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Biomedical subjects

S Patel

Publications and source records attributed to S Patel.

At least 379 records · Page 21Linked to original sources
PubMed1997

Ochratoxin A in wheat: certification of two reference materials.

The preparation of two wheat reference materials and the certification of their ochratoxin A content is described. The materials were prepared and certified within the European Commission. Measurements and Testing Programme (M&T). The first and second phases of this project, two intercomparisons of procedures for the determination of ochratoxin A in wheat, at levels of approximately 13 micrograms/kg, and 7 micrograms/kg, respectively have already been reported. This paper describes the work carried out in certification of the ochratoxin A content (mass fraction) of two wheat reference materials: a blank wheat CRM 471, and a contaminated wheat, CRM 472. These materials were prepared for use in the second intercomparison referred to above and reported previously. Reference material CRM 472 was prepared from naturally-contaminated wheat blended with the blank wheat (CRM 471). Details are given of the milling, blending and packaging procedure, and the checks to ensure homogeneity and stability of the material. The certification exercise was carried out by 15 laboratories using a variety of extraction and clean-up procedures, and the certified ochratoxin A content (mass fraction) of CRM 471 was < 0.6 microgram/kg. The value for CRM 472 was 8.2 micrograms/kg with an uncertainty of 1.0 microgram/kg.

Calibration↗
PubMed1997

Dysfunctional D1A receptor-G-protein coupling in proximal tubules of spontaneously hypertensive rats is not due to abnormal G-proteins.

BACKGROUND: Dysfunctional dopamine neurotransmission and defective D1A receptor-G protein coupling exist in renal proximal tubules (RPT) of the spontaneously hypertensive rat (SHR). OBJECTIVE: To determine whether the G proteins in SHR are abnormal, preventing formation of agonist high affinity sites in SHR. METHODS: We examined the expression levels of the alpha-subunits of G proteins, as well as D1A receptor receptor coupling to exogenously added normal G proteins, in RPT of SHR and the normotensive Wister-Kyoto (WKY) rat. RESULTS: In the presence of 110 mmol/l NaCl, the D1A dopamine receptor-selective agonist SKF R-38393 binds both to high- and to low-affinity sites on solubilized and reconstituted D1A receptors extracted from renal proximal tubules of normotensive Wistar-Kyoto (WKY) rats. In the spontaneously hypertensive rat (SHR), SKF R-38393 bound to a single site on the reconstituted receptor with affinity values corresponding to the low-affinity state of the receptor. Western blot analyses indicated that the alpha-subunit of the guanine nucleotide binding protein (G-protein), Gs, was expressed at similar levels, whereas G(o)alpha was not expressed in proximal tubule membranes from WKY rats and SHR. Pretreatment of proximal tubule membranes with the alkylating agent N-ethylmaleimide in the presence of SKF R-38393 inactivated alpha-subunits of endogenous G-proteins, but not D1A receptors, resulting in loss of high-affinity binding sites in WKY rats. These N-ethylmaleimide-treated D1A receptors from WKY rats, when reconstituted with exogenous sources of G-proteins, were able to couple to these exogenous G-proteins, with complete restoration of high-affinity sites. Moreover, the affinity values and the proportion of these hybrid sites were similar to those of untreated receptors, and these affinity sites were regulated by guanine nucleotide analogs. Reconstitution of D1A receptors from SHR with the same exogenous G-proteins failed to similarly induce formation of the high-affinity binding sites in the hybrid reconstituted systems, and SKF R-38393 continued to bind in a single low-affinity state of the receptor. CONCLUSION: These results demonstrate that the absence of G-protein coupling in SHR is due to intrinsic defects within the receptor protein, rather than to any abnormalities of the endogenous G-proteins themselves.

Alkylating Agents↗
PubMed1997

Effect of 0.4% benoxinate hydrochloride on corneal sensitivity, measured using the non-contact corneal aesthesiometer (NCCA).

The Non-Contact Corneal Aesthesiometer (NCCA) was used to monitor the effect of topical anesthesia on corneal sensitivity loss and recovery. In a single masked randomized study, one drop of nonpreserved 0.4% benoxinate hydrochloride was instilled in either the right, left, or both eyes of 20 subjects on separate days, and measurements were made of the central corneal sensitivity in the right eye over a 60-min period. As controls, 10 subjects were retested, without any anesthetic instillation, over the same time period. No significant differences were found between these two benoxinate treatments, nor between the contralateral treatment and controls. Results indicate that there is no contralateral effect of benoxinate anesthesia, and that full recovery does not occur until 60 min after instillation. No effect on the depth and duration of anesthesia was found for iris color.

