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Biomedical subjects

M W Smith

Publications and source records attributed to M W Smith.

At least 109 records · Page 6Linked to original sources
PubMed1995

Selective transport of microparticles across Peyer's patch follicle-associated M cells from mice and rats.

M cells are specialized structures in the Peyer's patch follicle-associated epithelium capable of taking up bacteria, viruses and other pathogens for later presentation to the gut-associated lymphoid tissue. The present work studies how coating microspheres with different proteins affects their ability to be taken up by M cells under near physiological conditions in vivo. The later appearance of microspheres in intestinal lymph has also been measured by flow cytometry. The protein preparations used in these experiments included bovine serum albumin (bSA), human immunoglobulin G (hIgG), secretory immunoglobulin A (hIgA), bovine growth hormone (bGH) and bGH complexed with an IgG antibody raised against bGH (bGH-Ab). Selectivity in binding of these microspheres to M cells, determined by confocal microscopy, was bGH < bSA < hIgG (mice) and bGH < bGH-Ab (rats and mice). A similar selectivity was seen for microsphere entry into M cells (bGH < bSA < hIgG; bGH < bGH-Ab). The appearance of protein-coated microspheres in rat mesenteric lymph showed a similar selectivity to that found for binding and entry into M cells (bGH < bGH-Ab). This latter selectivity was also found for hIgA-coated microspheres (bSA < hIgA). Preservation of transport selectivity throughout transcytosis highlights the unique importance of the M cell surface as being the primary site determining which type of antigen can be presented subsequently to the gut immune system. The possibility that this is a transient or phasic property of the M cell surface and that this could have physiological relevance is also discussed.

Animals↗
PubMed1995

Increases in [Ca2+]i mediated by the 92.5-kDa putative cell membrane receptor for HCMV gp86.

We determined that changes in intracellular Ca2+ concentration ([Ca2+]i) occur in human fibroblasts within the first hour of human cytomegalovirus (HCMV) infection when viral adsorption and fusion take place, and we investigated whether such changes also occur in response to monoclonal anti-idiotype antibodies (MAb2) that mimic HCMV gp86 and bind to a 92.5-kDa putative cell membrane receptor for gp86. Digitized image analysis of fura 2-loaded human embryonic lung fibroblasts indicated specific transient increases in [Ca2+]i beginning in some cells within the first 5 min of incubation with cross-linked MAb2 (70-750 nM, P < 0.01), which were similar in timing and intracellular distribution to those induced by HCMV. A primary source of Ca2+ appeared to be intracellular Ca2+ stores, since prior depletion of these stores with 30 nM thapsigargin inhibited the response (91.7 +/- 8.6%, P < 0.01); influx of Ca2+ from the extracellular medium was apparently necessary to maintain the intracellular Ca2+ stores. Transient increases in inositol trisphosphate (IP3) occurred in response to MAb2 (up to 3,329 +/- 84%, P < 0.001) or HCMV (92.8 +/- 19%, P < 0.01) during this same time period. These data suggest that the 92.5-kDa receptor for HCMV gp86 mediates an increase in IP3 and subsequent release of Ca2+ from intracellular stores.

Antibodies, Monoclonal↗
PubMed1995

Fate of the dominant follicle, embryonal survival, and pregnancy rates in dairy cattle treated with prostaglandin F2 alpha and progestins in the absence or presence of a functional corpus luteum.

Our objective was to examine the role of progestin type on serum concentrations of progesterone (p4) and estradiol-17 beta (E2), ovarian follicular dynamics, and fertility in cattle in the presence or absence of a corpus luteum (CL) in an estrus synchronization scheme using progestin and PGF2 alpha. In Exp. 1, 325 cows and heifers were given one injection of PGF2 alpha (d 0) and then assigned randomly within parity to five treatments: to receive a second PGF2 alpha injection 14 d later (control); to receive norgestomet (NORG) for 7 d beginning on d 8, with a second PGF2 alpha injection given either 1 d (NORG + no CL) or 6 d (NORG + CL) after insertion; or to receive a P4-releasing intravaginal device (PRID) in lieu of norgestomet at comparable times. Presence or absence of a CL was based on concentrations of serum P4 on d 14. Pregnancy rates after insemination were greater (P < .01) with luteal treatments than with nonluteal treatments. Embryonal survival between two stages of pregnancy was 87.6%. In Exp. 2, ovarian structures in 50 cows were examined daily using ultrasonography and the same five treatments. Diameter of the ovulatory follicle was greater (P < .05) with the nonluteal treatments (NORG and PRID + no CL) than with the control and luteal treatments (PRID and NORG + CL). Replacement of the dominant follicle during progestin treatment was altered by treatment (luteal status) and stage of the estrous cycle. Fertility was not enhanced by exogenous progestins when a CL was present. In the absence of a CL, progestin (P4 less than NORG at the doses used) reduced fertility by increasing E2 and the diameter of the ovulatory follicle and decreasing turnover of dominant follicles.

