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Biomedical subjects

G W Smith

Publications and source records attributed to G W Smith.

At least 73 records · Page 4Linked to original sources
PubMed1995

Detection of maternal cell contamination in amniotic fluid cell cultures using fluorescent labelled microsatellites.

A rapid PCR based assay was used to ascertain the presence of maternal cell contamination (MCC) in amniotic fluid cell cultures and to exclude MCC in cases where cytogenetic analysis was possible only from one primary cell culture. Six 6-carboxyfluorescein (FAM) and three 6-carboxyfluorescein hexachloride (HEX) labelled primer sets were used to amplify two tetra- and seven dinucleotide repeat polymorphisms. The PCR amplifications were multiplexed in (three) three primer set reactions and visualised on an Applied Biosystems 373A sequencer running Genescan 672 software. The microsatellite products obtained from 200 amniotic fluid cell cultures where the karyotype was female were compared against corresponding maternal blood PCR products. A single case of MCC was detected indicating the usefulness of such assays. We suggest that screening for MCC should be considered in instances where the amniotic fluid sample is bloodstained or was obtained with difficulty, or where the karyotype is female and chromosome analysis is not possible from more than one primary cell culture.

Amniocentesis↗
PubMed1995

Expression of messenger ribonucleic acid encoding tissue inhibitor of metalloproteinases-2 within ovine follicles and corpora lutea.

Metalloproteinase inhibitors, such as tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2), play a key role in the regulation of metalloproteinases that modify extracellular matrix composition. Although expression of TIMP-1 within ovarian tissues has been well characterized, little information is available regarding expression of TIMP-2. The objective of the present studies was to characterize the ontogeny and localization of TIMP-2 messenger RNA (mRNA) within ovine preovulatory follicles and luteal tissue. Total cellular RNA was isolated from preovulatory follicles collected before (presurge; n = 3), or 12-14 h after (post surge; n = 4) an LHRH-induced gonadotropin surge, and from luteal tissue collected on days 3, 7, 10, 13, and 16 post estrus (n = 5, 5, 4, 5, and 5, respectively). TIMP-2 mRNA was expressed by both presurge and postsurge follicles, and expression did not increase after the gonadotropin surge (P = 0.44). In situ hybridization localized TIMP-2 mRNA primarily to the thecal layer of post-surge follicles (n = 3). TIMP-2 mRNA was also localized in a heterogeneous distribution within corpora lutea collected on days 3 and 10 post estrus (n = 3 each). Concentrations of TIMP-2 mRNA (picograms per microgram tissue DNA) were greater in corpora lutea collected during the early luteal phase (days 3 and 7) than the late luteal phase (day 16; P < 0.05). TIMP-2 mRNA was detected in purified populations of both small (n = 4) and large (n = 3) luteal cells, and mRNA concentrations (picograms per microgram DNA) were greater in the large luteal cells (P < or = 0.0002). In addition, immunoreactive TIMP-2 (approximately 21,000 M(r)) was detected by Western blot analysis of ovine luteal cell secreted proteins. We conclude that 1) TIMP-2 mRNA is expressed by the thecal layer of ovine preovulatory follicles and expression is not increased by the preovulatory gonadotropin surge; 2) expression of TIMP-2 mRNA is maximal during the early luteal phase; and 3) expression of TIMP-2 mRNA is greatest in large luteal cells.

Animals↗
PubMed1995

Expression of messenger ribonucleic acid encoding cytochrome P450 side-chain cleavage, cytochrome p450 17 alpha-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave.

The objective of the present study was to investigate changes in the expression of messenger RNAs (mRNAs) encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) at different stages of the first follicular wave of the bovine estrous cycle. Groups of heifers (three to five per group) were ovariectomized on the day of initiation of the first follicular wave (as determined by ultrasonography, day 0) or on days 2, 4, 6, 8, and 10 after initiation of the first follicular wave following estrus. Expression of mRNAs encoding P450scc, P450c17, and P450arom was detected by in situ hybridization and quantified by image analysis. P450scc mRNA was localized to theca interna cells of large preantral follicles and also to granulosa cells of follicles 4 mm or greater in diameter. mRNA for P450c17 was localized exclusively to theca interna cells, whereas P450arom mRNA was localized to granulosa cells of follicles 4 mm or greater in diameter. There were changes in mRNA levels for all three enzymes in thecal and/or granulosa cells at different times of the first follicular wave. Before identification of the dominant follicle (i.e. on days 0 and 2), there was no change in expression of P450scc and P450c17 mRNAs, whereas expression of P450arom mRNA was higher on day 2 than on day 0. Maximal mRNA levels for all three enzymes were observed on day 4. By day 6, P450scc and P450c17 mRNA levels were reduced compared to those on day 4, whereas P450arom mRNA levels remained elevated. On day 8, mRNA levels for all three enzymes were reduced. After initiation of the second follicular wave (day 10), dominant follicles from the first wave were at an advanced stage of atresia. P450scc and P450arom mRNAs were undetectable in granulosa cells, and very low levels of P450scc and P450c17 mRNAs were observed in theca interna cells. Before identification of the dominant follicle, mRNA levels for all three enzymes were similar within a cohort of follicles. Therefore, expression of these enzymes may not be associated with the mechanism of selection of the dominant follicle during a follicular wave.

