Biomedical subjects

M Czerwinski

Publications and source records attributed to M Czerwinski.

29 records · Page 2Linked to original sources

PubMed1994

Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells.

Alterations in N- and O-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. The O-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. The O-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal beta 1-3GalNAc alpha 1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human erythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc alpha 1-Ser/Thr). Recombinant glycophorin A had the same O-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect its O-glycan composition. Deletion of the N-linked glycosylation site at Asn26, by introducing the Mi.I mutation (Thr28-->Met) by site-directed mutagenesis, did not markedly affect the O-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Restricted VH gene usage by murine hybridomas directed against the human N, but not M, blood group antigen.

The M and N human blood group antigens are complex glycopeptide determinants at the amino terminus of the red blood cell membrane glycoprotein, glycophorin A. The heavy and light chain variable region cDNA sequences were determined for seven murine monoclonal antibodies recognizing glycophorin A. Three of the antibodies were anti-M and four were anti-N. Each of the anti-M antibodies was composed of VH and VL regions derived from distinct germline gene families (VH1 (J558), VH4 (X24), VH5 (7183), VK5, VK8, and VK19). In contrast, all four anti-N heavy chains were composed of VH regions derived from the VH2 (Q52) germline gene family and all used the same J4 gene segment. In addition, two of the anti-N light chains were composed of VK regions from the VK8 germline gene family and used the J1 gene segment. Since each anti-N hybridoma was derived from different mice immunized by different protocols, these results suggest that the murine immune response to the N, but not the M, human blood group antigen is restricted.

Amino Acid Sequence↗

PubMed1993

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots. Identification of glycophorin and band 3 variant forms.

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with 125I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1992

Analysis of human cytochrome P450 catalytic activities and expression.

Cytochromes P450 are a large group of membrane-associated heme protein monooxygenases, most of which are responsible for metabolizing foreign compounds. Chemical carcinogens, which are ingested or absorbed into the body as inert forms, are metabolically activated by P450s to electrophilic metabolites capable of binding to and mutating DNA. Different P450 forms are responsible for activation of the various classes of chemical carcinogens including the arylamines, polycyclic aromatic hydrocarbons, nitrosamines and aflatoxins. Thus, the cellular constituency and levels of P450s could determine the fate of a particular carcinogen and the risk of humans to exposure. To study the catalytic activities of human P450s, human P450 cDNAs were cloned and expressed into active enzymes using cultured cells. By both transient and stable cDNA expression systems, several human P450s were found to be capable of metabolically-activating the human hepatocarcinogen aflatoxin B1. These cDNA expression systems can also be used to determine whether an unknown chemical will be activated by a human P450 and thus be toxic or mutagenic in humans. To assess the extent of interindividual variation in P450 expression, probes developed from P450 cDNAs are being used to quantify levels of P450 mRNAs in various human tissues. Studies using RNase protection revealed that the closely related CYP2B6 and CYP2B7 mRNAs could be independently quantified in liver and lung, respectively. This procedure can be used to examine expression of different P450 genes in banks of human tissue specimens.

Catalysis↗

PubMed1992

Automatization and training in visual search.

In several search tasks, the amount of practice on particular combinations of targets and distractors was equated in varied-mapping (VM) and consistent-mapping (CM) conditions. The results indicate the importance of distinguishing between memory and visual search tasks, and implicate a number of factors that play important roles in visual search and its learning. Visual search was studied in Experiment 1. VM and CM performance were almost equal, and slope reductions occurred during practice for both, suggesting the learning of efficient attentive search based on features, and no important role for automatic attention attraction. However, positive transfer effects occurred when previous CM targets were re-paired with previous CM distractors, even though these targets and distractors had not been trained together. Also, the introduction of a demanding simultaneous task produced advantages of CM over VM. These latter two results demonstrated the operation of automatic attention attraction. Visual search was further studied in Experiment 2, using novel characters for which feature overlap and similarity were controlled. The design and many of the findings paralleled Experiment 1. In addition, enormous search improvement was seen over 35 sessions of training, suggesting the operation of perceptual unitization for the novel characters. Experiment 3 showed a large, persistent advantage for CM over VM performance in memory search, even when practice on particular combinations of targets and distractors was equated in the two training conditions. A multifactor theory of automatization and attention is put forth to account for these findings and others in the literature.

Adult↗

PubMed1991

Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism.

