Biomedical subjects

S Patel

Publications and source records attributed to S Patel.

At least 343 records · Page 19Linked to original sources

PubMed1998

Reliability of the non-contact corneal aesthesiometer and its comparison with the Cochet-Bonnet aesthesiometer.

AIMS: To determine the repeatability of measurement of the corneal sensation threshold using the Non-Contact Corneal Aesthesiometer (NCCA); to evaluate the effect of the air-jet diameter and stimulus duration on the threshold; and to compare the sensation thresholds found with the NCCA and with the Cochet-Bonnet Aesthesiometer (C-BA), on the same group of normal, human subjects. METHODS: For all experiments, only the central corneal sensation thresholds were measured. (1) NCCA Repeatability; 17 subjects tested at the same time on five separate days. (2) NCCA Air-Jet Diameter; 15 subjects tested using four different air-jets: 0.35, 0.5, 1 and 2 mm. (3) NCCA Stimulus Duration; 15 subjects tested using three stimulus durations: 0.5, 0.9 and 1.5 sec. (4) NCCA and C-BA; 43 subjects tested using each instrument in turn. RESULTS: (1) No significant differences in mean sensation threshold were detected across the five days of testing, or between individual days. (2) 0.5 and 1 mm air-jets produce least variability in mean sensation threshold. (3) No significant difference in mean sensation thresholds was found between the three test durations. (4) For each subject, there was no correlation between each instrument's mean sensation threshold. CONCLUSIONS: (1) The NCCA was able to assess the corneal sensation threshold in an accurate and repeatable manner. (2) The optimum test parameters were found using the 0.5 mm air-jet, over a stimulus duration of 0.9 sec. (3) The C-BA has serious deficiencies in its design which limit its ability to accurately measure corneal sensitivity at low stimulus thresholds.

Anesthesiology↗

PubMed1998

Efficacy of a dentifrice containing zinc citrate for the control of plaque and gingivitis: a 6-month clinical study in adults.

The objective of this 6-month, double-blind, clinical study, conducted in harmony with American Dental Association (ADA) guidelines, was to evaluate the efficacy of a dentifrice containing 2% zinc citrate and 0.76% sodium monofluorophosphate in a silica base (zinc citrate dentifrice) for the control of supragingival plaque and gingivitis, compared to a control dentifrice containing 0.76% sodium monofluorophosphate in a silica base (control dentifrice). Adult men and women from the Atlanta, Georgia, area were entered in the study and stratified into two treatment groups, which were balanced for baseline Quigley-Hein Plaque Index scores and baseline Löe-Silness Gingival Index scores. Participants received an oral prophylaxis and were instructed to brush their teeth twice daily (morning and evening) for 1 minute with their assigned dentifrice, using a soft-bristled toothbrush. Examinations for supragingival plaque and gingivitis were conducted after 3 months and again after 6 months' use of the study dentifrices. Ninety-nine participants complied with the protocol and completed the entire 6-month clinical study. At both the 3- and 6-month study examinations, the zinc citrate dentifrice group exhibited statistically significant reductions in both plaque and gingivitis compared to the control dentifrice group, based on whole-mouth data. At the 6-month examination, the magnitude of these reductions met or exceeded 18% for both plaque and gingivitis (25.3% for plaque; 18.8% for gingivitis). The effect of the zinc citrate dentifrice was most pronounced on the more severe manifestations of plaque and gingivitis, indicating a statistically significant (50.2%) reduction in severe plaque and a statistically significant (66.7%) reduction in severe gingivitis over the control dentifrice after 6 months of use. Similar findings were observed for data obtained from proximal, lingual, and posterior sites. Among the sites that indicated a tendency toward high levels of plaque or gingivitis based on the baseline scores, substantially fewer sites tended to continue to present such high levels at follow-up exams in the zinc citrate dentifrice group than in the control dentifrice group. Thus, in accordance with the 1986 guidelines published by the ADA and the 1994 revision published by the Task Force on Design and Analysis in Dental and Oral Research, the results of this study support the conclusion that a dentifrice containing 2% zinc citrate and 0.76% sodium monofluorophosphate in a silica base is clinically efficacious for the control of supragingival plaque and gingivitis.

