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Biomedical subjects

D Marcus

Publications and source records attributed to D Marcus.

At least 19 recordsLinked to original sources
PubMed1992

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Acetylcholinesterase↗
PubMed1992

Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.

On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.

Antigens, CD↗
PubMed1992

[Nutritional changes caused by the germination of legumes commonly eaten in Chile].

The changes promoted by germination on phytates, oligosaccharides, crude protein, amino acids and riboflavin contents of black and white cultivars of beans, lentils, chicken-pea and peas, were studied. Seeds germination was carried out in darkness at 25 degrees C and 85% RH during 72 hours, previously soaked overnight in a solution of sodium hypochlorite at a concentration of 50 ppm. Germination capacity was assessed by determining hypocotyl and epicotyl lengths and percent of sprouted seed. The seeds were milled and freeze-dried for the chemical analysis. Germination promoted a significant increase in crude protein content and reduction also significant in phytates levels. These changes were attributed to an increase of proteases and phytase activities. In fact, this enzyme would make a solubilization of phytates and would release soluble protein and minerals. A significant reduction of flatulence oligosaccharides took place, which was also explained by an increase of alpha-galactosidase concentration. Sprouted seeds showed a higher content of almost all amino acid than crude legumes, although this change was variable. Significant increase of riboflavin was also found. Finally, germination decreased ashes and fat contents. These findings were determined in all legumes, although both cultivars of beans showed a higher response to the biochemical changes.

Amino Acids↗
PubMed1992

Five DNA tumor viruses undetectable in human retinoblastomas.

Retinoblastoma (RB) is a childhood eye cancer that arises when a retinal cell lacks a functional RB gene. Recent data indicate the transforming proteins of adenovirus, papillomavirus, and the polyomaviruses BK and JC all can bind to the product of the RB gene. Furthermore, adenovirus 12, JC virus, and simian virus 40 are able to induce RB-like tumors in rodents. In view of these findings, 50 human RBs were tested for the presence of five human DNA tumor viruses: adenovirus 12, BK virus, JC virus, and human papillomaviruses 16 and 18. Using the polymerase chain reaction, no viral sequences were detected in 50 RB DNAs. These data provide no evidence that these viruses have an etiologic role in human RB.

Base Sequence↗
PubMed1991

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Acetylcholinesterase↗
PubMed1991

Nutritional quality of lupine (Lupinus albus cv. Multolupa) as affected by lactic acid fermentation.

The effects of selected NRRL strains of Lactobacillus acidophilus, L. buchneri, L. cellobiosus and L. fermentum upon oligosaccharide, phytate and alkaloid contents, as well as on the nutritive value of lupine, were investigated. Lupine was processed to a 12% total solids suspension, inoculated with 1% (v/v) cultures and fermented until a final desired pH of 4.5. L. acidophilus B-2092 and L. buchneri B-1837 growth was related to a significant sucrose breakdown and decreases of phytates, whereas L. acidophilus B-1910 and L. fermentum B-585 reduced the content of flatulence oligosaccharides. The activity of L. acidophilus B-1910 was particularly associated with lowering of alkaloids and increase of riboflavin. Lactic acid fermentation produced slight changes in lysine and methionine contents. No significant differences in net protein ratio values and protein digestibility were found between fermented and unfermented lupine (P less than 0.05). A 1:1 ratio mixture of B-1910 and B-2092 strains of L. acidophilus lead to a final fermented lupine with nutritional advantages to those given by the individual cultures.

Alkaloids↗
PubMed1989

Anorexia nervosa reconceptualized from a psychosocial transactional perspective.

Anorexia nervosa is conceptualized as a number of very different instrumental actions that occur within particular family contexts. Six psychosocial transactional patterns--negativistic, attention centering, distracting, childlike, attractive, and self-punishing--are identified. This psychosocial transactional perspective is proposed as an alternative to approaches that focus on the "psychopathology" of young female anorexics.

Anorexia Nervosa↗
PubMed1989

Extended major histocompatibility complex haplotypes in patients with multiple sclerosis.

We derived complete haplotypes of the major histocompatibility complex for 33 patients with MS and their families. The DR2 allele and DR2-bearing extended haplotypes, in proportion, were overrepresented on chromosomes of MS patients compared with parental chromosomes not transmitted to MS offspring. We did not confirm previous reports that particular alleles at the BF locus are overrepresented in MS or that C2 hypocomplementemia is present. These results suggest that the DR2 allele is a risk factor for MS, and not merely a genetic marker of the population of origin.

Alleles↗
PubMed1988

Cerebral germinomas and Klinefelter syndrome. A review.

Patients with Klinefelter syndrome appear to be predisposed to the development of extragonadal cerebral germinomas. A case of a pineal region germinoma in a boy 15 years of age with Klinefelter syndrome is documented in this article. In view of three other cases of cerebral germinomas associated with Klinefelter syndrome in the literature, a hypothesis for this predisposition is suggested.

Adolescent↗
PubMed1988

Adolescent suicide and suicide attempts: a population study.