Administration, Intravaginal↗
PubMed1995

U-90042, a sedative/hypnotic compound that interacts differentially with the GABAA receptor subtypes.

U-90042 is a structurally novel compound that has comparable affinities for binding to three recombinant subtypes of the gamma-aminobutyric acidA receptor: alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2. The relatively high affinity for the alpha 6 beta 2 gamma 2 subtype is similar to the benzodiazepine (BZ) partial inverse agonist Ro 15-4513 and different from BZ sedative/hypnotics such as diazepam and zolpidem. In the present study, U-90042 (3 mg/kg i.p.) suppressed locomotor activity and impaired rotarod performance in mice. These effects were not antagonized by flumazenil. The sedative effect was further confirmed in rats (10 mg/kg i.p.) and monkeys (1 mg/kg p.o.) by an increase of behavioral sleep and a corresponding electroencephalographic frequency spectral shift. Unlike the BZ hypnotics, U-90042 (10 mg/kg i.p.) produced no amnesia in the one-trial passive avoidance response in mice but antagonized diazepam-induced amnesia. In rats trained to discriminate an injection of diazepam from saline, U-90042 produced predominantly vehicle-appropriate responses, even at depressant doses. The in vivo diazepam-antagonist effect of U-90042 is consistent with its low intrinsic activity and diazepam-antagonism at the gamma-aminobutyric acidA alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 receptor subtypes. The receptor mechanism for the sedative/hypnotic effect is not clear at this time.

Animals↗
PubMed1995

De novo malignancy after renal transplantation.

We reviewed the incidence of de novo malignancy in renal transplant recipients who received their grafts between 1979 and 1989, and had at least two years of graft function. Multiple attempts were made to contact 236 patients who were then surviving, or, in whom the medical history could be validated prior to death. There were 94 successful contacts, among whom 19 patients (20.12%) had developed 30 malignant lesions. There were 24 instances of skin and 6 of non-skin malignancy. Squamous cell carcinoma of the skin was the most common new malignancy observed.

Adolescent↗
PubMed1994

Substituted (S)-phenylpiperidines and rigid congeners as preferential dopamine autoreceptor antagonists: synthesis and structure-activity relationships.

A series of (S)-phenylpiperidines in which the substituents on the aromatic ring and nitrogen have been varied has been prepared. They have been evaluated pharmacologically to explore the importance of these substituents for the interaction with central dopamine (DA) receptors. On the basis of biochemical and behavioral data in rats, several of these compounds are characterized as centrally acting DA autoreceptor antagonists. (S)-Phenylpiperidines having an aromatic substituent with a high group dipole moment in the 3-position, i.e., meta with respect to the piperidine ring, and being N-substituted with a propyl group were found to be highly active in vivo on the synthesis and turnover of dopamine. However, they do not induce strong hypoactivity or catalepsy. Interestingly, the most active compounds in vivo were found to display only low affinity for DA D2 and D3 receptors in vitro. In addition, 7-triflate-substituted octahydrobenzo[f]quinolines and 6-triflate-substituted hexahydro-1H-benz[e]indoles have been prepared and pharmacologically evaluated. The trans isomers of these rigid structures were found to display a pharmacological profile similar to that of the flexible phenylpiperidines. The corresponding cis isomers were found to be inactive in vivo.

Animals↗
PubMed1994

Selective regulation of epithelial gene expression in rabbit Peyer's patch tissue.

The physiological mechanisms that regulate epithelial gene expression during enterocyte migration and differentiation are still poorly understood. The present study has used a combination of quantitative in situ hybridisation, immunohistochemistry and enzyme cytochemistry to examine epithelial cell differentiation in rabbit small intestine. We have measured and compared the levels of mRNA and enzyme activity of the enterocyte brush border markers alkaline phosphatase, amino-peptidase N and lactase in normal villus epithelia and in epithelial cells exposed directly to the Peyer's patch immune environment. All three genes appeared to be expressed in parallel, but in each epithelial population examined, the pattern of gene expression was different. The level of these mRNAs was markedly reduced in Peyer's patch-associated epithelia, this being most pronounced in the follicle-associated epithelium, compared with normal villi. The activities of alkaline phosphatase and aminopeptidase N approximated the expression of their genes, whereas additional post-transcriptional events were shown to clearly contribute to the level of lactase activity in these tissues. These findings demonstrate that the reduced brush border hydrolase activity in Peyer's patch tissue that has been observed previously, is due to a down-regulation of epithelial gene expression in this location. These observations have been used to discuss epithelial differentiation in Peyer's patch tissue and the possible role of local immune factors in regulating such events.

