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Biomedical subjects

A Dasgupta

Publications and source records attributed to A Dasgupta.

At least 145 records · Page 8Linked to original sources
PubMed1996

Stability of therapeutic drug measurement in specimens collected in VACUTAINER plastic blood-collection tubes.

Recently Becton-Dickinson marketed a plastic serum-separator tube that uses the same serum-separator gel as the glass tubes. We studied the stability of therapeutic drugs stored in plastic tubes by comparing it with the stability of drugs stored in glass serum-separator tubes and plain red-top glass tubes. We observed no absorption of caffeine, primidone, N-acetylprocainamide, procainamide, theophylline, tobramycin, ethosuximide, acetaminophen, amikacin, valproic acid, methotrexate, salicylate, and cyclosporine in either plastic or glass serum-separator tubes. On the other hand, concentrations of lidocaine, quinidine, phenobarbital, and phenytoin were reduced after storing in both plastic and glass serum-separator tubes, especially with prolonged storage and small sample volume. The reduction in concentrations were due to slow absorption of those drugs by serum-separator gel.

Blood Specimen Collection↗
PubMed1996

Interaction of valproic acid with nonsteroidal antiinflammatory drugs mefenamic acid and fenoprofen in normal and uremic sera: lack of interaction in uremic sera due to the presence of endogenous factors.

Valproic acid is an anticonvulsant that is strongly bound to serum albumin. The nonsteroidal antiinflammatory drugs mefenamic acid and fenoprofen are also strongly bound to albumin. We observed significant displacement of valproic acid from protein binding by mefenamic acid and fenoprofen at both therapeutic and slightly above therapeutic concentrations. The concentration of free valproic acid was higher in the uremic serum, as expected, but we observed no further displacement of valproic acid in the presence of mefenamic acid and fenoprofen. Known uremic compounds, hippuric acid and indoxyl sulfate, did not inhibit the interactions between valproic acid and mefenamic acid or fenoprofen. Treatment of uremic serum with activated charcoal at pH 3.0 removes endogenous interfering factors and corrects the binding defect of uremic serum for valproic acid. We observed significant displacement of valproic acid from protein binding by both mefenamic acid and fenoprofen in uremic serum after charcoal treatment. We conclude that endogenous factors are present in uremic sera that block interaction of valproic acid with mefenamic acid and fenoprofen.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity.

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.

Autoantigens↗
PubMed1996

Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro.

Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.

3C Viral Proteases↗
PubMed1996

DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease.

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.

3C Viral Proteases↗
PubMed1996

Diminished protein binding capacity of uremic sera for valproate following hemodialysis: role of free fatty acids and uremic compounds.

We previously reported higher free phenytoin concentration in predialysis serum compared to postdialysis serum. In our present study with valproic acid, we observed an opposite effect, where free valproic acid concentration was higher in postdialysis serum. The increased free valproic acid concentrations in postdialysis sera (15.4-77.4% higher in postdialysis sera) were probably related to a 2-to 4-fold increase in free fatty acid concentrations in postdialysis sera. On the other hand, when an aliquot of predialysis serum pool was subjected to in vitro equilibrium dialysis and then both undialyzed and dialyzed serum pools were supplemented with valproic acid and incubated, the concentration of valproic acid in dialyzed serum was significantly lower than the undialyzed serum. This is probably related to removal of some uremic compounds during in vitro dialysis. Treatment of both pre- and postdialysis sera with activated charcoal corrected the binding deficiency of valproic acid due to removal of both uremic compounds and free fatty acids.

Anticonvulsants↗
PubMed1996

Elimination of fluconazole interference in gas chromatography/mass spectrometric confirmation of benzoylecgonine, the major metabolite of cocaine using pentafluoropropionyl derivative.

Cocaine is a widely abused drug and causes death from overdose. Benzoylecgonine, the major metabolite of cocaine in urine is usually confirmed after derivatization by gas chromatography/mass spectrometry to demonstrate cocaine abuse. Recently, Wu et al. demonstrated that fluconazole coelutes with benzoylecgonine after conversion to trimethylsilyl analogs and causes false-negative result in the confirmation test. However, fluconazole did not interfere with the screening assay using a enzyme multiplied immunoassay technique. We demonstrated that by converting benzoylecgonine to the corresponding pentafluoropropionyl derivative, the interference of fluconazole can be completely eliminated. The pentafluoropropionyl derivative of benzoylecgonine eluted at 14.7 min while the derivatized fluconazole eluted at 15.6 min. The mass spectral fragmentation pattern of derivatized benzoylecgonine was distinctively different from the mass spectral features of derivatized fluconazole in both electron ionization and chemical ionization mode of operation of mass spectrometers. The quantitation of benzoylecgonine in positive urine specimens has not affected when the specimens were supplemented with 50 micrograms/mL of fluconazole.

Cocaine↗
PubMed1996

Methods of stem cell mobilization.

