Biomedical subjects

M L Frazier

Publications and source records attributed to M L Frazier.

At least 55 records · Page 3Linked to original sources

PubMed1992

Carbonic anhydrase II gene expression in mouse pancreatic duct cells.

Our goal is to create a transgenic mouse model for human pancreatic duct cell adenocarcinoma using the promoter/enhancer region of the carbonic anhydrase (CA) II gene to drive the expression of SV-40 T-antigen in pancreatic duct cells. This requires that the CA II gene be expressed in mouse pancreatic duct cells and not in other pancreatic cells, as has already been shown to be the case in the human and guinea pig pancreas. We have shown with an enzyme histochemical assay that mouse pancreatic duct cells contain CA activity in both intact pancreas and cultured interlobular duct epithelium. In addition, CA activity was detected with a biochemical assay in homogenates of cultured duct epithelium. The specific activity of duct cells was 2.75-fold greater than in whole pancreas, suggesting that a substantial amount of total pancreatic CA activity is contributed by duct cells. At least some of the CA in cultured duct cells was inferred to be CA II by Northern blot analysis of RNA extracted from the cells. The concentration of CA II mRNA in the cultured duct cells was substantially greater than in whole pancreas and would appear to account for the majority, if not all, of the CA II in the mouse pancreas.

Animals↗

PubMed1991

Predominant expression of mRNA coding for nonspecific cross-reacting antigen in colorectal carcinomas.

We have analyzed specific transcripts of the carcinoembryonic antigen (CEA) gene family expressed in 32 colorectal carcinomas (CRCs) and when possible, their corresponding adjacent (less than 1 cm) and distant (greater than 10 cm) mucosas. From 1 of the 32 patients, we obtained a corresponding tubulovillous adenoma. From 1 additional patient a Crohn's disease sample was also evaluated. CEA mRNA was detectable by northern blot hybridization in 97% of the CRCs examined. Of these, 41% expressed this transcript at higher levels in the tumor than in the corresponding distant mucosa. Nonspecific cross-reacting antigen (NCA) is another member of the CEA gene family. The NCA mRNA was detectable in 87% of the CRCs examined, with 92% of these expressing the NCA mRNA at higher levels than the tubulovillous adenoma and the specimen from Crohn's colitis, whereas CEA was easily detected. Our findings demonstrate that NCA mRNA is more consistently overexpressed in colon carcinoma relative to distant mucosa than CEA mRNA.

Adult↗

PubMed1991

Ectopic expression of mucins in colorectal cancer metastasis.

Cancer metastasis is an ectopic growth of malignant cells. In human colorectal cancer, it is hypothesized that, during the progression of the disease to an advanced stage, highly malignant and metastatic tumor cells arise within primary tumors and become predominant. Based on this hypothesis, molecules associated with metastatic cells have been sought by the comparison of surgical specimens from patients at various clinical stages. Colorectal carcinomas with increased metastatic potential and with poor prognosis have been characterized by a loss of an organ-specific mucin determinant (sulfomucin), by an increased expression of non-intestinal sialomucins, and by an ectopic expression of adhesion ligands (sialyl-dimeric Lex antigens) on mucins.

Adenocarcinoma↗

PubMed1990

Establishment of a new human pancreatic adenocarcinoma cell line, MDAPanc-3.

A new cell line was established from a liver metastasis of a human pancreatic adenocarcinoma. The cell line, MDAPanc-3, which arose from a moderately differentiated adenocarcinoma, produces carbonic anhydrase II mRNA, but no detectable levels of insulin or alpha amylase mRNA. The stem line chromosome number was determined to be 43, with six marker chromosomes. Growth of MDAPanc-3 is stimulated by cholecystokinin (CCK) fragment 26-33. The cell line will be useful in further studies on the mechanism(s) by which CCK stimulates growth of certain human pancreatic adenocarcinomas and normal human pancreatic exocrine tissue.

Adenocarcinoma↗

PubMed1990

Carbonic anhydrase II gene expression in cell lines from human pancreatic adenocarcinoma.