Adolescent↗

PubMed1998

Clinical efficacy of a dentifrice containing zinc citrate: a 12-week calculus clinical study in adults.

The objective of this double-blind clinical study, conducted following the Volpe-Manhold design for studies of dental calculus, was to investigate the efficacy of a dentifrice containing 2% zinc citrate and 0.76% sodium monofluorophosphate in a silica base (zinc citrate dentifrice) as compared to a control dentifrice containing 0.76% sodium monofluorophosphate in a silica base (control dentifrice). Adult men and women from the Northern New Jersey area were provided a full oral prophylaxis and assigned the use of a control (non-tartar-control) dentifrice for 8 weeks. At the completion of this initial period, participants were assessed for baseline Volpe-Manhold Calculus Index scores, provided another full prophylaxis, and stratified into two treatment groups that were balanced for age, sex, and baseline calculus. Participants were instructed to brush their teeth twice daily (morning and evening) for 1 minute with their assigned dentifrice, using a soft-bristled toothbrush. Examinations for dental calculus were again performed after 12 weeks' use of the study dentifrices. Seventy-five participants complied with the protocol and completed the entire study. At the 12-week examination, the zinc citrate dentifrice group exhibited a statistically significant 31.9% reduction in mean Volpe-Manhold Calculus Index score as compared to the control dentifrice group. Thus, the results of this clinical study support the conclusion that a dentifrice containing 2% zinc citrate and 0.76% sodium monofluorophosphate in a silica base is efficacious for the control of the development of supragingival calculus.

Adolescent↗

PubMed1998

Rapid, convenient radioimmunoassay of estrone sulfate.

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05-90 micrograms/L with a detection limit of 0.009 microgram/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 micrograms/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 micrograms/L, respectively. Linearity of dilution studies showed values of 80-105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were < 5%. When compared with a conventional assay (involving hydrolysis of E1S and indirect measurement of estrone), the present RIA showed excellent correlation (r = 0.99, slope = 1.54, Sy/x = 2.14, n = 71). Mean E1S concentrations measured with this RIA for normal men (n = 20) and women in follicular (n = 20) and luteal (n = 25) phases of their menstrual cycle were 0.96, 0.96, and 1.74 microgram/L, respectively. Mean E1S concentrations for oral contraceptive users (n = 20) and postmenopausal women without hormone replacement therapy (n = 21) or on hormone replacement therapy (n = 22) were 0.74, 0.13, and 2.56 micrograms/L, respectively. Serum concentrations of E1S in pregnant women in their first (n = 14), second (n = 17), and third (n = 15) trimesters were 20, 66, and 105 micrograms/L, respectively. Availability of this simple RIA should provide a useful tool for the assessment of estrogen status in women.

Cross Reactions↗

PubMed1998

The epidemiology of cryptosporidiosis: application of experimental sub-typing and antibody detection systems to the investigation of water-borne outbreaks.

A technique based on the analysis of banding patterns obtained by SDS-PAGE Western-blotting of an oocyst wall antigen obtained from faeces has been evaluated to subtype Cryptosporidium parvum Tyzzer, 1912. This technique appears to have sufficient stability to recognise multiple types of this parasite. A similar Western-blotting technique has also been used to assess antibody responses to cryptosporidial antigens in human sera. Two systems were developed: one against three antigens of apparent molecular weights 6, 14 and 17 kDa; the second against oocyst wall antigens of apparent molecular weights 57, 69, 75, 89, 128, 151 and 173 kDa. Antibodies to three antigens of apparent molecular weights 6, 14 and 17 kDa were most successful as diagnostic markers in that they were found in > 88% of convalescent phase sera from confirmed cryptosporidiosis patients and were uncommon (> or = 7%) in control subjects. Faecal samples from human and animal sporadic cases yielded a wide range of cryptosporidial antigen banding patterns. Samples from patients in a water-borne outbreak in South Devon (England) in 1995 also yielded a wide range of banding patterns including members within individual household family groups. These results are in contrast with those from samples collected from other defined geographical areas, including some from a second water-borne outbreak where much more homogeneous banding patterns were obtained. Sera collected for other purposes from apparently uninfected individuals 9 months after the South Devon 1995 outbreak were examined. Antibodies to the three antigens of molecular weights 6, 14 and 17 kDa were detected in 32-49% of individuals resident in the outbreak water supply area, and in 15-21% of those resident in an adjacent water supply area. The significance of these findings is discussed in relation to data obtained from epidemiological field studies.