Animals↗
PubMed1995

Regulation and regulatory role of proteinase inhibitors.

Excess or ill-timed proteolytic events have, of necessity, to be restrained. Therefore, there are partner proteinase inhibitors to most of the known mammalian enzymes that cleave peptide bonds, a situation that implicates the inhibitors in a myriad of normal as well as pathological processes. Here, what is currently known about the regulatory functions, the mode of action, and the control of gene expression of the commoner extracellularly acting proteinase inhibitors is reviewed. The review covers four families of serine proteinase inhibitors: the serpins, the Kunitz, Kazal, and leukoproteinase inhibitors, which overlap considerably in the range of their specificities but differ considerably in their respective structures and likely functions. There then follows a description of the growing family of metalloproteinase inhibitors (usually known by the acronym TIMP), the structurally varied cysteine proteinase inhibitors, and finally alpha 2-macroglobulin, a large multisubunit inhibitor, that is relatively catholic in its range of targets. The review concludes with two short sections, the first an overview of the acute phase response, which features several proteinase inhibitors from the different families discussed earlier, and the second, our prediction of where this research field is headed over the next decade.

Animals↗
PubMed1995

Physiological tests to predict long-term outcome of total abdominal colectomy for intractable constipation.

OBJECTIVE: Total abdominal colectomy (TAC) for intractable constipation has a variable reported success rate that decreases to 50% beyond 2 yr. We hypothesize that this inconsistent outcome can be explained by a more extensive intestinal involvement in some patients. DESIGN: A consecutive sample of patients with intractable constipation had preoperative evaluations that included both upper and lower GI studies. Stool frequency, constipation, diarrhea, abdominal pain, and laxative or enema requirements were compared before and after operation. The study took place in an academic referral center and included 37 consecutive referred patients with severe intractable constipation and colonic dysmotility documented by radiopaque marker studies. INTERVENTIONS: TAC, with ileoproctostomy in 34 patients and ileostomy in three. MAIN OUTCOME MEASURES: Patients with motility abnormalities only of the lower GI tract were diagnosed as having colonic inertia (CI). Those with motility disorders of both the upper and the lower GI tracts were considered to have generalized intestinal dysmotility (GID) with colon predominance. RESULTS: Twenty-one patients had CI, and 16 had GID. Ninety percent of CI patients undergoing TAC had a successful outcome with a mean of 23 bowel movements (BMs)/wk at a mean follow-up of 7.5 yr. Although 88% of GID patients had initial improvement, with a mean of 19 BMs/wk at 6 months, only 13% had prolonged relief. After 2 yr, nine of the GID patients had recurrent constipation, and three had severe diarrhea. CONCLUSIONS: This study has identified two distinct types of colonic dysmotility, CI and GID. It has demonstrated the long-term success of TAC for CI and the importance of upper GI physiological studies to identify colon-predominant GID, which has a poor long-term response to TAC.

Adult↗
PubMed1994

Molecular cloning of an ovine ovarian tissue inhibitor of metalloproteinases: ontogeny of messenger ribonucleic acid expression and in situ localization within preovulatory follicles and luteal tissue.