Administration, Topical↗
PubMed1997

Tazarotene-induced gene 2 (TIG2), a novel retinoid-responsive gene in skin.

Retinoids exert their biologic effects through two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which belong to the superfamily of steroid/thyroid hormone nuclear receptors. By using a subtraction hybridization approach, we have identified a cDNA sequence TIG2 (Tazarotene-induced gene 2), whose expression is up-regulated by the treatment of skin raft cultures by an RAR beta/gamma-selective anti-psoriatic synthetic retinoid tazarotene [AGN 190168/ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl] nicotinate]. The retinoid-mediated up-regulation in the expression of TIG2 was confirmed by Northern blot analysis. Upon sequencing, TIG2 was found to be a cDNA whose complete sequence was not in the GenBank and EMBL data bases. The TIG2 cDNA is 830 bp long and encodes a putative protein product of 164 amino acids. TIG2 is neither expressed nor induced by tazarotene in primary keratinocyte and fibroblast cultures. Thus, TIG2 is expressed and induced by tazarotene only when keratinocytes and fibroblasts form a tissue-like 3-dimensional structure. We further demonstrate that RAR-specific retinoids increase TIG2 mRNA levels. In contrast, neither RXR-specific retinoids nor 1,25-dihydroxyvitamin D3 increased TIG2 levels. Finally, we demonstrate that TIG2 is expressed at high levels in nonlesional psoriatic skin but at lower levels in the psoriatic lesion and that its expression is up-regulated in psoriatic lesions after topical application of tazarotene.

Administration, Topical↗
PubMed1997

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Alleles↗
PubMed1997

PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying mycobacteria.

A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5' fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization to a panel of mycobacterial species-specific capture probes, which were immobilized via their 5' biotin ends to a streptavidin-coated microtiter plate. Specific hybridization of a 5' fluorescein-labelled strand to a species probe was detected colorimetrically with an anti-fluorescein enzyme conjugate. The assay was able to identify 10 Mycobacterium spp. A probe able to hybridize to all Mycobacterium species (All1) was also included. By a heminested PCR, the assay was sensitive enough to detect as little as 10 fg of DNA, which is equivalent to approximately three bacilli. The assay was able to detect and identify mycobacteria directly from sputa. The specificities of the capture probes were assessed by analysis of 60 mycobacterial strains corresponding to 18 species. Probes Avi1, Int1, Kan1, Xen1, Che1, For1, Mal1, Ter1, and Gor1 were specific. The probe Tbc1 cross-hybridized with the Mycobacterium terrae amplicon. Analysis of 35 strains tested blind resulted in 34 strains being correctly identified. This method could be used for rapid identification of early cultures and may be suitable for the detection and concurrent identification of mycobacteria within clinical specimens.

DNA Probes↗
PubMed1997

Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative effects of retinoids in Kaposi's sarcoma.

All-trans-retinoic acid (RA) is active in the treatment of Kaposi's sarcoma (KS), and retinoids inhibit KS cell growth in vitro. To understand the mechanism of retinoid action in KS, we studied the expression of autocrine growth factors of KS cells after RA treatment. We demonstrate that RA and its synthetic analogs inhibit the proliferation of KS cells by inhibiting the mRNA and protein levels of interleukin-6 (IL-6), an autocrine growth factor for KS cells. We further demonstrate that nuclear retinoid receptors (RA receptors [RARs] and retinoid X receptors [RXRs]) inhibit IL-6 promoter action by antagonizing the enhancer action of NF-IL6, a basic domain leucine zipper transcription factor belonging to the family of CAAT enhancer binding proteins. Furthermore, RARs and RXRs do not bind in vitro to an NF-IL6 binding site. However, the secondary folded structure of the DNA binding domain of RAR and RXR is obligatory for inhibiting NF-IL6 activity. Thus, NF-IL6 is a potential therapeutic target for the treatment of KS. Finally, using receptor-selective synthetic retinoids, we demonstrate that NF-IL6 antagonism and transactivation are separable functions of RAR alpha, thus indicating that synthetic retinoids with properties of NF-IL6 antagonism but lacking transactivation capabilities can be synthesized. Such retinoids might increase therapeutic potential in KS.

CCAAT-Enhancer-Binding Proteins↗
PubMed1997

Changes in bone mineral density in patients with Paget's disease treated with risedronate.