Alkaline Phosphatase↗
PubMed1994

Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack.

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.

Adenine Nucleotides↗
PubMed1994

Differential regulation of vitamin D receptor and intestinal calcium transport occurring during sexual maturation in the fowl (Gallus domesticus).

Quantitative measurements of intestinal vitamin D receptor (VDR) and calcium transport taking place in individual villus-attached enterocytes have been compared during the onset of sexual maturity to test for a direct involvement of VDR in controlling calcium transport across the chick small intestine. Chickens of all ages showed VDR to be present in crypt and villus enterocytes, the amounts of VDR declining only slightly during enterocyte migration from the crypts to the tips of villi. Calcium transport corrected for initial adsorption was lowest in the crypt and highest in villus tip enterocytes. These results are consistent with VDR initiation and possible later maintenance of calcium transport across differentiating enterocytes. The total amount of VDR determined along the crypt-villus axis was significantly less in immature 11-week-old compared with 17-week-old sexually mature non-laying and 25-week-old laying chickens. Calcium transport was significantly greater in 25-week-old compared with 17- and 11-week-old birds. This unexpected up-regulation of VDR in 17-week-old birds was not affected by dietary restriction of calcium. Increased VDR expression in 17-week-old sexually mature birds is probably initiated by estrogen acting indirectly to increase 1,25 (OH)2D3 production in the kidney. Increased calcium transport in 25-week-old laying chickens could involve estrogen interacting with estrogen receptors as well as 1,25 (OH)2D3 interacting with vitamin D receptors to promote gene transcription in the intestine.

Animals↗
PubMed1994

Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene.

The role of cytosolic Ca2+ ([Ca2+]i) and protein kinases in the hsp70 induction following heat shock was investigated in cultured rat proximal tubular epithelial (PTE) cells. Changes in [Ca2+]i were measured by digital imaging fluorescence microscopy using fura 2. Steady state levels of hsp70 mRNA were examined by either Northern or dot blot analyses. [Ca2+]i increased within 10 minutes and continued to increase following heat shock. The increases in [Ca2+]i were reduced in nominally Ca(2+)-free media with or without EGTA. [Ca2+]i also increased within 0.5 minutes following ionomycin, but then declined to normal levels by 1.0 to 1.5 minutes. Heat shock induced hsp70 mRNA within 15 minutes, which continued to increase up to three hours. Ionomycin also induced hsp70 mRNA, which peaked at 30 minutes, and gradually decreased thereafter. The hsp70 induction following heat shock was attenuated when extracellular Ca2+ was reduced. Chelation of [Ca2+]i by quin-2 also reduced the hsp70 induction. Inhibitors of protein kinases, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), calphostin C, genistein, and 2-aminopurine, also had inhibitory effects on the hsp70 induction. In contrast, a calmodulin inhibitor, chlorpromazine, had little effect. These results suggest that heat shock increases [Ca2+]i in rat PTE cells and that [Ca2+]i and protein kinases are involved in the hsp70 induction following heat shock.

Animals↗
PubMed1994

Genomic sequence sampling: a strategy for high resolution sequence-based physical mapping of complex genomes.

We present a simple and efficient method for constructing high resolution physical maps of large regions of genomic DNA based upon sampled sequencing. The physical map is constructed by ordering high density cosmid contigs and determining a sequence fragment from each end of every clone. The resulting map, which contains 30-50% of the complete DNA sequence, allows the identification of many genes and makes possible PCR amplification of virtually any part of the genome. We apply this strategy to the automated analysis of the genome of the primitive eukaryote Giardia lamblia and evaluate its applicability to the physical mapping and DNA sequencing of the human genome.

Amino Acid Sequence↗
PubMed1994

Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene.

We isolated a soybean (Glycine max) cDNA encoding the heme and chlorophyll synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nodules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a common lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosymbiont ancestor of chloroplasts and that the Alad gene was transferred to the nucleus during plant evolution.

Amino Acid Sequence↗
PubMed1994

Pharmacology of a human dopamine D4 receptor expressed in HEK293 cells.