To clarify the epidemiology of adolescent suicide, a retrospective study was undertaken of suicides (1978 to 1982) and hospitalized suicide attempts (1979 to 1983) by adolescents aged 10 to 19 years in an affluent suburban area. Data included date of injury, demography (for both suicides and suicide attempts), and recorded personal and social history (available for attempts only). There were 11 deaths due to suicide (definite or possible) in the five years reviewed: seven male, 10 aged 15 to 19 years. The mean annual rate for suicide deaths (definite and possible, based on ICD codes) was 10.3 per 100,000 15 to 19 year olds, with male rates exceeding female rates. Male rates were lower in the study area than in Chicago, Illinois or the United States, but female rates were higher. Suicides represented an unusually high proportion of all adolescent deaths. Atypically, there were no firearm suicide deaths. Two hundred ten suicide attempts were studied: 77% aged 15 to 19 years, 70% female, and 82% white; 83% involved ingestion of medications or poisons. The mean annual suicide attempt rate was approximately 140 per 100,000 for 15 to 19 year olds, and 45 per 100,000 for 10 to 14 year olds, with female rates exceeding male rates. There was an association between suicide attempt dates and occurrence of holidays, and there was a peak in attempts at the end of the school year. Detailed analysis of personal and social attributes associated with suicide attempts was prevented by poor recording of relevant factors in the medical record.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Disintegration of microorganisms.

The most common methods for the large-scale disintegration of microorganisms are high-pressure homogenization and wet milling. The most-used homogenizer is produced by Manton Gaulin and the common ball mill is the Dyno-Mill. Other manufacturers are now producing similar equipment (e.g., Rannie homogenizers and Netzsch ball mills). However, only relatively limited information on these systems has been published so far. An additional system that might become available for large-scale disintegration is microfluidization. When coming to choose optimal equipment and conditions for cell disintegration, it might be useful to consider some relevant topics that were compiled by the Retsch company for particle disruption and are presented, with modification, in Table XIII. Obviously, the importance of the above points will vary in different processes, but all of them should be considered in any biotechnological large-scale application. It is important to realize that although the mechanical disintegration methods are of general use, it is essential to define the optimal disintegration conditions for each microorganism and/or product. As a starting point, the published data for a similar product should be consulted. This should be followed by laboratory-scale experiments carried out with equipment and conditions that can be easily scaled up.

Cell Fractionation↗
PubMed1988

Genetically determined polymorphism of the circulating human breast cancer-associated DF3 antigen.

The murine monoclonal antibody (MAb) DF3 was prepared against a human breast carcinoma. Previous studies have demonstrated that DF3 antigen levels are elevated in plasma of patients with breast cancer. Furthermore, MAb DF3 reacts with circulating glycoproteins of different molecular weights ranging from approximately 300 to 450 kd. The present study demonstrates that plasma DF3 antigen is comprised of at least four moieties with slow (S), intermediate (I), rapid (R) and very rapid (VR) electrophoretic mobilities. The electrophoretic mobility patterns for circulating DF3 antigen differ among individuals. Moreover, DF3 antigen is detectable in urine, and the electrophoretic mobility of the urinary moieties is similar, but not identical, to that in the plasma. Studies in family members suggest that the electrophoretic heterogeneity of plasma DF3 antigen is determined by codominant expression of multiple alleles at a single locus. This locus may code for the core protein of DF3 antigen. These findings thus identify a genetically determined polymorphism of a circulating tumor-associated glycoprotein.

Antibodies, Monoclonal↗
PubMed1987

Production of carcinoembryonic antigen from a human colon adenocarcinoma cell line. II. Use of monoclonal antibodies to carcinoembryonic antigen for antigen purification and characterization.

Monoclonal antibodies (McAbs) were prepared against carcinoembryonic antigen (CEA), derived from human metastases to the liver of primary colon adenocarcinoma. A stable clone, secreting monoclonal anti-CEA antibody (Ab), was selected. The hybridoma cells were grown in BALB/C mice, forming ascites fluids containing 2-5 mg/ml of the relevant McAbs. Ascites fluids were purified using a DEAE-Sephacel column. The purified immunoglobulin fraction was characterized as IgG2b, having a dissociation constant of 2 X 10(-9)M. The potential use of these McAbs for CEA diagnosis and purification was evaluated: the IgG2b fraction, when bound to Sepharose 4B, facilitated the purification of CEA from spent media of a human colon adenocarcinoma cell line, resulting in iodination grade CEA; the anti-CEA McAbs demonstrated differential histochemical staining of various sections, both fresh and fixed; the McAbs were also found to be suitable as tagging Abs in a CEA RIA.

Adenocarcinoma↗
PubMed1987

Production of carcinoembryonic antigen from a human colon adenocarcinoma cell line. I. Large-scale cultivation of carcinoembryonic antigen-producing cells on cylindric cellulose-based microcarriers.

A continuous cell line from a human colon carcinoma, designated HuCC1-14, was successfully grown on cyclindrical cellulose microcarriers (MC) charged by DEAE. The cells grow on these carriers as large cell-MC aggregates and maintain high cell densities in submerged conditions for extended periods. HuCC1-14 cells secrete into the culture medium significant amounts of carcinoembryonic antigen (CEA). Maximum efficiency in CEA secretion occurred after the cells switched from logarithmic growth to stationary phase. At this state, high CEA levels could be obtained with low-serum medium which greatly facilitates subsequent product purification. The described method provides a system which can be scaled-up and produce this tumor-associated antigen in essentially unlimited amounts and reproducible quality for detection and monitoring of cancer patients.

Adenocarcinoma↗
PubMed1985

Identification of a novel ganglioside on erythrocytes with blood group Cad specificity.

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.

Animals↗
PubMed1985

"Natural termination": some comments on ending analysis without setting a date.

This paper is a re-examination of the rule of date setting in termination. It reviews the literature and presents an analytic case which was terminated in what the patient called a "natural" manner, i.e., without the setting of a date. Some ideas about the nature of the rules used in organizing analysis are discussed.

Adult↗