Animals↗

PubMed1998

Artificial neural networks and logistic regression as tools for prediction of survival in patients with Stages I and II non-small cell lung cancer.

The prognosis of patients with Stage I and II non-small cell lung cancer (NSCLC) can be estimated but cannot be definitively ascertained by use of current clinicopathologic criteria and tumor marker studies. The potential value of probabilistic neural networks (NNs) with genetic algorithms and multivariate logistic regression to predict the survival of NSCLC patients has not been previously evaluated. Multiple prognostic factors (age, sex, cell type, stage, tumor grade, smoking history, and immunoreactivity to c-erbB-3, bcl-2, Glut1, Glut3, retinoblastoma gene and p53 were correlated with 5-year survival in 63 patients with Stage I or II NSCLC, treated solely by surgical excision at Baylor Medical College, Houston, Texas. Several probabilistic NNs with genetic algorithm models were developed using the prognostic features as input neurons and survival at 5 years (free of disease/dead of disease) as output neurons. The probabilistic NN yielded excellent classification rates for dependent variable survival. The best model was trained with 52 cases and classified all 11 "unknown" test cases correctly. Several statistically significant logistic regression models were fitted using 50 cases to build the models and 13 cases as "hold-out" test cases. These multivariate statistical models provide various cutoff values that predict/classify the probability of survival at 5 years. In conclusion, probabilistic NNs and logistic regression models can be useful in estimating the prognosis of patients with Stage I and II NSCLC using multiple clinicopathologic and molecular variables. These multivariate predictive models need to be validated with much larger groups of patients to assess their potential clinical value.

Adult↗

PubMed1998

The AAA team: related ATPases with diverse functions.

A new family of related ATPases has emerged, characterized by a highly conserved AAA motif. This motif forms a 230-amino-acid domain that contains Walker homology sequences and imparts ATPase activity. Homology between AAA-family members is confined mostly to the AAA domain, although additional homology outside the AAA motif is present among closely related proteins. AAA proteins act in a variety of cellular functions, including cell-cycle regulation, protein degradation, organelle biogenesis and vesicle-mediated protein transport. The AAA domain is required for protein function, but its exact role and the specific activity that it confers on AAA proteins is still unclear. This review describes current understanding of the AAA protein family.

Adenosine Triphosphatases↗

PubMed1998

Low and maximally phosphorylated levels of the retinoblastoma protein confer poor prognosis in newly diagnosed acute myelogenous leukemia: a prospective study.

A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression was prognostic for survival in acute myelogenous leukemia (AML). Individuals with no/low RB protein expression were considered to have loss of RB function, and those with maximally phosphorylated (maxphos) RB were also felt to have nonfunctional RB. To confirm this, we prospectively investigated whether the level of RB expression was prognostic in AML in a larger cohort of patients. RB level was measured by Western blot and immunohistochemical analysis on peripheral blood samples from 210 newly diagnosed AML patients. Patients were divided into three groups based on the level of RB protein expression (i.e., no or low, elevated, and maxphos) or into two groups on the basis of presumed RB function, altered function (AF-RB, low and maxphos RB), versus normal function (NF-RB, elevated RB). By combined results of Western blot and immunohistochemical analysis, 20%, 65%, and 15% of patients had low, elevated, and maxphos RB, respectively. Most patients with acute promyelocytic leukemia (APL) with a French-American-British classification of M3 were in the low RB group, likely reflecting a lower proliferative rate of promyelocytes. Analysis was performed with and without these APL patients. The median survival was significantly shorter for both patients with low RB expression (48 weeks, P = 0.05, including APL patients; 34 weeks, corrected P = 0.008, with APL patients excluded) and maxphos RB expression (51 weeks, P = 0.007) compared to those with elevated RB expression (122 weeks including and 98 weeks excluding APL patients). Differences were greatest among patients with nonfavorable prognosis cytogenetics (median survival, 34 weeks versus 85 weeks; corrected P = 0.001 for AF-RB versus NF-RB). Remission duration was also significantly shorter for non-APL patients with AF-RB versus NF-RB (median survival, 36 weeks versus not reached; corrected P = 0.02). In multivariate analyses, including cytogenetics, performance status, age, antecedent hematological disorder, and RB status, with and without APL patients included, no/low and maxphos-RB protein expression were independent predictors for poorer survival. This prospective study confirms that the level of expression of RB is a strong prognostic factor in AML, with an inferior survival experience being associated with no/low RB and maxphos RB expression. Therefore, therapeutic decisions based on the level of RB expression may be indicated, and protocols to incorporate this are currently under development.