Secretion of a tissue inhibitor of metalloproteinases (TIMP-1) is initiated by ovine preovulatory follicles after the gonadotropin surge. In addition, TIMP-1 is a major secretory product of ovine corpora lutea. We have isolated an approximately full-length cDNA clone for TIMP-1 from an ovine luteal cDNA library. The 887-basepair cDNA obtained was 95%, 86%, and 77% identical to the reported nucleotide sequences of bovine, human, and mouse TIMP-1 cDNAs, respectively. Total cellular RNA was isolated from preovulatory follicles collected before (presurge; n = 5) or 12-14 h after (postsurge; n = 4) a LHRH-induced gonadotropin surge (36 h after prostaglandin F2 alpha-induced luteolysis); from luteal tissue collected on days 3, 7, 10, 13, and 16 postestrus (n = 5, 5, 4, 5, and 5, respectively); and from purified populations of small (n = 4) and large (n = 3) luteal cells. Concentrations of TIMP-1 mRNA (picograms per micrograms tissue DNA) were increased in preovulatory follicles after exposure to a gonadotropin surge (P < or = 0.01). TIMP-1 mRNA was localized primarily to the granulosa layer of postsurge follicles by in situ hybridization. Concentrations of TIMP-1 mRNA in luteal tissue did not differ throughout the luteal phase (P = 0.07). However, TIMP-1 mRNA was localized predominantly to specific cells located in the connective tissue surrounding and within day 3 corpora lutea. In situ hybridization of day 10 corpora lutea localized TIMP-1 mRNA predominantly to specific cells that were randomly dispersed throughout the luteal tissue. TIMP-1 mRNA was expressed by purified populations of both small and large luteal cells collected from day 10 corpora lutea. Concentrations of TIMP-1 mRNA (picograms per micrograms DNA) were greater in the large luteal cell populations (P < or = 0.0001). We conclude that 1) expression of TIMP-1 mRNA by the granulosa layer of ovine preovulatory follicles increased after the gonadotropin surge, whereas TIMP-1 mRNA concentrations during the luteal phase remained constant; 2) during the luteal phase, TIMP-1 mRNA was localized to specific cells surrounding (day 3) or located within (day 10) the corpus luteum; and 3) expression of TIMP-1 mRNA was greatest in large luteal cells.

Amino Acid Sequence↗
PubMed1994

Pattern of protein production by bovine corpora lutea during luteolysis and characterization of expression of two major secretory products of regressing corpora lutea.

Although the decrease of progesterone in serum and in luteal tissue during luteal regression is well characterized, relatively little is known about changes in proteins produced by the corpus luteum during this time. The first objective was to examine changes in patterns of protein secretion that might be associated with functional and structural luteal regression. The second objective was to characterize the expression of two major secretory products of regressing corpora lutea. Thirty normally cyclic heifers were randomly assigned at day 15-16 of the oestrous cycle (oestrus = day 0) to be ovariectomized at 0 h (no PGF2 alpha; n = 5) or at 4, 8, 12, 24 or 48 h after PGF2 alpha-induced luteal regression (n = 5 per time point). Total cellular RNA was isolated from tissue frozen at the time of ovariectomy. Thin slices (< 1 mm) of tissue were placed in methionine-deficient minimum essential media with [35S]methionine and placed in a humidified CO2 incubator at 38 degrees C. Media and tissues were collected 6 h later. Changes in profiles of secreted proteins were analysed by one-dimensional SDS-PAGE. A number of proteins (relative molecular mass ranging from 14,300 to 200,000) were produced by luteal tissue at each time point (0-48 h). The major secretory proteins had relative molecular masses of approximately 21,500, 28,200, 43,700 and 46,000. Secretion of the relative molecular mass 46,000 protein(s) increased (P < 0.05) between 4 and 24 h after PGF2 alpha injection compared with the 0 h group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Production of tissue inhibitor of metalloproteinases-1 by porcine follicular and luteal cells.

The objectives of the first experiment were to characterize the pattern of protein secretion by 1) porcine follicles before and after the preovulatory gonadotropin surge and 2) corpora lutea on d 4 and 11 after estrus (d 0 = estrus). Gilts were ovariectomized 1) on d 20 after estrus and before the preovulatory gonadotropin surge (pre-estrus group; n = 3), 2) 18 to 36 h after the onset of estrus and after the preovulatory gonadotropin surge (estrus group; n = 3), 3) on d 4 after estrus (n = 3), and 4) on d 11 after estrus (n = 4). Changes in pattern of protein secretion were determined by densitometric scanning of fluorographs. In the pre-estrus group, the major proteins secreted by follicular shells (FS) and granulosa cells (GC) had relative molecular masses (M(r)) of approximately 40,000, 46,000, and 55,000. In the estrus group (FS and GC), secretion of a M(r) 40,000 protein was decreased (P < .05) and secretion of a M(r) 30,000 protein was increased (P < .05) relative to the pre-estrus group. A predominant M(r) 30,000 protein was also secreted by corpora lutea on d 4 and 11. The objective of Exp. 2 was to determine whether synthesis of the M(r) 30,000 protein could be increased during the follicular phase by treatment with hCG, suggesting a role for the preovulatory gonadotropin surge in altering the pattern of protein secretion. Gilts were injected (i.m.) with saline (n = 3) or 500 IU of hCG (n = 3) on d 18 of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Mechanisms associated with corpus luteum development.