Current evidence suggests that carbonic anhydrase II (CA II) is produced by pancreatic duct cells but not by pancreatic acinar or islet cells. The aim of this study was to determine whether CA II homologous RNA and CA II immunoreactive protein are produced by cell lines established from human pancreatic adenocarcinomas. A 1.7-Kb CA II homologous RNA was detected in poly(A+) RNA isolated from normal human pancreas, normal human liver, and to varying degrees in the cell lines examined. The CA II immunoreactivity corresponding to approximately 30 kD (consistent with the established molecular mass of CA II) was also detected by immunoblotting in normal human pancreas, normal human liver, and some of the cell lines. We also found that the levels of CA II homologous RNA increase in the pancreatic adenocarcinoma cell lines following treatment with the differentiating agent, retinoic acid.

Adenocarcinoma↗

PubMed1989

Hypomethylation and ADA gene expression in mouse CAK cells.

The adenosine deaminase (ADA) locus appears to be under complex transcriptional control since levels of ADA enzyme activity vary greatly between different tissues and stages of development. Evidence that a trans-acting factor may be involved with the regulation of this locus came from previous experiments where fusion of ADA-negative human JEG cells and mouse ADA-positive cells led to the trans-activation of human ADA in a hybrid nucleus. Here, we demonstrate that the near euploid mouse embryo fibroblast cell line, CAK, also lacks detectable ADA enzyme activity due to altered gene regulation. We further demonstrate that ADA in CAK cells is not amenable to activation by somatic cell fusion. Following treatment with 5-azacytidine and Xyl-A selection (for ADA), however, CAK clones were obtained that stably express the ADA gene. Molecular analysis of the parental CAK cells and the ADA-positive derivative clones demonstrated that both 5' and 3' regions of the ADA gene had become hypomethylated in the ADA+ clones. We conclude that methylation is another element involved with the transcriptional control of the ADA gene and that ADA might serve as a useful model for studying the interaction of cis- and trans-acting regulational elements.

Adenosine Deaminase↗

PubMed1989

Type II diabetes mellitus and polymorphism of insulin-receptor gene in Mexican Americans.

Resistance to insulin action is a well-established feature of type II (non-insulin-dependent) diabetes and is believed by many to contribute to the etiology of this condition. We therefore characterized restriction-fragment-length polymorphisms of the insulin-receptor gene with the restriction enzyme Rsa 1 in 242 Mexican Americans and non-Hispanic Whites with type II diabetes and 202 age-, sex-, and ethnicity-matched control subjects who participated in a population-based study in San Antonio. Alleles of 6.7 kilobases (kb) (A allele), 6.2 kb (B allele), and 3.4 kb (C allele) were identified. The C allele was observed in Mexican Americans only, where its frequency among nondiabetic control subjects was 17.7%. Diabetic Mexican Americans were twice as likely as control subjects to be homozygous for the C allele. The crude odds ratio for diabetes in CC homozygotes compared with the other two genotypes was 2.22, although this result was not statistically significant (chi 2 = 1.57, P = .21). The Mantel-Haenszel odds ratio, adjusting for age, however, indicated a 4.71-fold increased risk of diabetes among Mexican Americans with the CC genotype compared with Mexican Americans without this genotype (chi 2 = 5.38, P = .020). The age of onset of diabetes was also slightly younger in CC homozygote cases (45.4 +/- 9.2 yr) than in CX or XX cases (47.7 +/- 9.0 and 48.6 +/- 9.6 yr, respectively), although this difference was not statistically significant (P .467).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Restriction fragment length polymorphism of the human insulin receptor gene among Mexican Americans.

Recently, we examined a restriction fragment length polymorphism (RFLP) at the insulin receptor locus in Mexican Americans. This RFLP can be detected using the restriction endonuclease Rsa I, and has three alleles of 3.4 kb, 6.2 kb, and 6.7 kb. Our data suggested that the 3.4 kilobase pair (kb) allele may be associated with type II (non-insulin dependent) diabetes mellitus in Mexican Americans. We initiated studies to identify additional polymorphisms at the insulin receptor locus that might be useful in further studies on the association of type II diabetes and the insulin receptor gene. During the course of these studies we observed that the 6.7 kb and 6.2 kb alleles of the RsaI polymorphism appears to be due to an insertion or deletion of DNA sequences, so that DNA fragments of different lengths are generated when DNA from heterozygous individuals is digested with selected restriction endonucleases that cut on either side of this region. The 3.4 kb allele is apparently due to a site specific polymorphism. In the present report, results of these findings are presented as well as a description of additional polymorphisms that we have identified among Mexican Americans.