OBJECTIVES: To study changes in bone mineral density (BMD) in patients with Paget's disease of bone treated with risedronate. METHODS: Whole body dual energy x ray absorptiometry (DXA) scans were carried out on 20 patients with Paget's disease treated with oral risedronate. DXA scanning was carried out at baseline and 11 months. Whole body bone mineral content (BMC) was measured. In addition, regions of interest were drawn around the skull, individual lumbar vertebrae, hemipelvis, femora, and tibiae to obtain BMD for these sites. An uncoupling index was also calculated as the area under the curve for serum alkaline phosphatase (ALP) divided by the area under the curve for hydroxyproline excretion (HYPRO) for the period of treatment. RESULTS: Median whole body BMC increased from 3057 g to 3156 g (p < 0.001) resulting from an increase in pagetic and non-pagetic BMD. From the analysis of regions of interest it was found that pagetic trabecular bone showed the largest increase in BMD. The pretreatment HYPRO and the uncoupling index were significantly related to the change in BMD for all pagetic sites for a patient (r = 0.65, p < 0.01 and r = 0.57, p < 0.05 respectively). CONCLUSION: Bisphosphonate treatment of Paget's disease results in an increase in BMD of pagetic bone without redistribution of mineral from non-pagetic bone. The remodelling space and extent of uncoupling are significantly related to increases in BMD at pagetic sites.

Absorptiometry, Photon↗
PubMed1997

Pitfalls in the echocardiographic diagnosis of aortic dissection.

The authors studied 25 patients with transthoracic echocardiography (TTE) and/or transesophageal echocardiography (TEE) for suspected aortic dissection. Of these, aortic dissection was diagnosed correctly in 19 patients, but in 6 the echocardiographic findings for dissection were atypical or false-positive. In patient 1, the TEE revealed a dilatated proximal aortic root. TEE showed a possible flap but was nondiagnostic. The diagnosis was made by computed tomography (CT) and confirmed at surgery to be type 1 dissection. In patient 2, the TTE depicted flail aortic cusps, questionable vegetations, and dilatated aortic root. In patient 3, TTE demonstrated moderate pericardial effusion with hematoma but no dissection. Type 1 dissection was subsequently confirmed at autopsy in both. Patient 4 had TEE diagnosis of type 3 dissection. However, reevaluation of the study by a senior sonographer just prior to surgery led to the correct diagnosis of type 1 dissection. Patients 5 and 6 had dilatated ascending aortas with linear echoes within the lumen on TEE and were reported as having type 1 dissections. CT and/or angiography did not reveal dissection in either patient. In conclusion, TTE and TEE are vaulable tests in diagnosing aortic dissection. However, atypical features, misdiagnosis of the site of dissection, or false-positive studies can occur.

Adult↗
PubMed1997

Overexpression of hormone-sensitive lipase in Chinese hamster ovary cells leads to abnormalities in cholesterol homeostasis.

Hormone-sensitive lipase (HSL) is an intracellular enzyme that functions as both a neutral triglyceride and cholesteryl ester hydrolase. In order to explore the effects of HSL on cholesterol homeostasis, Chinese hamster ovary (CHO) cells were transfected with rat HSL and several different stable cell lines that overexpress HSL mRNA, HSL protein, and HSL activity approximately 600-fold were isolated. Cells transfected with HSL contained less cholesteryl esters and unesterified cholesterol than control cells. HSL transfectants expressed 20-60% fewer LDL receptors than control cells when grown in lipid-depleted media or in the presence of mevinolin, as assessed by binding and degradation of LDL and immunoblotting of LDL receptors. In contrast, the rate of cholesterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were increased 3- to 14-fold in HSL transfectants grown in sterol replete media. The rate of cholesterol synthesis and the activity of HMG-CoA reductase increased when cells were grown in lipid-depleted media, and remained markedly elevated compared to control cells. These results show that the regulation of LDL receptor expression and cholesterol synthesis can be dissociated through the actions of HSL and suggest multiple control mechanisms for sterol-responsive genes.

Animals↗
PubMed1997

Biological profile of L-745,870, a selective antagonist with high affinity for the dopamine D4 receptor.