Adolescent↗

PubMed1998

Molecular characterisation of Cryptosporidium parvum from two large suspected waterborne outbreaks. Outbreak Control Team South and West Devon 1995, Incident Management Team and Further Epidemiological and Microbiological Studies Subgroup North Thames 1997.

Polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the Cryptosporidium parvum outer wall protein (COWP) gene was applied to specimens collected from 95 patients with cryptosporidiosis associated with two suspected waterborne outbreaks, 46 sporadic human cases and 62 infected livestock from other areas, and 12 patients infected with other gastrointestinal parasites. Ninety-six per cent of C. parvum isolates from patients linked to the two suspected waterborne outbreaks were of genotype 1; all the isolates from livestock were of genotype 2. Isolates from 59% of the sporadic human infections were of genotype 1 and 35% were of genotype 2. Specimens from two patients yielded both genotypes. Specimens from patients infected with other parasites yielded no amplicons.

Animals↗

PubMed1997

Human herpesvirus 8 variants in sarcoid tissues.

BACKGROUND: The cause of sarcoidosis is unknown, although mycobacteria have been implicated. We examined sarcoid tissues for human herpesvirus 8 (HHV-8) in addition to mycobacterial genomic sequences. METHODS: Biopsy samples from 17 patients with sarcoidosis were studied (eight transbronchial, 27 lymph node, two skin, and two oral mucosa). We used tissues (n = 137) from 96 patients without sarcoidosis as negative controls. A nested PCR was applied to amplify a segment of open reading frame (ORF) 26 of the HHV-8 genome, and a heminested PCR was to amplify a segment of ORF 25 of HHV-8 and of the 16 S rRNA gene of mycobacteria. Differences in base sequences of the amplified fragments were resolved with single-strand conformation polymorphism and dideoxy sequencing. FINDINGS: HHV-8 ORF 26 DNA was detected in significantly higher proportions of sarcoid than of non-sarcoid tissue samples from lung (8/8 vs 0/54; p < 0.0001), lymph nodes (26/27 vs 6/29; p < 0.0001), skin (2/2 vs 0/17; p = 0.006), and oral tissues (2/2 vs 1/13; p = 0.029). 31 (82%) of the 38 ORF 26 DNA-positive sarcoid specimens were also positive for ORF 25 DNA. For mycobacteria-like 16 S rRNA DNA, the proportion positive was significantly higher in sarcoid than non-sarcoid tissues for lymph node samples (11/27 vs 2/29; p = 0.003) but not for other tissues (lung 3/8 vs 22/54; skin 2/2 vs 15/17; and oral tissues 1/2 vs 0/13). Overall, the prevalence of HHV-8 ORF 26 sequences was higher in sarcoid tissues than in non-sarcoid tissues (p < 0.0001). When patients whose tissues were included in a masked phase of the study were treated as units of analysis, eight of eight sarcoidosis patients were positive for HHV-8 ORF 26 DNA, compared with three of 56 control patients (p < 0.0001); for mycobacteria-like sequences, three of eight sarcoidosis patients were positive, compared with four of 56 controls (p = 0.0464). The HHV-8 ORF 26 sequences, ten of which were unique, could be segregated into four groups according to peptide motifs. In seven of nine patients from whom biopsy samples were taken from various sites, different sequences were recovered. The mycobacterial sequences amplified from sarcoid tissues were also varied, but none was homologous to those of known species. INTERPRETATION: Variant HHV-8 DNA sequences are found in a wide range of sarcoid but not non-sarcoid tissues. Mycobacteria-like 16 S rRNA sequences are more frequently present in sarcoid lymph nodes and not in other tissue types, but do not indicate infection by a particular mycobacterial species.