Autoradiography↗

PubMed1988

Structure and evolution of somatostatin genes.

A bovine pancreatic preprosomatostatin cDNA clone has been isolated and sequenced. Although it encodes a predicted 116 amino acid preprosomatostatin that is very similar in primary structure to those deduced from other mammalian preprosomatostatin cDNAs, there are some differences in amino acid composition. Hybridization of this clone to Northern blots of fetal bovine pancreatic poly(A+) RNA reveals a mRNA of 700 nucleotides. Evolution of the preprosomatostatin genes was studied by statistical analysis of anglerfish, catfish, bovine, rat, and human cDNA sequences. The results suggest that the two somatostatin genes present in both anglerfish and catfish were the result of a gene duplication event in a common ancestor of anglerfish and catfish.

Animals↗

PubMed1987

Cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for bovine preproinsulin.

A cDNA library was prepared from poly(A+) RNA isolated from fetal bovine pancreas. Bacterial colonies were screened for sequences homologous to a rat preproinsulin I cDNA probe. Ten positive clones were selected at random and further studied. Northern blot analyses revealed that seven of these clones hybridized to a single RNA species, of approximately 400 nucleotides. Sequence analysis of one of these clones (pbI2885) revealed the entire structural region of bovine preproinsulin mRNA including a 72 nucleotide region encoding a signal peptide enriched in hydrophobic residues. The overall nucleotide homology between bovine and human preproinsulin mRNA was 76% for the preregion, 89% for the A chain, 83% for the B chain, and 68% for the C peptide (including a 15 nucleotide deletion).

Amino Acid Sequence↗

PubMed1987

Localization of an abundant myeloid mRNA to individual leukocytes in mixed cell populations.

Differential screening of cDNA libraries with radiolabeled RNAs isolated from various sources provides a convenient way to identify cDNA clones representing RNAs that are more (or less) abundant in selected tissues. This strategy was previously used to isolate cDNA clones representing poly(A+)RNAs (mRNA) that are abundant in leukocytes from chronic myelogenous leukemia (CML) patients. One limitation with the initial experiments was that the RNAs were isolated from heterogenous cell populations and it was impossible to distinguish whether all of the cells were producing the abundant mRNAs or if a subset of the cell population was responsible for the majority of the RNA species. To resolve this important issue, we have directly hybridized radiolabeled cDNAs to the cellular RNAs of intact, morphologically distinguishable, primary hematopoietic cells. In the present study clone pC-A3, which represents an mRNA species that is abundant in the chronic phase of CML, was used to examine three samples from normal bone marrow, one sample from normal peripheral blood, and four samples from peripheral blood of leukemic patients (one Ph1 + AL, two CML in compensated phase, and one CML in accelerated phase). The results show that while C-A3 gene expression is detectable in cells from the granulocytic, monocytic, and lymphoid lineage, its relative abundance peaks at the level of the neutrophilic myelocytes and promyelocytes. Earlier myeloid precursors like myeloblasts or mature neutrophilic granulocytes show less labeling. Further, all maturation stages of eosinophils (Eos) and basophil (Baso) are highly labeled. This finding reinforces recent evidence that Eos and Baso share a common progenitor and suggests that these two cell types may have a stronger role than previously noted in the prominent myeloproliferative response that is characteristic of CML.

Basophils↗

PubMed1986

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma.

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.

Animals↗

PubMed1986

Restriction fragment length polymorphism of the human insulin gene region among type II diabetic Mexican-Americans and Tunisians.

The human insulin gene is flanked by a polymorphic locus that is located approximately 500 base pairs (bp) from the 5' end of the point where transcription begins (Bell et al. 1981; Bell et al, 1982). Its occurrence is due to an insertion-deletion region which gives rise to two major classes of alleles: those containing small insertions of 0-600 bp and those containing larger insertions of 1,600-2,200 bp (Owerbach and Nerup, 1982). Insertions of 600-1,600 bp are rare (Rotwein et al., 1983). The larger insertions have previously been reported to be associated with type 2 diabetes (Owerbach and Nerup, 1982). We have conducted studies on a Mexican-American population in Starr County, Texas (98% Mexican-American) and a Tunisian population in Tunis, Tunisia, to determine if the frequency distribution of these classes of insulin gene alleles are similar to the previously reported frequency distributions and if any of the classes of alleles are associated with type 2 diabetes in these populations. We conclude that none of the classes of insulin gene alleles are associated with type II diabetes among Mexican-Americans or Tunisians, and that the frequency distributions of the insulin gene alleles do not vary significantly between the Tunisians, Mexican-Americans, or the aggregate data resulting from combining the insulin gene frequencies of several of the populations described thus far (Bell et al., 1984).

