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E P Bennett

Publications and source records attributed to E P Bennett.

34 records · Page 2Linked to original sources
PubMed1996

cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3.

The glycosylation of serine and threonine residues during mucin-type O-linked protein glycosylation is carried out by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferase). Previously two members, GalNAc-T1 and -T2, have been isolated and the genes cloned and characterized. Here we report the cDNA cloning and expression of a novel GalNAc-transferase termed GalNAc-T3. The gene was isolated and cloned based on the identification of a GalNAc-transferase motif (61 amino acids) that is shared between GalNAc-T1 and -T2 as well as a homologous Caenorhabditis elegans gene. The cDNA sequence has a 633-amino acid coding region indicating a protein of 72.5 kDa with a type II domain structure. The overall amino acid sequence similarity with GalNAc-T1 and -T2 is approximately 45%; 12 cysteine residues that are shared between GalNAc-T1 and -T2 are also found in GalNAc-T3. GalNAc-T3 was expressed as a soluble protein without the hydrophobic transmembrane domain in insect cells using a Baculo-virus vector, and the expressed GalNAc-transferase activity showed substrate specificity different from that previously reported for GalNAc-T1 and -T2. Northern analysis of human organs revealed a very restricted expression pattern of GalNAc-T3.

Amino Acid Sequence↗
PubMed1995

Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F.L., Hollanders, T., Lehman, D.J., Thomsen, D.R., and Elhammer, A.P. (1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.

Amino Acid Sequence↗
PubMed1995

Genomic cloning of the human histo-blood group ABO locus.

A P1 phage clone containing the near complete protein coding region of the human histo-blood group ABO gene locus was isolated and characterized. The insert was estimated at approximately 41 kp, and an approximately 20 kbp BamHI fragment contained the near complete coding region as evaluated by Southern blot analysis. Intron/exon boundaries were determined and the coding region was found to span at least seven exons. In situ hybridization to metaphase chromosomes confirmed the predicted localization to 9q34.

ABO Blood-Group System↗
PubMed1994

Blood group ABO-related glycosylation of urothelial cell lines: immunocytological, enzymatic, and genetic characterization.

Three immortalized, human urothelial cell lines were characterized with respect to their ABO-related carbohydrate phenotypes using a panel of monoclonal antibodies directed to a series of carbohydrate epitopes (Lac, sialylated Lac, Le(a), sialylated Le(a), Le(x), sialylated Le(x), H types I and II, Ley, Leb, A monofucosylated types I and II, ALey, Aleb, and A type III). The glycosyltransferases forming some of these epitopes (beta 1-3/4 galactosyltransferase, alpha 1-2 fucosyltransferase, alpha 1-3 galactosyltransferase, and alpha 1-3-N-acetyl-galactosaminyltransferase) were determined by enzyme assays. The ABO gene complex was analyzed by Southern blotting, Northern blotting, and polymerase chain reaction across the O deletion and across base differences between the A and B alleles. The immunocytochemical stainings showed marked differences between the three cell lines; the high grade (tumorigenic, metastatic) cell line showed difucosylated types I and II structures, and the low grade (nontumorigenic, nonmetastatic) cell lines showed monofucosylated types I and II structures. Polymerase chain reaction genotyping of the cell lines indicated that one was OO, one was AA, and one was A plus a mutated allele. Northern blotting showed RNA encoding the A transferase. However, even though both of the A cell lines seemed to have an intact gene, which could produce A transferase and transcribed RNA, none of them showed any activity of the A gene encoded enzyme or any A-structures at the cell surface. In contrast, the three other examined glycosyltransferases were active. The three urothelial cell lines reflect in vivo findings in humans. They represent a competent system for in vitro studies of the different carbohydrate transferase genes responsible for the carbohydrate structures expressed on the cell surface in bladder tumors.

ABO Blood-Group System↗
PubMed1994

Evaluation of histo-blood group ABO genotyping in a Danish population: frequency of a novel O allele defined as O2.

Traditional blood group ABO serology is based on immunoreactivity with the carbohydrate determinants A, B and H antigens. Recent advances at the DNA level of the ABO genes have provided a molecular genetic model for the ABO polymorphism. This genetic model has to date only been tested on a limited basis. The present study was initiated to evaluate the universality of the proposed genetic model on a larger group of serologically defined ABO phenotypes. Three hundred healthy Danish blood donors were analysed (A:50, B:50, AB:50, O:150) by PCR amplification followed by diagnostic restriction enzyme cutting. In all cases A, B, and AB at least one allele of correctly predicted status was found. However, in O phenotype individuals, 11 out of 150 carried one allele discordant to the proposed genetic model. This novel O allele (3.7% allele frequency) was further characterized by diagnostic restriction enzyme analysis in two positions divergent between A and B alleles and by DNA sequencing of the two major exons. The novel O allele is termed O2 as it typed as B in nucleotide position 526 and as A in positions 703, 796, and 803, in contrast to the most predominant O allele termed O1, which types as A in all 4 positions. The structural defect in the O2 allele appears to be an additional substitution at nucleotide position 802. The results clearly demonstrate that with the addition of the two distinctly different O alleles, O1, O2, the previously proposed molecular genetic basis of the ABO polymorphism is quite valid.(ABSTRACT TRUNCATED AT 250 WORDS)