L-745,870,(3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H- pyrollo[2,3-b] pyridine, was identified as a selective dopamine D4 receptor antagonist with excellent oral bioavailability and brain penetration. L-745,870 displaced specific binding of 0.2 nM [3H] spiperone to cloned human dopamine D4 receptors with a binding affinity (Ki) of 0. 43 nM which was 5- and 20-fold higher than that of the standard antipsychotics haloperidol and clozapine, respectively. L-745,870 exhibited high selectivity for the dopamine D4 receptor (>2000 fold) compared to other dopamine receptor subtypes and had moderate affinity for 5HT2, sigma and alpha adrenergic receptors(IC50 < 300 nM). In vitro, L-745,870 (0.1-1 microM) exhibited D4 receptor antagonist activity, reversing dopamine (1 microM) mediated 1) inhibition of adenylate cyclase in hD4HEK and hD4CHO cells; 2) stimulation of [35S] GTPgammaS binding and 3) stimulation of extracellular acidification rate, but did not exhibit any significant intrinsic activity in these assays. Although standard antipsychotics increase dopamine metabolism or plasma prolactin levels in rodents, L-745,870 (</=30 mg/kg p.o.) had no effect in these assays. The lack of a suitable in vivo assay for D4 receptor activation prompted the use of in vivo surrogate marker assays which confirmed that doses of 5-60 microg/kg L-745,870 would be sufficient to occupy 50% D4 receptors in the brain. These results show that dopamine D4 receptor antagonism in the brain does not result in the same neurochemical consequences (increased dopamine metabolism or hyperprolactinemia) observed with typical neuroleptics.

Animals↗
PubMed1997

Constitutive modulation of Raf-1 protein kinase is associated with differential gene expression of several known and unknown genes.

BACKGROUND: Raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Consistent with this notion is the fact that constitutive modulation of expression and/or activity of Raf-1 protein kinase modifies cell growth, proliferation, and cell survival. Although these effects are controlled at least in part by transcriptional mechanisms, the role of Raf-1 in the regulation of specific gene expression is unclear. MATERIALS AND METHODS: Differential display of mRNA was used to identify the genes differentially expressed in human head and neck squamous carcinoma cells (PCI-06A) transfected with either the antisense c-raf-1 cDNA (PCI-06A-Raf(AS)), or a portion of cDNA coding for the kinase domain of Raf-1 (PCI-06A-Raf(K)). The differentially expressed fragments were cloned and sequenced, and they were used as probes to compare the expression patterns in parent transfectants by Northern blot analysis. In addition, expression patterns of the novel genes were examined in normal tissues and cancer cell lines. RESULTS: Six differentially expressed cDNA fragments were identified and sequenced. Northern blot analysis revealed that four of these fragments representing human alpha 1-antichymotrypsin (alpha 1-ACT), mitochondrial cytochrome c oxidase subunit II (COX-II), and two as-yet unidentified cDNAs (KAS-110 and KAS-111) were relatively overexpressed in PCI-06A-Raf(AS) transfectants compared with PCI-06A-Raf(K) transfectants. The other two cDNA fragments representing human elongation factor-1 alpha (HEF-1 alpha) and ornithine decarboxylase antizyme (OAz) were overexpressed in PCI-06A-Raf(K) transfectants compared with PCI-06A-Raf(AS) transfectants. The KAS-110 (114 bp) and KAS-111 (202 bp) cDNAs did not show significant matches with sequences in the GenEMBL, TIGR, and HGS DNA databases, and these may represent novel genes. The KAS-110 and KAS-111 transcripts, approximately 0.9 kb and approximately 0.5 kb, were observed in most normal tissues and several cancer cell types, indicating their housekeeping function. CONCLUSIONS: This study reports novel components of the Raf-1 signaling pathway. alpha 1-ACT, HEF-1 alpha, COX-II, and OAz have been previously implicated in diverse cellular responses including transformation, energy metabolism, and cell survival. Our data suggest that expression of these genes may play a role in the Raf-1-mediated biological activity of PCI-06A cells. The KAS-110 and KAS-111 cDNAs represent unknown genes, and further investigations are necessary to determine their role in the cellular response. Identification of specific targets may provide useful markers for prognosis and therapy selection in squamous cell carcinoma.

Base Sequence↗
PubMed1997

L-745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavioral tests.

