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Biomedical subjects

A Dasgupta

Publications and source records attributed to A Dasgupta.

At least 127 records · Page 7Linked to original sources
PubMed1997

Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro.

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. Previous studies from our laboratory showed that activated transcription from a cyclic AMP-responsive element (CRE)-containing promoter was severely inhibited in extracts prepared from poliovirus-infected HeLa cells compared to those from mock-infected cells. Here we demonstrate that the CRE-binding protein, CREB, is specifically cleaved by the poliovirus-encoded protease 3Cpro both in vitro and in virus-infected cells. The proteolytic cleavage of CREB leads to a significant loss of its DNA binding as well as transcriptional activity. Additionally, we demonstrate that the phosphorylated, transcriptionally active form of CREB is cleaved by the viral protease in vitro. The results presented here suggest that a direct cleavage of CREB by the viral protease 3Cpro leads to inhibition of CREB-activated transcription in poliovirus-infected HeLa cells.

Amino Acid Sequence↗
PubMed1997

Poliovirus-encoded protease 2APro cleaves the TATA-binding protein but does not inhibit host cell RNA polymerase II transcription in vitro.

Transient expression of the poliovirus-encoded protease 2APro in eukaryotic cells results in inhibition of both cellular transcription and translation. The inhibition of transcription observed in cells expressing 2APro could be due to a primary effect or secondary effect caused by inhibition of translation. Because transcriptional activity of the TATA-binding protein (TBP) is drastically reduced in poliovirus-infected cells, we determined if 2APro is able to cleave TBP in vitro. We demonstrate here that 2APro directly cleaves the single tyrosine-glycine bond at position 34 of TBP. This cleavage is also seen in poliovirus-infected HeLa cells. Surprisingly, despite TBP cleavage 2APro was unable to inhibit RNA polymerase II transcription in vitro. Under similar conditions, however, 2APro inhibited translation of a capped cellular mRNA in vitro. Thus, cleavage of TBP at position 34 does not alter its transcriptional activity in vitro. These results suggest that inhibition of host cell RNA polymerase II transcription seen in cells transiently transfected with 2APro is due to host cell translational shutoff.

Amino Acid Sequence↗
PubMed1997

A novel derivatization of phenol after extraction from human serum using perfluorooctanoyl chloride for gas chromatography-mass spectrometric confirmation and quantification.

Phenol (carbolic acid) is widely used as a disinfectant as well as in the chemical industry as an intermediate in the synthesis of a variety of chemicals. Phenol is also the major metabolite of benzene which is used in many commercial solvents. Phenol is toxic and caustic and may cause death even from dermal absorption. Therefore, measurement of phenol in postmortem blood is essential. The concentration of phenol in blood can be measured by gas chromatography with flame ionization or mass spectrometry. Phenol can also be analyzed by high performance liquid chromatography. However, in forensic toxicology, unambiguous confirmation of phenol by mass spectrometry is as important as quantification in blood. Here we describe a novel derivatization of phenol after extraction with chloroform from human serum using perfluorooctanoyl chloride. The perfluorooctanoyl derivative of phenol showed a strong molecular ion at m/z 490 (relative abundance: 23%) whereas the base peak was observed at m/z 77. The derivative of the internal standard 3,4-dimethylphenol showed a very strong molecular ion at m/z 518 (relative abundance: 56%) and the base peak was observed as m/z 121. The derivative of p-cresol, a chemically related phenolic compound, showed a strong molecular ion at 504 m/z (relative abundance: 54%) and a base peak at m/z 107. We observed baseline separation between derivatized phenol (retention time: 6.1 min), p-cresol (retention time: 7.8 min), and the internal standard (retention time: 9.4 min). We observed no interferences in our assay from grossly hemolyzed serum. Within and between run precision was studied using a serum standard containing 25 mg/L of phenol. The within run precision was 6.6% (mean = 24.3, SD = 1.6 mg/L, n = 8) whereas the between run precision was 8.6% (mean = 25.5, SD = 2.2 mg/L, n = 8). The assay was linear for serum phenol concentrations of 10-200 mg/L. The detection limit was 1 mg/L of serum phenol concentration. The average recoveries were 92.1% to 94.0% for various serum phenol concentrations.

Caprylates↗
PubMed1997

Gas chromatographic-mass spectrometric identification and quantitation of benzyl alcohol from human serum and postmortem blood after derivatization with 4-carbethoxy hexafluorobutyryl chloride: a novel derivative.