ABO Blood-Group System↗
PubMed1993

Biosynthetic basis of incompatible histo-blood group A antigen expression: anti-A transferase antibodies reactive with gastric cancer tissue of type O individuals.

The expression of incompatible A carbohydrate antigens in some adenocarcinomas may provide an explanation for the generally observed lower incidence of adenocarcinoma among types O and B versus type A individuals. The chemistry and genetic basis of incompatible A expression is largely unknown. Here, we have screened 31 cases of gastric tumors of phenotype O for the expression of blood group A gene-defined glycosyltransferase by immunohistology on frozen sections using newly developed monoclonal antibodies to the transferases. Three cases were positive, and transferase expression was confirmed by enzyme analysis of extracts from the specimens. Blood group A carbohydrate antigens were also identified immunohistologically in these three cases as well as in five other cases. Thin-layer chromatography immunostaining analysis of glycolipid extracts from the three cases did not confirm the chemical presence of A antigen. The ABO genotype of all patients was found to be OO, showing that all carried O alleles with a structural defect at nucleotide position 261 leading to a shift in the reading frame. The data suggest that incompatible A antigen expression is a result of transferase expression derived from the ABO genes.

ABO Blood-Group System↗
PubMed1992

Carbohydrates of the cell surface: molecular aspects of glycosyltransferases and their genes.

With the development of monospecific antibodies to transferase proteins as well as cDNA probes to cloned transferase genes, the study of glycosylation and its regulatory mechanisms holds great promise for the future. Here we provide a brief overview of the present knowledge of glycosyltransferases, their primary structure, cellular topography, regulation, and the basis of the genetic polymorphisms as revealed by blood groups.

Animals↗
PubMed1981

Serum calcium in blood pressure regulation during hemodialysis.

Frequent hypotensive episodes developed in a 56-year-old man, who was receiving long-term hemodialysis therapy, at the time he had a uremic pericardial effusion. During dialysis with an inadvertent calcium infusion, however, the mean blood pressure (BP) was sustained and even rose from 93 to 133 mm Hg. During continued dialysis to restore the serum calcium level from a peak of 17.4 to 12.0 mg/dL, mean arterial pressure decreased back to 93 mm Hg. In contrast, BP had fallen in three of six preceding dialysis treatments and five of nine subsequent dialysis treatments, all at the same ultrafiltration rate. This report suggests the importance of serum calcium to BP regulation during hemodialysis.

Blood Pressure↗
PubMed1976

Enzyme induction by oral testosterone.

Six normal male volunteers ingested a dose of 400 mg free testosterone daily as tablets over 21 days. By the end of treatment intravenous antipyrine half-life had decreased significantly from 8.0 +/- 2.7 to 5.7 +/- 2.6 hr. The subjects eliminated testosterone from serum more rapidly on the twenty-first day of testosterone ingestion than on the first day. Serum albumin, bilirubin, prothrombin, alanine-amino-transferase, and alkaline phosphatases were unchanged during the experiment. It is concluded that oral testosterone treatment induces the hepatic drug-metabolizing system including that of testosterone.

Administration, Oral↗
PubMed1976

Rise in dehydroepiandrosterone and oestrogens during clomiphene administration in normal men.

Serum concentrations of LH, FSH and testosterone, and urinary excretion of individual 17-ketosteroids, oestrogenic substances and free cortisol was investigated in 12 normal men after a daily administration of 50 mg clomiphene citrate for 14 days. A significant increase in the serum concentrations of LH, FSH and testosterone was seen after 8 days with a further increase when measured a week later. The percentual increase in urinary excretion of oestrogens and dehydroepiandrosterone was of the same order as the increase in serum testosterone concentrations whereas the increase in androsterone and a aetiocholanolone was significantly lower. The excretion of free cortisol was unaltered. The study that measurements of oestrogens in the urine might be used as a parameter of the stimulatory effect of clomiphene on the hypothalamic - pituitary - gonadal axis in man. In addition, of the results support the hypothesis of an independent effect of clomiphene on adrenal steroidogenesis, not related to the production of glucocorticoids.

17-Ketosteroids↗