The transition of a preovulatory follicle into a corpus luteum is a complex process involving mechanisms similar to wound healing and tumor formation. The objective of this review is to focus on mechanisms associated with corpus luteum development with specific attention to the follicular lineage of luteal cells, mechanisms associated with luteinization, and neovascular changes during luteal development. Corpora lutea are a continuation of follicular maturation and form from granulosal and theca interna cells. There is morphological and immunological evidence in ruminant species for the differentiation of granulosal and theca interna cells into large and small steroidogenic luteal cells, respectively. Different morphological, physiological, and biochemical characteristics of large and small luteal cells may reflect different follicular lineages with separate embryological origins. Following the preovulatory gonadotropin surge, follicular cells begin morphological, endocrinological, and biochemical changes associated with luteinization. Luteinization involves the transition of a preovulatory follicle into a highly vascular corpus luteum capable of secreting large quantities of progesterone. In addition, various cell types undergo hyperplasia, hypertrophy, and(or) migration during corpus luteum formation. An essential component of corpus luteum development is the recruitment of a blood supply. The development of a new microcirculatory bed involves breakdown of the follicular basement membrane, endothelial cell migration, endothelial cell proliferation, and development of capillary lumina. This process is regulated by the interaction of angiogenic and antiangiogenic substances. Further clarification of the preceding mechanisms may result in the development of improved methodologies for controlling the time of ovulation and(or) increasing pregnancy rates.

Animals↗
PubMed1994

Cardiovascular and renal hemodynamic effects of A-68930 in the conscious dog: a comparison with fenoldopam.

The renal hemodynamic and cardiovascular effects of the novel dopamine (DA) D1 receptor agonist A-68930 (5-6-dihydroxy-3-phenyl-1-aminomethyl- isochroman hydrochloride) were studied in the chronically instrumented conscious dog. Intravenous infusion of A-68930, fenoldopam or DA produced a fall in renal vascular resistance and a rise in renal blood flow (RBF), with hypotension and tachycardia. Both compounds were more potent than DA at increasing RBF. By the i.v. route, A-68930 was calculated to be 12 times more potent than fenoldopam. At the end of a submaximal infusion, the effects of A-68930 on RBF declined with a half-time of 14.8 +/- 2.5 min, significantly longer than that of fenoldopam (2.9 +/- 0.5 min) or DA (1.4 +/- 0.4 min). After p.o. dosing, A-68930 was calculated to be 84 times more potent than fenoldopam at increasing RBF. Furthermore, the A-68930-induced increase in RBF took longer to return to base line (5-6 hr) than after an equieffective p.o. dose of fenoldopam (1-2 hr). The effects of p.o. A-68930 were blocked by the DA1 antagonist, SCH 23390 [7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- tetrahydro-(1H)-3-benzazepine hydrochloride], consistent with an action at the DA1 receptor. These data indicate that A-68930 is a potent, p.o.-active DA1 agonist in the conscious dog with a longer duration of action than fenoldopam.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1993

99mTc-MDP scintigraphy in ankylosing spondylitis.

99mTechnetium-MDP bone scintigrams in 11 patients with ankylosing spondylitis were reviewed. Increased activity in sacroiliac joints was present in five of 11 cases, all of whom had symptoms of less than 5 years duration. Patients with longstanding disease had normal or low sacroiliac joint activity. In the spine, appearances included diffuse symmetrical, unifocal or multifocal asymmetrical increased uptake involving the costovertebral, costotransverse and facet joints as well as the spinous processes. In advanced disease with extensive ankylosis, the lumbar spine was featureless on scintigraphy, except for focal increased activity at the site of previous fracture in one patient. Of six available views of the sternum, increased uptake was present in five at the manubriosternal joint and five at the sternoclavicular joints. Increased peripheral uptake was mainly in the hips and knees in advanced cases. Plain radiographic changes correlated poorly with scintigraphic changes, scintigraphy detecting considerably more lesions than radiography. Awareness of the scintigraphic appearances of ankylosing spondylitis may lead to diagnosis before the development of radiographic changes and avoid confusion with other pathology. Clinical indications for bone scintigraphy in ankylosing spondylitis are suggested.