This study examined the high-affinity, selective dopamine D4 receptor antagonist, L-745,870 (3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2, 3-b]pyridine) in rodent behavioral models used to predict antipsychotic potential and side-effect liabilities in humans. In contrast to the classical neuroleptic, haloperidol, and the atypical neuroleptic, clozapine, L-745,870 failed to antagonize amphetamine-induced hyperactivity in mice or impair conditioned avoidance responding in the rat at doses selectively blocking D4 receptors. Furthermore, L-745,870 failed to reverse the deficit in prepulse inhibition of acoustic startle responding induced by the nonselective dopamine D2/3/4 receptor agonist apomorphine, an effect which was abolished in rats pretreated with the D2/3 receptor antagonist, raclopride (0.2 mg/kg s.c.). L-745,870 had no effect on apomorphine-induced stereotypy in the rat but did induce catalepsy in the mouse, albeit at a high dose of 100 mg/kg, which is likely to occupy dopamine D2 receptors in vivo. High doses also impaired motor performance; in rats L-745,870 significantly reduced spontaneous locomotor activity (minimum effective dose = 30 mg/kg) and in mice, L-745,870 reduced the time spent on a rotarod revolving at 15 rpm (minimum effective dose = 100 mg/kg). Altogether these results suggest that dopamine D4 receptor antagonism is not responsible for the ability of clozapine to attenuate amphetamine-induced hyperactivity and conditioned avoidance responding in rodents. Furthermore, the lack of effect of L-745,870 in these behavioral tests is consistent with the inability of the compound to alleviate psychotic symptoms in humans.

Amphetamine↗
PubMed1997

Microleakage of dentin-bonded crowns placed with different luting materials.

PURPOSE: To assess the microleakage of dentin-bonded porcelain crowns placed with three different luting materials. MATERIALS AND METHODS: Standardized full-coverage crown preparations were made on 45 extracted premolar teeth of a design which was considered typical of the minimal preparation indicated for dentin-bonded crowns. Half of the margin of the crown was placed below the cemento-enamel junction. Impressions of the prepared teeth were made and individual crowns constructed in feldspathic porcelain. The interior surface of each crown was etched with Mirage Superetch and treated with silane bond-enhancing solution. The crowns were cemented using one of the following combinations: Group A, Scotchbond Multi-Purpose Plus (SBMP)/3M Ceramic Luting Kit; Group B, Mirage ABC/FLC, and Group C, All-Bond 2/DuoLink. The teeth were stored for 2 weeks at 37 degrees C, subsequently thermocycled, and the restorations examined for leakage using buffered methylene blue as the marker. The teeth were sectioned bucco-lingually through the restoration and the degree of microleakage assessed microscopically. RESULTS: All groups showed leakage at the dentin and enamel margins and at the restoration-cement interface. There were significant differences in leakage at the enamel margins for the three groups (P = 0.007) but not at the margins finished in dentin (P = 0.762). There was a significant difference in leakage between Groups B and C at the enamel margin (P = 0.009) and, within Group B, significantly greater leakage at the enamel-restoration interface than the dentin-restoration interface (P = 0.033). There were no other significant interactions. At the restoration-adhesive interface there was no significant difference in leakage among the groups.

Bicuspid↗
PubMed1997

Protein phosphatase-2A associates with the cytoskeleton to maintain cell spreading and reduced motility of nonmetastatic Lewis lung carcinoma cells: the loss of this regulatory control in metastatic cells.

Metastatic Lewis lung carcinoma (LLC-LN7) variants have previously been shown to have reduced levels of protein phosphatase-2A (PP-2A) activity as compared to the nonmetastatic LLC-C8 cells. The present study showed that inhibition of PP-2A in the nonmetastatic LLC-C8 cells caused a rapid change from a spread to a rounded morphology and increased their in vitro invasiveness through laminin. In contrast, the metastatic LLC-LN7 cells were rounded and invasive, which was not affected by inhibition of PP-2A. To determine whether these differences could be attributed to alterations in PP-2A association with the cytoskeleton, the extent of PP-2A colocalization with microtubules was tested. Immunostaining for tubulin showed prominent filamentous fibers in nonmetastatic LLC-C8 cells and small foci of PP-2A immunostaining along these microtubules. In contrast, the tubulin staining was diffuse throughout the metastatic LLC-LN7 cells and there was little evidence of association with PP-2A. Western blot analyses showed that this reduced level of PP-2A association with microtubules in metastatic LLC-LN7 cells was not due to differences in levels of the PP-2A subunits. Instead, it may be due to the reduced association of the subunits into the heterotrimeric form of the PP-2A holoenzyme. These studies show the importance of PP-2A in maintaining a spread morphology and in restricting invasiveness, and a loss of this regulatory control in metastatic cells. This loss of PP-2A regulatory control in metastatic cells may be due to a reduction in the trimeric form of the PP-2A holoenzyme.

Animals↗