometrium elucidated. The disappointing results of simple oestrogen supplementation were confirmed. Three broad areas of research were considered to be important for future studies. The first of these is not related to molecular and cellular function but rather to understanding better the attitudes of women to vaginal bleeding in the multicultural diverse situations in which this event occurs. It was recognized that a broader constituency was not interested in this problem with the increasing use of continuous combined regimes of hormone replacement therapy in the developed world. Studies that listen to and educate women about this problem are needed to ensure that these techniques are felt to be better owned by the women themselves. Molecular and cellular studies were identified that attempted to define the factors necessary for the development and maintenance of healthy, non-leaky vessels. These included studies of the pro- and anti-angiogenic agents expressed in endometrium and a definition of their interactions with matrix metalloproteinases in maintaining vessel integrity. Finally there was recognition of the need to understand how neutrophils traffic through the endometrium and to determine how they effect endometrial bleeding.

Contraceptive Agents, Female↗