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high performance liquid chromatography. We describe a novel derivatization of benzyl alcohol using 4-carbethoxyhexafluorobutyryl chloride after extraction from human plasma, and subsequent analysis by gas chromatography-mass spectrometry (GC/MS). The derivative was eluted at a significantly higher temperature and the method was free from interferences from more volatile components in serum and hemolyzed specimens. However, with postmortem specimens, we observed multiple peaks which were eluted at a very high temperature, long after derivatized benzyl alcohol and the internal standard. Therefore, baking the column at 310 degrees C is recommended after analysis of a postmortem specimen. Another advantage of this derivatization technique is the conversion of low molecular weight benzyl alcohol (MW 108) to a high molecular weight derivative (MW 358). The positive identification of benzyl alcohol can be easily achieved by observing a distinct molecular ion at m/z 358 as well as other characteristic ions at m/z 107 and 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 50 mg/L) were 2.2% (mean = 50.6, SD = 1.1 mg/L), and 6.9% (mean = 50.8, SD = 3.5 mg/L). The assay was linear for the serum benzyl alcohol concentrations of 5 mg/L to 200 mg/L and the detection limit was 1 mg/L. We observed no carry-over problem in our assay as when 2 microL ethyl acetate was injected into the GC/MS after analyzing serum specimens containing 200 mg/L of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.

Anesthetics↗
PubMed1997

Determination of phenmetrazine in urine by gas chromatography-mass spectrometry after liquid-liquid extraction and derivatization with perfluorooctanoyl chloride.

Phenmetrazine is a central nervous system stimulant and is currently used as an anorectic agent. The drug is abused and reported to cause death from overdose. We describe a liquid-liquid extraction protocol for phenmetrazine from urine using 1-chlorobutane and subsequent derivatization using perfluorooctanoyl chloride for gas chromatography-mass spectrometric confirmation. Quantitation of urinary phenmetrazine can be easily achieved by using N-propylamphetamine as an internal standard. The perfluorooctanoyl derivative of phenmetrazine showed a weak molecular ion at m/z 573 and a characteristic strong peak at m/z 467 in the electron ionization mass spectrometry thus aiding unambiguous identification. The perfluorooctanoyl derivative of the internal standard did not show any molecular ion, but showed strong characteristic peaks at m/z 482 and 440. The within run and between run precisions of the assay were 1.7% and 3.2% at a urinary phenmetrazine concentration of 20 microgram/mL. The within run and between run precisions were higher (9.4% and 10.8%) at a urinary phenmetrazine concentration of 1.0 microgram/mL, which was very close to the detection limit of the assay. The assay was linear for urinary phenmetrazine concentration of 1 to 100 micrograms/mL with a detection limit of 0.5 microgram/mL.

Amphetamines↗
PubMed1997

Breast feeding practices in a teaching hospital of Calcutta before and after the adoption of BFHI (Baby Friendly Hospital Initiative).

A comparative study has been made on two groups of 102 mothers each who delivered children in the postnatal ward of obstetrics and gynaecology department of Calcutta National Medical College before and after the introduction of BFHI (Baby Friendly Hospital Initiative). The study revealed that only 14.3% of the babies who were delivered normally were given their first breast feed in time, the ideal time of half an hour, while not a single baby delivered by caesarean section were given their breast feed within the stipulated time period of 4-6 hours. However, there has been a significant overall reduction in the time gap between the birth and the first breast feed in all types of delivery. BFHI has also made significant reduction of prelacteal feeds and in-between feeds in the newborns especially those delivered normally. The fact that babies of first order and those delivered by caesarean section are lagging behind as far as exclusive breast feeding is concerned has been highlighted in the study.

Breast Feeding↗
PubMed1997

Calcium and vitamin D status of pregnant teenagers in Maiduguri, Nigeria.

This study investigates parameters related to calcium and bone metabolism by determining the concentrations of total calcium, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, and phosphorous in young pregnant women. The patient population was 30 pregnant Nigerian teenage women grouped by trimester (10 per group), 10 women immediately following delivery, and 21 healthy age-matched controls. On the basis of serum prealbumin levels, the general nutrition of the pregnant women was found to be significantly below that of the more privileged and better-educated nonpregnant controls. The mean total calcium concentration in sera of the third-trimester women was 8.83 mg/dL, which was significantly below that of the controls (9.77 mg/dL) and the first-trimester group (9.30 mg/dL). Despite the 10% to 15% decline in the serum level of total calcium during pregnancy, the parathyroid hormone level decreased markedly from 0.60 to 0.61 ng/mL in the first and second trimesters to 0.41 ng/mL in the third trimester. Serum vitamin D and 1,25-dihydroxyvitamin D levels in the second and third trimesters were within the normal range. These data indicate that toward the end of gestation, pregnant teenagers in northern Nigeria appear to become calcium deficient and do not exhibit the expected increase in serum parathyroid hormone levels normally seen in pregnant women.

