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Shalini Gupta

Publications and source records attributed to Shalini Gupta.

At least 19 recordsLinked to original sources
PubMed2006

Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum.

An in vitro cell-based bioassay was developed and validated to assess the pharmacokinetic profiles of two novel therapeutic recombinant proteins (EP1 and EP2) with erythropoiesis stimulating properties in Sprague-Dawley rats. While immunoassays are the standard choice for evaluating the pharmacokinetic parameters of drugs, no immunoassay was available for EP2, necessitating the need for a quantitative bioassay capable of measuring both EP1 and EP2 separately so that appropriate comparisons could be made. The bioassay described here utilizes a sub clone of the murine 32D cell line transfected with the gene encoding for the human erythopoietin (HuEPO) receptor. Erythropoietin (EPO), EP1 and EP2 exert their proliferative effect on the cell line by signaling through the HuEPO receptor. The proliferation induced by the erythropoietic proteins was measured by [methyl-(3)H]thymidine incorporation into the cellular DNA. The assay was conducted in 96-well microtiter plates and had relatively high throughput. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of the different assay parameters including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity. The robustness of the bioassay is demonstrated by the lack of an effect of age of the 32D cell culture on the performance of the EP2 bioassay. The bioassay demonstrated good linearity, yielding a coefficient of determination of 0.99 or higher for both EP1 and EP2. The assay showed reproducible dose-response curves for EP1 in the range of 0.039-2.5 ng/mL and for EP2 in the range of 0.125-8 ng/mL. The accuracy estimates ranged between 98% and 108% for EP1 and between 90% and 110% for EP2 in the reproducible range mentioned above. Intermediate precision (within-plate R.S.D.) in the same range was within 26% and 17% for the EP1 and EP2 bioassays, respectively. The validated bioassays for EP1 and EP2 were utilized to quantitatively analyze serum samples from a pharmacokinetic study conducted to compare the profiles of the two compounds in Sprague-Dawley rats.

Animals↗
PubMed2006

Breast cancer CADx based on BI-RAds descriptors from two mammographic views.

In this study we compared the performance of computer aided diagnosis (CADx) algorithms based on Breast Imaging Reporting And Data System (BI-RADS) descriptors from one or two views. To select cases for the study with different mediolateral (MLO) and craniocaudal (CC) view descriptors, we assessed the agreement in BI-RADS lesion descriptors, BI-RADS assessment, and subtlety ratings for 1626 cases from the Digital Database for Screening Mammogrpahy (DDSM) using kappa statistics. We used 115 mass caseswith different descriptors for the two views to design linear discriminant analysis (LDA) based CADx algorithms. The CADx algorithms used BI-RADS descriptors and patient age as features. Thealgorithms based on BI-RADS descriptors from both the views performed marginally betterthan algorithms based on BI-RADS descriptors from a single view. A system that averaged theresults of two classifiers trained separately on the MLO and CC views displayed the best performance (Az=0.920 +/- 0.027). Thus, some improvement in performance of BI-RADS based CADx algorithms may be achieved by combining information from two mammographic views.

Breast↗
PubMed2005

Risk assessment of p53 genotypes and haplotypes in tobacco-associated leukoplakia and oral cancer patients from eastern Idia.

The role of 3 p53 polymorphisms (16 bp duplication at intron 3, codon 72 Arg/Pro and intron 6 NciI RFLP at np 13494) as potential markers for indicating cancer risk remains inconclusive. In our case-control study consisting of 197 leukoplakia and 310 oral squamous cell carcinoma (SCC) patients and 348 controls, genotype frequencies at these 3 p53 loci were determined by PCR-RFLP method and analyzed by multiple logistic regression to determine the risks of the diseases. The 2/2 genotype at codon 72 of p53 was at risk for developing leukoplakia (OR = 1.6, 95% CI 1.1-2.3), whereas the combination of 1/2 and 2/2 genotypes at intron 3 and 1/1 and 1/2 genotypes at intron 6 conferred a protective effect against leukoplakia and oral SCC development, respectively (OR = 0.5, 95% CI 0.4-0.8 and OR = 0.6, 95% CI 0.5-0.9, respectively). When subjects were stratified according to specific tobacco habit, the risk/protection estimates improved significantly in some cases. Specifically, the exclusive smokers with p53 codon 72 2/2 genotype showed a higher risk of developing leukoplakia (OR = 2.7, 95% CI 1.2-6.3). Furthermore, a particular p53 haplotype 1-2-2 was at risk for both tobacco-associated leukoplakia and oral SCC (OR = 1.5, 95% CI 1.1-1.9 and OR = 1.3, 95% CI 1.1-1.7, respectively). Our results show that both specific p53 genotype and haplotype can indicate risk of tobacco-associated leukoplakia, but risk of development of tobacco-associated oral SCC can be predicted by specific p53 haplotype only.