Adult↗

PubMed1997

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% +/- 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 +/- 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Animals↗

PubMed1997

Incremental value of rubidium-82 positron emission tomography for prognostic assessment of known or suspected coronary artery disease.

Myocardial perfusion imaging using positron emission tomography (PET) may be more accurate for the diagnosis of coronary artery disease (CAD) than conventional imaging. The purpose of this study was to evaluate the prognostic implications of perfusion abnormalities in 685 patients (age 62 +/- 11 years, 199 women) studied by PET, and to assess the incremental value of these data in relation to prognostic implications of clinical and angiographic findings. Rubidium (Rb)-82 PET was performed before and after dipyridamole stress. Transient defects were detected in 227 patients (33%), and were moderate or greater in severity (> 15% of the left ventricle) in 84 (12%). Resting defects were present in 435 (64%) and were moderate or greater in severity in 216 (32%). The total extent of abnormally perfused myocardium was small (< 15% of the left ventricle) in 198 (29%), moderate in 216 (32%), and extensive in 105 (15%). Clinic review or standardized phone interview in 657 patients (96%) identified 151 cardiac events, including 81 cardiac deaths, 16 patients with myocardial infarction, 7 with unstable angina, and 47 with late revascularization (> 3 months after PET). Normal scans had a 90% event-free survival, compared with 87% in patients with small, 75% with moderate, and 76% with extensive defects (log rank chi-square 30, p <0.0001). Functional class, extent of CAD, and the presence and extent of perfusion defects (both at rest and during stress) were independent predictors of cardiac death and total cardiac events. In sequential Cox proportional-hazards models, the results of PET were incremental to those of clinical and angiographic evaluation. Thus, the presence and extent of damaged and jeopardized myocardium are independent and incremental predictors of outcome in patients undergoing Rb-82 PET.

Aged↗

PubMed1997

Molecular interactions between Hb alpha-G Philadelphia, HbC, and HbS: phenotypic implications for SC alpha-G Philadelphia disease.

We show here that alpha2(G-Phila.) beta2(C) has an increased rate of crystal nucleation compared to alpha2 beta2(C) (HbC). We conclude from this finding that position alpha68, the mutation site of alpha2(G-Phila.) beta2 (HbG(Philadelphia)), is a contact site in the crystal of HbC. In addition, that HbS enhances HbC crystallization (additive to the effect of alpha(G-Phila.) as shown here) and that alpha(G-Phila.) inhibits polymerization of HbS are pathogenically relevant previously known facts. All of these findings help explain the phenotype of an individual simultaneously heterozygous for the betaS, betaC, and the alpha(G-Phila.) genes (SC alpha-G Philadelphia disease). This disease is characterized by a mild clinical course, abundant circulating intraerythrocytic crystals, and increased folded red cells. This phenotype seems to be the result of increased crystallization and decreased polymerization brought about by the opposite effects of the gene product of the alpha(G-Phila.) gene on the betaC and betaS gene products. Some of the intraerythrocytic crystals in this syndrome are unusually long and thin, resembling sugar canes, unlike those seen in SC disease. The mild clinical course associated with increased crystallization implies that, in SC disease, polymerization of HbS is pathogenically more important than the crystallization induced by betaC chains. The SC alpha-G Philadelphia disease is an example of multiple hemoglobin chain interactions (epistatic effect among globin genes) creating a unique phenotype.

Adult↗

PubMed1997

5-(Piperidin-2-yl)- and 5-(homopiperidin-2-yl)-1,4-benzodiazepines: high-affinity, basic ligands for the cholecystokinin-B receptor.

The design, synthesis, and biological activity of a series of high-affinity, basic ligands for the cholecystokinin-B receptor are described. The compounds, which incorporate a piperidin-2-yl or a homopiperidin-2-yl group attached to C5 of a benzodiazepine core structure, are substantially more basic (e.g., 9d, pKa = 9.48) than previously reported antagonists based on 5-amino-1,4-benzodiazepines (e.g., 5, pKa = 7.1) and have improved aqueous solubility. In view of their basicity, it would be tempting to speculate that the present series of compounds might be binding to the CCK-B receptor in their protonated form. Compounds such as 9d, e and 10d showed high affinity for this receptor (IC50 < 2.5 nM) and very good selectivity over CCK-A (CCK-A/CCK-B > 2000), even as the racemates. Additionally, a significantly improved in vivo half-life was observed for a selection of compounds compared to the clinical candidate L-365, -260 (1).