Adult↗

PubMed1985

Ontogeny of immunoreactive insulin in the fetal bovine pancreas.

The aim of this study was to characterize the development of immunoreactive insulin (IRI) in the fetal bovine pancreas. Pancreatic IRI was acid extracted, and both pancreatic and serum IRI were quantitated by RIA. The amount of pancreatic IRI per wet tissue wt in first trimester fetuses was similar to that in the adult animal (8.2 +/- 0.7 and 5.9 +/- 1.7 U/g pancreas, respectively). IRI increased progressively during gestation, attaining 39.2 +/- 6.5 U/g pancreas in the third trimester, 7-fold higher than that in the adult. When pancreatic IRI concentrations were standardized for protein content of the extracts, a decrease was noted between the midsecond and third trimesters. This is most likely the result of dilution of the endocrine portion of the pancreas by the rapidly growing exocrine pancreas. IRI was also detectable in fetal sera from all three trimesters. In contrast to the profile for pancreatic concentrations of IRI, serum concentrations remained constant throughout gestation at approximately 20 microU/ml. Poly(A+)RNA was isolated from adult and fetal pancreata, and the relative levels of preproinsulin mRNA were assessed by DNA/RNA filter hybridization. There was a 2- to 3-fold increase in the relative level of preproinsulin mRNA in fetal pancreata between the first and second trimesters which was maintained through the third trimester. In the adult pancreas, preproinsulin mRNA levels were similar to those in the first trimester fetus. This profile for the ontogeny of pancreatic preproinsulin mRNA was similar to that for pancreatic IRI (units per pancreas) during fetal maturation. We conclude that in the bovine fetus: the endocrine pancreas synthesizes IRI during all three trimesters of development; pancreatic (units per g pancreas), but not serum, concentrations of IRI increase progressively as development proceeds; and the ontogeny of preproinsulin mRNA is paralleled by that of pancreatic IRI (units per pancreas).

Animals↗

PubMed1984

Evolution of glucagon genes.

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(-9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.

Animals↗

PubMed1984

Cell-free synthesis of rat epidermal growth factor-like polypeptide.

Poly(A+)RNA isolated from the adult male rat salivary gland has been found to direct the synthesis of immunoreactive EGF polypeptide in the wheat germ cell-free translation system. When the immunoreactive translation products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single radioactive peak which co-migrated with the standard 125I-EGF (mouse) was observed.

Animals↗

PubMed1983

Efficient extraction of RNA from mammalian tissue.

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.

Animals↗

PubMed1983

Mammalian pancreatic preproglucagon contains three glucagon-related peptides.

We have isolated cDNA clones encoding bovine pancreatic preproglucagon. Twenty-five putative preproglucagon clones were selected by screening 3,100 clones of a fetal bovine pancreas cDNA library with a synthetic oligodeoxynucleotide probe. The probe was a mixture of synthetic 17-base DNA oligomers constructed to correspond to the six carboxyl-terminal amino acids (residues 24-29) of mature glucagon. Restriction mapping of six of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1,200-base-pair DNA insert revealed that it contained an essentially full-length copy of glucagon mRNA. Analysis of the cDNA suggested a protein coding sequence of 540 nucleotides and 5'- and 3'-untranslated regions of 90 and 471 nucleotides, respectively. This cDNA sequence encoded a 20-amino acid signal sequence followed by one for glicentin, a 69-amino acid polypeptide containing an internal glucagon moiety that has been found in porcine intestines. Glicentin is followed by two additional glucagon-like peptides, each flanked by paired basic amino acids (Lys, Arg) characteristic of prohormone processing. These polypeptide sequences show striking homology with those for glucagon and other members of the glucagon family of peptides.

Amino Acid Sequence↗