Adolescent↗
PubMed1997

Argyrophilic nucleolar organiser regions (AgNORs) in breast lesions.

Nucleolar organiser regions (NORs) demonstrated by argyrophilia of NOR-proteins are indicator of cellular proliferative activity. The NORs can be identified in the nuclei as brown or black dots with silver colloidal staining technique in formalin fixed paraffin sections and in cytology smears. Seventy-five cases including 45 tissue sections and 30 fine needle aspiration cytology (FNAC) smears of benign and malignant lesions of breast have been studied to evaluate the significance and practical application of AgNOR count per nucleus. Out of 45 tissue sections 15 belonged to fibrocystic disease, 10 fibro-adenomas and 20 carcinomas and of the 30 FNAC smears, 10 were fibrocystic disease, 8 fibro-adenomas and 12 carcinomas. In fibrocystic disease the mean AgNOR count was 1.60 (FNAC group-0.75, tissue section-1.61). In fibro-adenomas it was 1.61 (FNAC-1.63, tissue section-1.59). The mean count in carcinoma was 12.10 (FNAC-12.08, tissue section-12.10). The difference in AgNOR count in fibrocystic disease and fibro-adenoma was not significant, but that between benign breast lesion and carcinoma was significant. No difference was observed between FNAC and tissue section groups in benign or malignant lesions. The simple staining technique can be used as an additional criterion to differentiate the benign and malignant lesions of breast.

Biopsy, Needle↗
PubMed1996

Apoptosis is associated with an increase in saturated fatty acid containing phospholipids in the neuronal cell line, HN2-5.

Two widely different paradigms of stress, hypoxia and nutrient deprivation, were observed to trigger apoptosis in a clonal neuronal (hippocampal) cell line (HN2-5) as judged by DNA laddering analysis and chromatin condensation. Since according to its original definition, apoptosis is concomitant with a dramatic change in cell morphology, the composition of major phospholipids that determine morphological properties of cells was analyzed in this study. We observed a significant increase (10-20%) in the proportion of saturated fatty acid side chains only in phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) but not in phosphatidylcholine (PC). This increase was independent of the paradigm of stress used (hypoxia or nutrient deprivation) to initiate apoptosis. Since phospholipids constitute the fabric of both plasma membranes as well as the membranes surrounding the intracellular organelles such as the lysosomes, endoplasmic reticulum and the nuclei, such an apoptosis-associated, selective change in phospholipid composition could cause a dramatic and overall change in membrane fluidity, thus producing the typical shrinking, deformation and porosity of both nuclear as well as plasma and lysosomal membranes as observed in apoptotic cells. Leakiness of lysosomal and nuclear membranes could cause diffusion of deoxyribonucleases into cell nuclei, thus resulting in the characteristic cleavage and laddering of chromosomal DNA which accompany apoptosis.

Antibodies↗
PubMed1996

O-linked glycosylation modifies CD44 adhesion to hyaluronate in colon carcinoma cells.

CD44 alternative splicing patterns differ between normal and malignant tissue, and accordingly, modulation of CD44 splicing has received the most attention in studies that have examined the role of CD44 in tumor progression. Many investigators have examined functional differences between individual CD44 alternative splice variants. However, specific CD44 isoforms function uniquely depending on the type of cell on which they are expressed, thereby suggesting that additional tissue-specific mechanisms regulate CD44 function. In the present study we have demonstrated that colon carcinoma cells modify CD44 with O-linked glycosyl groups, and blockade of this glycosylation enhances their CD44-mediated adhesion to hyaluronate. This enhancement is attributable principally to CD44H (CD44s) rather than high molecular weight CD44 variants. Use of site-directed mutant CD44H cDNA transfectants demonstrated that CD44 O-linked glycosylation modulates interaction between hyaluronate and the B loop domain of CD44. The influence of glycosylation on CD44 function in colon carcinoma cells is specific to the presence of O-linked sugars; inhibition of N-linked glycosylation had minimal influence on CD44 function. These findings indicate that O-linked glycosylation may be as important as alternative splicing in the regulation of CD44 function and the broad spectrum of biological processes attributed to it, including normal development, tumor metastases, and lymphocyte function.

Alternative Splicing↗
PubMed1996

Keratan sulfate modification of CD44 modulates adhesion to hyaluronate.

CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function.

Alternative Splicing↗
PubMed1996

Significantly different fatty acid profiles in various phospholipid head groups of neuroblastoma cell lines.

We studied lipid profiles of hybrid cells derived from fusion of sympathetic ganglia and neuroblastoma cells (NCB 20, F 11) and also CHO (Chinese hamster ovary) cells. The proportion of saturated to unsaturated fatty acids changed significantly in different phospholipid fractions. We observed 35.4% of total fatty acids as saturated fatty acids in the phosphatidyl ethanolamine (PE) fraction of NCB-20 cell lines while 47.2% of total fatty acids were saturated in the phosphatidyl inositol (PI) fraction. In general, in neuroblastoma cell lines, we observed the lowest proportion of saturated fatty acids in the PE fraction while the other lipid fraction showed a much higher proportion of saturated fatty acids. On the other hand the PE fraction of a non neuronal cell line, CHO, showed 57.4% saturated fatty acids in the PE fraction. This significant difference in saturated to unsaturated fatty acid ratios in different phospholipid head groups may be linked to the different biological functions of those hybrid cells.

Animals↗
PubMed1996

Estimating concentrations of total digoxin and digoxin-like immunoreactive substances in volume-expanded patients being treated with digoxin.

High concentrations of digoxin-like immunoreactive substances (DLIS) artificially increase serum digoxin concentrations. However, DLIS are absent in the protein-free ultrafiltrate because of their strong binding with serum macromolecules, whereas approximately 75% of digoxin can be found in the ultrafiltrate. Using regression analysis, we devised equations by which total digoxin concentration can be calculated from free digoxin and albumin concentrations in serum. We used two different assays, fluorescence polarization and chemiluminescence, for measuring total and free-digoxin concentrations in sera. Both equations were very similar. Because measured concentrations of digoxin in the serum exhibit DLIS interferences, the measured concentrations were sometimes higher in volume-expanded patients than the calculated digoxin concentrations. We also estimated the extent of interferences from DLIS by subtracting the calculated digoxin concentration from the measured digoxin concentration in volume-expanded patients.

Cardenolides↗
PubMed1996

In vitro displacement of phenytoin from protein binding by nonsteroidal antiinflammatory drugs tolmetin, ibuprofen, and naproxen in normal and uremic sera.

Displacement of phenytoin (90% bound to albumin) by other highly albumin-bound drugs like salicylate has been well documented. Other widely used nonsteroidal antiinflammatory drugs like tolmetin, ibuprofen, and naproxen are also strongly bound to albumin and can potentially displace phenytoin. However, phenytoin-ibuprofen interaction has been poorly studied in the past, and interaction of phenytoin with tolmetin or naproxen has not been studied before. For normal serum pool (albumin 3.7 g/dl), we observed significant increases in free phenytoin concentrations only with antiinflammatory drug concentrations at the upper end of therapeutic or above therapeutic concentrations. However, for the uremic pool (albumin 2.9 g/dl), displacement of phenytoin was significant even at the lower end of therapeutic concentrations of those antiinflammatory drugs. Of the three antiinflammatory drugs we studied, ibuprofen caused the highest displacement of phenytoin.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Displacement of valproic acid and carbamazepine from protein binding in normal and uremic sera by tolmetin, ibuprofen, and naproxen: presence of inhibitor in uremic serum that blocks valproic acid-naproxen interactions.

Displacement of valproic acid (90-95% bound to albumin) and carbamazepine (80% bound to albumin) by salicylate, leading to higher concentrations of pharmacologically active free drugs, has been reported. We studied the possibility of displacement of valproic acid and carbamazepine by other strongly albumin-bound nonsteroidal antiinflammatory drugs tolmetin, ibuprofen, and naproxen. We observed statistically significant displacement of carbamazepine from protein binding in uremic serum at higher therapeutic concentrations of all three antiinflammatory drugs we studied, whereas in normal serum, we observed statistically significant displacement only with 75 micrograms/ml of naproxen. For valproic acid, we observed significant displacement even at lower therapeutic concentrations with all three drugs when the study was conducted using a normal serum pool. In the uremic serum pool, we observed significant displacements only with tolmetin and ibuprofen, whereas we observed no significant displacement of valproic acid even with higher concentrations of naproxen. We conclude that tolmetin, naproxen, and ibuprofen can displace both carbamazepine and valproic acid from protein binding, but uremic serum contains an inhibitor that blocks valproic acid-naproxen interaction.

Binding, Competitive↗