Adult↗
PubMed1993

Intraoperative fluid management.

The surgeon should be cognizant of both the intraoperative and postoperative consequences of intraoperative fluid administration. Optimal fluid management should take into consideration the patient's overall condition and should not be based solely on the cardiovascular response to volume loading. The selection of a particular fluid for resuscitation solution should be tailored to the patient's individual situation. No single fluid preparation will be appropriate for all clinical situations. Crystalloids, colloids, and hypertonic saline solutions have all been shown to be effective in restoring intravascular volume. Each has its own relative advantages and disadvantages and will be appropriate in differing situations. Colloid preparations should not be avoided for fear of inducing pulmonary edema, and the use of hypertonic solutions should not be precluded by fear of potential metabolic complications. The judicious use of both hypertonic solutions and colloids is safe. For the vast majority of routine surgical cases, where the patient is hemodynamically stable and postoperative fluid overloading is not a significant problem, isotonic crystalloids such as lactated Ringer's are both sufficient and cost effective.

Colloids↗
PubMed1993

Identification of a 30,000 M(r) polypeptide secreted by cultured ovine granulosa cells and luteal tissue as a tissue inhibitor of metalloproteinases.

Previous studies have revealed that the gonadotropin surge initiates, via transcriptional mechanisms, synthesis and secretion of a 30,000 M(r) polypeptide by the granulosa cells of ovine preovulatory follicles. This polypeptide also appears to be secreted by luteal tissue and has been tentatively identified as a tissue inhibitor of metalloproteinases (TIMP; 68% NH2-terminal amino acid sequence identity to a human TIMP). Therefore, the objective of the present study was to confirm that TIMP is produced by granulosa cells and luteal tissue in the ewe. A series of experiments was conducted to determine whether the 30,000 M(r) polypeptide secreted by granulosa cells and luteal cells is similar to TIMP in biochemical properties (degree of N-linked glycosylation), in biological activity (as ascertained by gelatin zymographic analysis), and in immunoreactivity (as ascertained by Western blot analysis). Incubation of granulosa cells or luteal cells with tunicamycin revealed that the 30,000 M(r) polypeptide is glycosylated. The form lacking N-linked chains had an M(r) (approximately 20,000) similar to that of the unglycosylated form of TIMP in other species. Gelatin zymographic analysis detected significant metalloproteinase inhibitor activity associated with polypeptides of M(r) approximately 21,000 and 30,000 secreted by granulosa cells and luteal cells. Northern hybridization of granulosa cell RNA and Day 10 luteal RNA with an anti-sense TIMP oligonucleotide probe detected an approximately 900-base transcript, which is similar in size to that reported for TIMP mRNA in other species. Finally, Western blot analysis with a rabbit anti-human TIMP antiserum detected immunoreactive polypeptides of M(r) 30,000 secreted by granulosa cells and luteal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effect of bacterial products including endotoxin on neutrophil function in infected urine.

Experiments were performed to determine the effects of products of bacterial growth (including endotoxin) on phagocytosis and intracellular killing by polymorphonuclear leucocytes (PMNL) in urine. Bacteriologically filtered supernates of two strains of Escherichia coli grown in urine were added in varying amounts to mixtures of PMNL and E. coli, also in urine. Phagocytosis of the two strains was reduced from > 90% in controls to 66% and 48%, respectively, in the presence of undiluted culture filtrate (containing endotoxin 2-2.5 micrograms/ml). Intracellular killing was also decreased and was abolished by dilutions corresponding to endotoxin concentrations of 0.6 and 0.75 micrograms/ml. When PMNL exposed to these inhibitory dilutions were resuspended in fresh urine, their phagocytic ability was fully restored and 13-24% of their killing activity was regained. A minimum concentration of commercially purified E. coli endotoxin of 200 micrograms/ml was required to abolished PMNL killing, with phagocytosis uninhibited. The results strongly suggest that bacterial growth metabolites, not endotoxin, are responsible for the depression of phagocytosis and intracellular killing in infected urine. A moderate dilution of the bacterial products in urine permits good PMNL function. Extrapolating this to the clinical situation, diluting the urine by water loading (as recommended for patients with urinary infections) should ensure efficient activity of PMNL under in-vivo conditions providing urinary pH and osmolality are not adversely affected.

Bacteriuria↗