Animals↗

PubMed1997

4-Heterocyclylpiperidines as selective high-affinity ligands at the human dopamine D4 receptor.

5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.

Cell Line↗

PubMed1997

A simple SDS-PAGE immunoblotting technique using an enhanced chemiluminescence detection system to identify polyclonal antibody responses to complex cryptosporidial antigen preparations following a monoclonal antibody retest and image overlay technique.

SDS-PAGE immunoblotting is a standard immunochemical technique. We describe a modification of this technique to facilitate the identification of polyclonal antibody responses to complex antigen preparations. The technique utilises an enhanced chemiluminescence (ECL) detection technique where an immunoblot is initially tested with polyclonal antibodies. The polyclonal antibodies are 'stripped' from the nitrocellulose support and the immunoblot is retested with a monoclonal antibody. Polyclonal antibody responses to individual components also recognised by the monoclonal antibody are identified by superimposing the two photographic-film images. This technique has the advantage that antigens can be identified across an individual blot despite distortions which may have occurred. An example of the use of this technique is described in which antibody responses in human sera against antigens from the coccidian parasite Cryptosporidium are identified.

Animals↗

PubMed1997

Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes.

Adipose tissue is an important storage depot for retinol, but there are no data regarding retinol mobilization from adipose stores. To address this, dibutyryl cAMP was provided to murine BFC-1beta adipocytes and its effects on retinol efflux assessed. High performance liquid chromatography analysis of retinol and retinyl esters in adipocytes and media indicated that cAMP stimulated, in a time- and dose-dependent manner, retinol accumulation in the culture media and decreased cellular retinyl ester concentrations. Study of adipocyte retinol-binding protein synthesis and secretion indicated that cAMP-stimulated retinol efflux into the media did not result from increased retinol-retinol-binding protein secretion but was dependent on the presence of fetal bovine serum in the culture media. Since our data suggested that retinyl esters can be hydrolyzed by a cAMP-dependent enzyme like hormone-sensitive lipase (HSL), in separate studies, we purified a HSL-containing fraction from BFC-1beta adipocytes and demonstrated that it catalyzed retinyl palmitate hydrolysis. Homogenates of Chinese hamster ovary cells overexpressing HSL catalyzed retinyl palmitate hydrolysis in a time-, protein-, and substrate-dependent manner, with an apparent Km for retinyl palmitate of 161 microM, whereas homogenates from control Chinese hamster ovary cells did not.

Adipocytes↗

PubMed1997

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast.

Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy. In this paper, we report the characterization of the C. albicans TOP2 gene and its use to develop a yeast system that allows the identification and study of anti-fungal topoisomerase II inhibitors in vivo. The gene, specifying a 1461-residue polypeptide with only 40% identity with human topoisomerase IIalpha and beta isoforms, was isolated from C. albicans on a 6.3 kb EcoRI fragment that mapped to chromosome 4. It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57-1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter. The plasmid rescued the lethal phenotype of a temperature-sensitive S. cerevisiae DNA topoisomerase II mutant allowing growth at 35 degrees C. Yeast cells, bearing ISE2 permeability and rad52 double-strand-break-repair mutations the growth of which at 35 degrees C was dependent on C. albicans topoisomerase II, were killed by the known topoisomerase II inhibitors amsacrine and doxorubicin. Parallel experiments in yeast expressing human topoisomerase IIalpha allowed the relative sensitivities of the fungal and host topoisomerases to be examined in the same genetic background. To compare the killing in vivo with drug inhibition in vitro, the recombinant C. albicans topoisomerase II protein was expressed and purified to near-homogeneity from S. cerevisiae yielding a 160 kDa polypeptide that displayed the expected ATP-dependent DNA-relaxation and DNA-decatenation activities. The enzyme, whether examined in vitro or complementing in S. cerevisiae, was comparably sensitive to amsacrine and doxorubicin. Our results suggest that potential topoisomerase II-targeting anti-fungal inhibitors can be identified and studied in S. cerevisiae.

Amino Acid Sequence↗