The rapid rescue of hematopoiesis following transplantation of peripheral blood stem cells (PBSC) has facilitated expanded application of high-dose chemotherapy regimens for several malignancies. A variety of regimens have been described to enhance the circulation of PBSC, including the use of hematopoietic growth factors, either alone or following myelosuppressive chemotherapy. Such improvements of PBSC mobilization can increase the number of hematopoietic progenitors for transplantation and reduce the number of leukapheresis procedures required. We review several approaches to the collection of PBSC, including our experience with the use of cytokines in addition to chemotherapy. In addition, we discuss the investigation of novel cytokine combinations and in vitro expansion techniques, which may lead to further improvements in both the efficiency of PBSC collection and hematopoietic recovery following transplantation.

Antineoplastic Agents↗
PubMed1996

Prevention of occupational cyanide exposure in autopsy prosectors.

Autopsy prosectors examining individuals with cyanide poisoning are at risk for occupational cyanide exposure. No protective autopsy precautions to mitigate this risk have been published. We report an autopsy on an individual with cyanide poisoning where the procedure was performed in a negatively pressured isolation room and the stomach was opened under a biosafety cabinet hood. None of the three autopsy prosectors had measurable cyanide in pre or post procedure blood specimens. We recommend that similar precautions be taken in all autopsies where cyanide is suspected as a possible cause of death.

Adult↗
PubMed1995

Amino terminal regions of poliovirus 2C protein mediate membrane binding.

The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.

Amino Acid Sequence↗
PubMed1995

Gas chromatographic-mass spectrometric identification and quantitation of ethylene glycol in serum after derivatization with perfluorooctanoyl chloride: a novel derivative.

Ethylene glycol poisoning is a common clinical problem and identification as well as quantitation of ethylene glycol in serum is important for medical and legal purposes. Most investigators described determination of ethylene glycol by gas chromatography without derivatization or derivatives forming a molecular ion < 200. We describe a novel derivatization technique of ethylene glycol using perfluorooctanoyl chloride, after extraction from serum using acetone. This derivative has a molecular mass of 854 and produces a base peak at m/z 441 and other diagnostic strong peaks for unambiguous identification. Moreover, this derivative is less volatile and is free from interferences from endogenous serum components. Quantitation can be achieved by using 1,4-butanediol as an internal standard. The assay showed within-run and between-run precision of 7.2% and 8.0%, respectively, and linearity over the serum ethylene glycol concentration range 70-2240 micrograms/ml with a detection limit of 5 micrograms/ml.

Caprylates↗
PubMed1995

Development of ulcerative colitis-associated anti-idiotype antibody using a novel monoclonal antibody against a colonic autoantigen.

Earlier studies have established reactivity of ulcerative colitis colon-bound IgG antibody with an autoantigen recognized by a novel monoclonal antibody, 7E12H12 (IgM isotype). Purified 7E12H12-IgM was used to raise anti-idiotype antibody in the rabbit, and the presence of disease-specific idiotype(s) was evaluated in patients with ulcerative colitis. F(ab')2 fragments were prepared from serum-IgG of the rabbit immunized with 7E12H12-IgM and extensively absorbed against unrelated mouse IgM and purified serum IgG from 4 normal subjects. The reactivity of the absorbed F(ab')2 fragment was tested in an ELISA against the serum from 19 patients with ulcerative colitis, 15 patients with Crohn's disease, and 10 normal subjects. The OD values with ulcerative colitis sera were significantly (P < 0.0005) higher than those with Crohn's disease and normal sera. Blood serum from 84% of patients with ulcerative colitis contains antibodies that are reactive to the F(ab')2 fragments of the immune rabbit serum-IgG. Further studies of the anti-idiotype antibodies may elucidate autoimmune processes in ulcerative colitis and may have diagnostic and therapeutic values.

Animals↗
PubMed1995

Confirmation and quantitation of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene by gas chromatography/mass spectrometry. Use of microwave irradiation for rapid preparation of trimethylsilyl and T-butyldimethylsilyl derivatives.

The authors developed a technique by which cocaine metabolites as well as cocaethylene can be identified and quantitated using gas chromatography/mass spectrometry (GC/MS), and deuterated standards of benzoylecgonine, cocaine, ecgonine methyl ester, and cocaethylene as internal standards. The authors used a solid phase extraction (Clean Screen, Worldwide Monitoring Corporation, (Horsham, PA)) technique, and rapid derivatization of metabolites using microwave irradiation before GC/MS analysis. The t-butyldimethylsilyl derivatives (prepared by microwave irradiation in 2.5 minutes, 640 Watts [W]) offered better baseline separation and chromatography than conventional trimethylsilyl derivatives. The assay was linear between 0.87-17.3 mumol/L of benzoylecgonine (250-5,000 ng/mL) concentration, 0.83-16.6 mumol/L of cocaine (250-5,000 ng/mL), 0.79-15.8 mumol/L of cocaethylene (250-5,000 ng/mL) and 1.25-25.1 mumol/L of ecgonine methyl ester (250-5,000 ng/mL). The yields are comparable between the microwave technique and conventional heating.

Cocaine↗
PubMed1995

A novel derivatization of ethylene glycol from human serum using 4-carbethoxyhexafluorobutyryl chloride for unambiguous gas chromatography-chemical ionization mass spectrometric identification and quantification.