4-Ipomeanol is a pulmonary toxin in cattle and rodents that is metabolically activated by cytochromes P-450 (P-450s). P-450-mediated activation of 4-ipomeanol to DNA binding metabolites was evaluated using a vaccinia virus complementary DNA expression system and an in situ DNA-binding assay. Twelve human P-450s and two rodent P-450s were expressed in human hepatoma Hep G2 cells and examined for their abilities to metabolically activate this toxin. Three forms, designated CYP1A2, CYP3A3, and CYP3A4, were able to catalyze significant production of DNA-bound metabolites of 20-, 8-, and 5-fold, respectively, above binding catalyzed by Hep G2 cells infected with wild-type vaccinia virus. These enzymes, with highest activities, are not known to be expressed in human or rodent lung. CYP2F1 and CYP4B1, two enzymes that are expressed in lung, display only modest 3- and 2-fold respective increased abilities to metabolically activate 4-ipomeanol. Two human forms were inactive and seven other human forms showed activities ranging from 0.5- to 2-fold above control level. Surprisingly, rabbit complementary DNA-expressed CYP4B1 was the most active enzyme (180-fold above control) among all P-450s tested in producing DNA-binding metabolites from this mycotoxin. These studies demonstrate a species difference in 4-ipomeanol metabolism and suggest caution when attempting to extrapolate rodent data to humans.

Biotransformation↗

PubMed1990

Metabolic activation of 4-ipomeanol in human lung, primary pulmonary carcinomas, and established human pulmonary carcinoma cell lines.

4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [14C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [14C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 549 +/- 60). In lung cancer tissue, values ranged from 0 to 2.566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 60, P greater than .3). When patients were divided into smokers and current non-smokers (no tobacco products smoked for greater than 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P greater than .1 in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10; P greater than .30). The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted.

Biotransformation↗

PubMed1990

Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal lung tissue and for altered gene regulation in primary pulmonary carcinomas.

The major polycyclic aromatic hydrocarbon inducible-cytochrome P4501A1 gene (CYP1A1) is presumed to be important in pulmonary carcinogenesis and toxicology because its product, the cytochrome P4501A1-dependent (CYP1A1-dependent) monooxygenase, transforms selected xenobiotics (including polycyclic aromatic hydrocarbon procarcinogens in cigarette smoke) to potent carcinogenic metabolites. CYP1A1 messenger RNA (mRNA) expression has not, however, been previously demonstrated in human pulmonary tissue. This report defines CYP1A1 gene expression in normal lung tissue and primary pulmonary carcinoma tissue obtained at thoracotomy from 56 patients with lung cancer. When Northern blot hybridization analyses were performed, 17 of 19 (89%) and zero of five (0%) samples of normal lung tissue from active cigarette smokers and nonsmokers, respectively, expressed the normal 2.8-kilobase CYP1A1 mRNA. In addition, a time-dependent decrease in expression of the CYP1A1 gene was noted in normal lung tissue from individuals who were former smokers, with a decrease in expression occurring as early as 2 weeks following cessation of cigarette smoking. Expression became undetectable in all patients who had stopped smoking more than 6 weeks prior to study. When CYP1A1 gene expression was evaluated in lung cancers, mRNA levels were detectable in one of four (25%) tumors from nonsmokers; two of 24 (8%) tumors from former smokers; and seven of 15 (47%) tumors from cigarette smokers. In addition, an approximately 10-kilobase CYP1A1 RNA species, which was not detectable in normal lung tissue, was observed in five of ten (50%) of the lung cancers that expressed the CYP1A1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P-450 CYP1A1↗

PubMed1989

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

Adenocarcinoma↗

PubMed1985

Computer-assisted studies of molecular structure-biological activity relationships.

Computer-assisted methods can be used to investigate the relationships between the molecular structures of compounds and their biological activity. A number of approaches have been reported in the literature, including correlations of activity with substituent constants, conformational analysis and display, quantum mechanical methods, and methods relying on discriminant development and pattern-recognition techniques. Application areas for this technology include drug design, agricultural chemical design, and studies of chemical toxicity and genetic toxicity (mutagenic or carcinogenic potential). These structure-activity methods are introduced, and citations are given. Several current structure-activity relationship (SAR) studies using pattern recognition are presented as examples of typical projects that are feasible with this approach. These include the investigation of a set of 122 antiinflammatory steroids, a study of 153 retinoids for cancer prevention, and a study of chemicals that have been tested in a sister chromatid exchange mutagen screen.

Animals↗

PubMed1984

Hemispheric asymmetry in use of semantic category information.

When two pictures of common objects were presented sequentially, the second was named more quickly if both were members of the same semantic category. This semantic priming effect occurred only when both pictures went directly to the left hemisphere. If the target or prime stimulus was presented to the left visual field--right hemisphere, no priming effect was observed. These results suggest that semantic category information is activated and used by the left hemisphere of the brain.

Adult↗