Carcinoma, Squamous Cell↗
PubMed2005

Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry.

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.4, 3.1, 4.5, or 6.7M trifluoroacetic acid at 37, 65, or 95 degrees C for 30min to 24h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.

Acetylglucosamine↗
PubMed2005

Capturing SDS-treated biotinylated protein and peptide by avidin functional affinity electrophoresis with or without SDS in the gel running buffer.

Avidin functional affinity electrophoresis (AFAEP) is a new method of affinity electrophoresis. In this technique, bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution to embed avidin within the gel matrix by interaction with the amino/amide groups. Samples are heated with triglycine sodium dodecyl sulfate (SDS) sample buffer to ensure that biotinylated proteins biotinylated peptides are negatively charged and migrate electrophoretically toward the cathode through the avidin zone regardless of their pI values. The AFAEP method allows the capture and concentration of biotinylated proteins or biotinylated peptides irrespective of the use of SDS in both the sample buffer and the gel running buffer.

Animals↗
PubMed2005

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis.

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.

Antibodies↗
PubMed2005

Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin-biotinylated peptide complexes was excised from a 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95 degrees C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65 degrees C for 20 min. However, some AFAEP-captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time.

Avidin↗
PubMed2005

Characterization of oligosaccharide moieties of glycopeptides by microwave-assisted partial acid hydrolysis and mass spectrometry.

Microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to study oligosaccharide structures of glycopeptides. Tryptic N-glycosylated peptides of horseradish peroxidase, with MH+ ions at m/z 2533, 2612, 3355, 3673, and 5647, were used as test cases. Within a microwave exposure with trifluoroacetic acid of 2 min, partial cleavages of the oligosaccharides of these tryptic N-glycosylated peptides were observed. The data showed that the most labile group within the oligosaccharides is the fucose (Fuc) residue, and that a majority of the end cleavage products are peptides with one N-acetylglucosamine (GlcNAc) residue linked to asparagine (Asn). In addition, the glycopeptides with m/z 3355 and 3673 carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, the glycopeptide at m/z 5647 carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and the glycopeptides at m/z 2612 and 2533 carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the m/z 2612 peptide at Asn286 is partially occupied. This simple and rapid method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.

Acids↗
PubMed2005

Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry.

A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.

Acids↗
PubMed2005

Correspondence in texture features between two mammographic views.

It is well established that radiologists are better able to interpret mammograms when two mammographic views are available. Consequently, two mammographic projections are standard: mediolateral oblique (MLO) and craniocaudal (CC). Computer-aided diagnosis algorithms have been investigated for assisting in the detection and diagnosis of breast lesions in digitized/digital mammograms. A few previous studies suggest that computer-aided systems may also benefit from combining evidence from the two views. Intuitively, we expect that there would only be value in merging data from two views if they provide complementary information. A measure of the similarity of information is the correlation coefficient between corresponding features from the MLO and CC views. The purpose of this study was to investigate the correspondence in Haralick's texture features between the MLO and CC mammographic views of breast lesions. Features were ranked on the basis of correlation values and the two-view correlation of features for subgroups of data including masses versus calcification and benign versus malignant lesions were compared. All experiments were performed on a subset of mammography cases from the Digital Database for Screening Mammography (DDSM). It was observed that the texture features from the MLO and CC views were less strongly correlated for calcification lesions than for mass lesions. Similarly, texture features from the two views were less strongly correlated for benign lesions than for malignant lesions. These differences were statistically significant. The results suggest that the inclusion of texture features from multiple mammographic views in a CADx algorithm may impact the accuracy of diagnosis of calcification lesions and benign lesions.

Algorithms↗
PubMed2004

Catching and separating protein ligands by functional affinity electrophoresis.

A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.