Ethylene glycol poisoning is a common problem and identification of ethylene glycol in serum is important for both medical and legal purposes. Other glycols like propylene glycol, 2,3-butanediol, and diethylene glycols are also abused. Common derivatization techniques to form cyclic ester of ethylene glycol work only for 1,2-diols and cannot derivatize other glycols like diethylene glycol. The authors describe novel derivatization technique of ethylene glycol (Molecular weight 62) using 4-carbethoxyhexafluorobutyryl chloride that produces a distinct protonated molecular ion peak at m/z 563 in chemical ionization mode, thus aiding in unambiguous confirmation of ethylene glycol. The authors' novel derivatization technique is not limited to 1,2-diols and can derivatize any glycol. It yields less volatile derivatives than any conventional derivatives and is free from interference from the serum matrix. Moreover, those glycols produce distinctively different mass spectra compared to ethylene glycol. Quantitation of ethylene glycol can be easily achieved by using 1,2-butanediol as an internal standard. The assay showed a within run and between run precision of 6.7% and 8.2% and a linearity from 1.1-36.1 mmol/L (70-2,240 micrograms/mliters) of serum ethylene glycol concentration.

Ethylene Glycols↗
PubMed1995

Carbamazepine-salicylate interaction in normal and uremic sera: reduced interaction in uremic sera.

Displacement of phenytoin and valproic acid by salicylate have been described. We studied carbamazepine-salicylate interactions in normal and uremic sera, which have not been studied. Salicylate caused significant displacement of carbamazepine from protein binding, leading to higher concentrations of free carbamazepine. The concentrations of free carbamazepine were always significantly higher in uremic sera than in normal sera. However, when uremic sera were supplemented with both carbamazepine and salicylate, we observed a much lower displacement of carbamazepine and only a slight increase in free carbamazepine concentration. Treatment of uremic sera with activated charcoal corrected the binding deficiency for carbamazepine. Known uremic compounds like hippuric acid and indoxyl sulphate can only partly explain the observed displacement of carbamazepine in uremic sera. We conclude that salicylate displaces carbamazepine from protein binding in normal sera, but this interaction is significantly reduced in uremic sera.

Carbamazepine↗
PubMed1995

Expression of a unique protein on colon cancer cells that reacts with a novel monoclonal antibody and ulcerative colitis serum.

We earlier developed a MoAb, 7E12H12 (IgM isotype), against a protein present in normal colonic epithelial cells. To examine if 7E12H12-reactive protein is expressed in colon cancer cells and is recognized by ulcerative colitis (UC)-associated autoantibody, we investigated several colon cancer cell lines. 7E12H12 reactivity against the cells was examined by indirect immunofluorescence assay and whole cell ELISA against six colon cancer cell lines HT-29, LoVo, COLO 205, DLD-1, LS 180 and SW 1116. A competitive ELISA was developed using 7E12H12 MoAb and patients' serum to examine the cross-reactive antibodies in the serum. Among the six colon cancer cell lines only LS 180, DLD-1 and SW 1116 reacted with 7E12H12 MoAb, while others did not. The mean (+/- s.e.m.) inhibition of the binding of 7E12H12 MoAb to LS 180 cells by UC serum (n = 51) was 42 +/- 2.1%, whereas in normal subjects (n = 17) it was 14 +/- 2.6%, in Crohn's disease (n = 19) it was 15.3 +/- 2.5%, in infectious diarrhoea (n = 10) it was 11% +/- 3%, and in systemic lupus erythematosus (n = 10) it was 2% +/- 0.6%. The inhibition by the UC group was significantly (P < 0.001 - < 0.0001) higher than any of the non-UC groups, and this inhibition was mainly by IgG1 antibody. The protein in the specific colon cancer cells recognized by the 7E12H12 MoAb cross-reacts with UC-IgG1 antibody and may provide an in vitro system to examine the autoimmune mechanisms in UC.

Adolescent↗
PubMed1995

Adsorption of charged non-albumin bound drugs by amorphous silica.

We investigated the in vitro drug adsorption of PQ 10150 sodium silicate gel (AIS, Santa Clara, CA) with particle size of 230 microns and surface area of 400 m2/g. We observed 99% to 88% adsorption of gentamicin; a mean 91% of disopyramide; a mean 89% of quinidine at low concentration, falling to 75% at higher concentration. Insulin was 88% adsorbed at low concentrations but less so (65%) at higher concentrations. We observed a mean 83% adsorption of procainamide, a mean 84% of N-acetyl procainamide, 74% of lidocaine, 73% of amitriptyline, and 44% of desipramine. We found an average of 14% reduction of total digoxin concentration when serum containing digoxin (2 to 33 ng/mL) was exposed to sodium silicate, while the reduction in free digoxin concentration was 16%. Five percent ethosuximide was also removed. The adsorption of theophylline, phenobarbital, acetaminophen, phenytoin, ethylene glycol, methotrexate, salicylate, thiocyanate and diazepam was minimal and not significant. We conclude that significant amounts of charged, non-albumin bound drugs can be removed by PQ 10150 sodium silicate gel.

Adsorption↗