Dopamine receptors play an important role in neurotransmission within the central nervous system, as evidenced by their affinity to and apparent site of action of a variety of psychotropic agents. The cloning of dopamine receptor subtypes makes possible the discovery of subtype-selective agonist and antagonist compounds useful for the study of receptor physiology and for the development of more effective treatments for disturbances stemming from aberrant dopamine neurotransmission. We describe in this report the receptor pharmacology of a human D4-dopamine receptor expressed in HEK293 cells from a synthetic gene. The affinities of monoaminergic antagonists in competition with [3H]spiperone-labeled sites in these cells matched very closely their rank order potency measured by other investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell investigators for D4 receptors expressed in COS-7 cells. Further, the sites expressed in the HEK293-D4-24 line met six specific pharmacological criteria by which the D4 subtype has been distinguished from other D2-like dopamine receptors. The site expressed in this cell line thus bears the earmarks of a human D4-dopamine receptor. Competition of several agonists best fit a two-site binding model. This result, along with the measured 10-fold GTP-shift for dopamine, strongly suggests functional coupling of this receptor to endogenous G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Characterization of U-92016A as a selective, orally active, high intrinsic activity 5-hydroxytryptamine1A agonist.

The purpose of the present study was to characterize U-92016A [(+)-R)-2-cyano-N,N-dipropyl-8-amino-6,7,8,9-tetrahydro-3H-benz[e] indole] as a 5-hydroxytryptamine (5-HT)1A receptor agonist and to compare its activity with that of standard 5-HT1A receptor agonists. U-92016A binds with high affinity to human 5-HT1A receptors expressed in Chinese hamster ovary cells (Ki = 0.2 nM). Radioligand binding studies also indicate that U-92016A is selective for the 5-HT1A receptor over other biogenic amine receptors. In Chinese hamster ovary cells expressing the human 5HT1A receptor, U-92016A decreased the forskolin-induced increase in cyclic AMP synthesis and had an intrinsic activity of 0.82 relative to 5-HT. U-92016A potently decreased rectal temperature in mice. The maximum temperature decrease was significantly greater than that observed for 8-hydroxy-di-n-propyl aminotetralin, buspirone, gepirone, ipsapirone or flesinoxan. U-92016A also elicited the 5-HT-mediated syndrome in rats and resulted in a dose-related decrease in 5-hydroxytryptophan accumulation. The compound also decreased arterial blood pressure in spontaneously hypertensive rats and inhibited sympathetic nerve activity in cats. In these assays U-92016A displayed excellent potency and a long duration of action. U-92016A also inhibited the firing of dorsal raphe 5-HT neurons and was active in two social interaction assays. The p.o. bioavailability of U-92016A was calculated to be 45%. Taken together, these data indicate that U-92016A is a metabolically stable, p.o. active 5-HT1A receptor agonist with an exceptionally high degree of intrinsic activity.

5-Hydroxytryptophan↗
PubMed1994

Excitation of noradrenergic cell firing by 5-hydroxytryptamine1A agonists correlates with dopamine antagonist properties.

The 5-HT1A receptor agonists buspirone, 8-hydroxy-N,N-dipropyl-2-aminotetralin, gepirone and ipsapirone were evaluated for their receptor binding profiles and their effects on firing rates of 5-HT, dopamine (DA) and noradrenaline (NA) neurons in the dorsal raphe, substantia nigra pars compacta and the locus ceruleus, respectively. All agents bound to 5-HT1A receptors with high affinities. All agents also bound to dopamine D2 receptors but, with the exception of buspirone, affinities were usually much lower than for 5-HT1A receptors. All agents depressed 5-HT neurons, with 8-hydroxy-N,N-dipropyl-2-aminotetralin having a potency about 8 to 12 times those for the other three. All agents also antagonized the inhibition of DA neurons by amphetamine, an index of DA antagonist properties. Buspirone reversed amphetamine's effects with doses similar to those for depressing 5-HT neurons, but the remaining three required much higher doses to affect DA neuron function. All four 5-HT1A agonists excited NA neurons. In each case, doses required for excitation of NA cells were similar to those reversing amphetamine's effects on DA cells, but not to those for depressing 5-HT cells. Haloperidol also stimulated NA cells. It is concluded that excitation of NA neurons by 5-HT1A agonists may be due to interactions with dopaminergic, rather than serotonergic, receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1993

Substituted 3-phenylpiperidines: new centrally acting dopamine autoreceptor antagonists.

The (+)-and (-)-enantiomer of compounds 4 and 5 were synthesized and tested for central dopamine (DA) receptor stimulating activity, using biochemical and behavioral tests in rats. Based on the available data the (-)-enantiomers of 4 and 5 are characterized as centrally acting DA autoreceptor antagonists with oral activity. They display a similar pharmacological profile as the prototype DA autoreceptor antagonists (+)-1 and (+)-2 and show a certain preference for the D3 DA receptor antagonist binding site.

Animals↗