Antibodies, Monoclonal↗
PubMed2004

Catching protein antigens by antibody affinity electrophoresis.

A new kind of affinity electrophoresis called antibody affinity electrophoresis is a technique used to capture protein antigens based on their interactions with specific monoclonal or polyclonal antibodies incorporated in the polyacrylamide gel. Polyclonal anti-glutathione-S-transferase (anti-GST), monoclonal anti-bovine serum albumin (anti-BSA), and polyclonal anti-human alpha-lactalbumin are embedded in distinct areas of a 7.5% native polyacrylamide gel. Some of the embedded antibodies get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these antibodies do not show significant electrophoretic mobility, as compared to their specific protein antigen analytes. We observed that electrophoretic migration of GST, BSA, and protein G ceases when they encounter anti-GST, anti-BSA, and immunoglobulin G, respectively.

Acrylic Resins↗
PubMed2004

Development and validation of a cell-based bioassay for the detection of neutralizing antibodies against recombinant human erythropoietin in clinical studies.

An in vitro cell-based bioassay capable of detecting neutralizing antibodies (NAb) to recombinant human erythropoietin (rHuEPO) in clinical samples was developed and validated. The bioassay uses the IL-3-dependent murine 32D cell line transfected with human EPO receptors (EPOR). This cell line responds to rHuEPO with proliferation measurable by [methyl-3H] thymidine incorporation into the cellular DNA. The reduction of rHuEPO-induced cell proliferation response indicates the possible presence of anti-rHuEPO NAb. In addition, a specificity assay using murine IL-3 (mIL-3) induced proliferation of the same cell line was developed and validated. The specificity assay allowed testing of samples that inhibited the biologic activity of rHuEPO to evaluate whether the inhibition was specific and not attributable to cytotoxicity of the serum sample. Both assays are conducted in a 5% human serum matrix in 96-well microtiter plates. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of different assay parameters including analytical recovery, precision, sensitivity, specificity, selectivity, and robustness. The anti-rHuEPO NAb assay is capable of detecting concentrations of NAb equivalent to 500 ng/ml of the positive control antibody in undiluted human serum. The anti-rHuEPO NAb assay yielded consistent results with cells cultured for up to 30 days. The positive control antibody maintained its ability to inhibit the biologic activity of rHuEPO upon freezing and thawing. The presence of free rHuEPO in serum samples interfered with the detection of the antibody. The validated assay was sensitive, specific and robust and was successfully used to monitor NAb development in patients.

Antibodies↗
PubMed2003

Effect of labor on glucocorticoid receptor (GR(Total), GRalpha, and GRbeta) proteins in ovine intrauterine tissues.

OBJECTIVE: Activation of the fetal hypothalamic-pituitary-adrenal axis and a concurrent increase in plasma cortisol concentration plays a crucial role in parturition in sheep. The effects of cortisol on intrauterine tissues are mediated, in part, by glucocorticoid receptors (GR). In the present study, we determined the temporal and tissue-specific expression of GR(Total), GRalpha, and GRbeta isoforms in ovine placenta, fetal membranes, and uterus during the onset and progression of labor. METHODS: Intrauterine tissues were collected from following three groups of pregnant ewes at term (147-149 days of gestation): animals not in labor (NIL; 140-145 days; n = 5), early labor (EL; 143-149 days; n = 6), and active labor (L; 145-149 days; n = 6). They were analyzed for GR(Total), GRalpha, and GRbeta by immunohistochemical and Western blot analyses. RESULTS: GR(Total), GRalpha, and GRbeta proteins were present in trophoblast cells but not maternal tissue of the placentome. GRalpha and beta were distinctly expressed in trophoblast nuclei and cytosol, respectively. GR(Total), GRalpha, and GRbeta were also detected in amnion epithelial cells and chorionic epithelium of the fetal membranes, and the maternal endometrial epithelium. GR(Total) and GRalpha were also present in amnion mesenchymal and glandular epithelium. GR(Total) and GRalpha (95 kDa) protein levels were significantly (P < .05) higher during labor; GRbeta (90 kDa) levels remained unchanged. CONCLUSION: The temporal and tissue-specific pattern of GR expression within ovine intrauterine tissues during labor implicates GR in regulating cortisol action to induce enzymatic changes controlling labor in sheep.

Amnion↗