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M Heffernan

Publications and source records attributed to M Heffernan.

35 records · Page 2Linked to original sources
PubMed1993

Branching beta 1-6N-acetylglucosaminetransferases and polylactosamine expression in mouse F9 teratocarcinoma cells and differentiated counterparts.

beta-All-trans-retinoic acid (RA)-induced endodermal differentiation of mouse F9 teratocarcinoma cells is accompanied by changes in glycoprotein glycosylation, including expression of i antigen (i.e. polylactosamine) and leukophytohemagglutinin-reactive oligosaccharides (i.e. -GlcNAc beta 1-6Man alpha 1-6-branched N-linked). We have used the F9 teratocarcinoma cells as a model to study developmental regulation of glycosyltransferase activities which are responsible for the biosynthesis of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides and polylactosamine. Growth of F9 cells in the presence of 10(-6) M RA for 4 days increased core 2 GlcNAc transferase and GlcNAc transferase V activities by 13- and 6-fold, respectively, whereas the activities of GlcNAc transferase I, beta 1-3GlcNAc transferase (i), beta 1-4Gal transferase, and beta 1-3Gal transferase increased 2-4-fold. Induction of glycosyltransferase activities by RA was dose-dependent and showed a biphasic response with approximately half of the increase observed 3 days after RA treatment and the remainder occurred by day 4. PYS-2, a parietal endoderm cell line, showed levels of glycosyltransferase activities similar to those of RA-treated F9 cells. Glycosyltransferase activities in the RA-resistant F9 cell line (RA-3-10) were low and showed only a small induction by RA. These observations suggest that differentiation of F9 cells is closely associated with induction of multiple glycosyltransferase activities, with most pronounced increases in GlcNAc transferase V and 2',5'-tetradenylate (core 2) GlcNAc transferase. The increase in GlcNAc transferase V was also reflected by the 4-6-fold increase in the binding of 125I-leukophytohemagglutinin to several cellular glycoproteins, which occurred after 3 days of RA treatment. The endo-beta-galactosidase-sensitive polylactosamine content of membrane glycoproteins and, in particular, the LAMP-1 glycoprotein was markedly increased after RA treatment of F9 cells. Consistent with these observations, fucosylated polylactosamine (i.e. dimeric Lex) was also increased in RA-treated cells. Analysis of the aryl oligosaccharides produced by F9 cells cultured in the presence of aryl alpha-D-GalNAc showed that RA treatment enhanced the synthesis of disialyl core 2 O-linked oligosaccharides and increased the polylactosamine content of the aryl oligosaccharides by > 20-fold. The results suggest that differentiation of F9 cells into endoderm is closely associated with increased GlcNAc transferase V and core 2 GlcNAc transferase activities, enzymes which control the level of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides, the preferred substrates for polylactosamine addition.

Amino Sugars↗
PubMed1993

Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin.

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatin-substrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60-90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN2, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60-90-kDa proteinases are cathepsin L-like proteinases.

Animals↗
PubMed1993

Short-term effects of recombinant human insulin-like growth factor I on metabolic control of patients with type II diabetes mellitus.

Recombinant human insulin-like growth factor I (rhIGF-I) lowers blood glucose, serum insulin, C-peptide, and lipid levels in healthy and diabetic animals and humans. We hypothesized that rhIGF-I might control blood glucose levels and concomitantly reduce pancreatic insulin secretion in patients with type II diabetes. If true, rhIGF-I might serve as a therapeutic agent that could mitigate some of the detrimental effects of hyperinsulinemia secondary to insulin resistance in these patients. In this study, we treated 12 patients with type II diabetes mellitus twice daily for 5 days with sc rhIGF-I in doses of 90, 120, or 160 micrograms/kg body weight. Metabolic parameters in the fasting and postprandial states were assessed during a 3-day baseline period, the rhIGF-I treatment period, and a 3-day follow-up period, respectively. Administration of rhIGF-I significantly reduced mean (+/- SD) concentrations of fasting blood glucose (12.3 +/- 4.5 to 9.1 +/- 2.6 mmol/L), serum insulin (98 +/- 52 to 56 +/- 27 pmol/L), and C-peptide (993 +/- 298 to 728 +/- 232 pmol/L). It also decreased postprandial (area under the curve) blood glucose (32.5 +/- 12.7 to 23.9 +/- 8.1 mmol/L.h), serum insulin (1102 +/- 707 to 467 +/- 332 pmol/L.h), and C-peptide (5958 +/- 2747 to 3442 +/- 1523 pmol/L.h). The administration of rhIGF-I was also associated with a small but significant reduction in serum triglycerides (6.76 +/- 3.45 to 5.32 +/- 2.59 mmol/L) and total cholesterol (6.13 +/- 1.25 to 5.66 +/- 1.20 mmol/L), 24-h creatinine clearance increased significantly (85 +/- 30 to 133 +/- 51 mL/min), and microalbuminuria was unchanged. Although rhIGF-I was reasonably well tolerated, side effects included low-grade edema, mild and mainly asymptomatic orthostatic hypotension, and bilateral temporomandibular tenderness. We conclude that short-term treatment of type II diabetic patients with rhIGF-I favorably affects metabolic control and enhances kidney function. An assessment of the risk/benefit ratio of rhIGF-I administration to this group of patients awaits extended experiments.

Aged↗
PubMed1991

Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively.

Animals↗
PubMed1989

Molecular characterization of P2B/LAMP-1, a major protein target of a metastasis-associated oligosaccharide structure.

Sialylated and GlcNAc beta 1-6Man alpha 1-6Man beta 1 (beta 1-6 branched) complex-type oligosaccharides linked to asparagine residues of membrane glycoproteins in metastatic murine tumor cells have been associated with efficient tumor cell metastasis. A large proportion of these oligosaccharide structures, in several unrelated malignant cell lines, have been shown to be associated with a glycoprotein termed P2B, with a molecular weight of 130,000. This glycoprotein has recently been purified from the metastatic MDAY-D2 cell line and shown to be biochemically similar to a lysosomal associated membrane glycoprotein (LAMP-1). We report here the details of a 2147 nucleotide complementary DNA encoding the entire murine P2B polypeptide which was immunoselected from a lambda gt11 expression library and sequenced. The sequence is similar to a complementary DNA coding for mouse LAMP-1 with the exception of a 5' untranslated region, a leader signal-sequence, and various insertions, deletions, and substitutions in the 3' untranslated domain. An open reading frame of 405 amino acids encodes a mature polypeptide of 382 residues with a predicted molecular weight of 42,000. P2B/LAMP-1 possesses 20 asparagine-linked glycosylation sites separated into equal halves by a central, putative hinge region and is anchored by a carboxy, membrane-spanning, domain. Topological considerations dictate that cell surface expression of P2B/LAMP-1 exposes the bulk of the glycoprotein into the extracellular compartment. Immunofluorescent staining of fibroblast cells indicated that P2B/LAMP-1 was associated with lysosomal membranes and, to a lesser degree, select surfaces of plasma membrane. An amino acid comparison of the murine sequence with its recently cloned rat, human, and chicken counterparts shows a conservation of 17 of 20 asparagine-linked glycosylation consensus sites, eight of eight cysteine residues, and other selected protein domains. The interspecies conservation of these domains suggests that they are important for the structure and function of the P2B/LAMP-1 glycoprotein. Northern analysis revealed that P2B/LAMP-1 is widely expressed in normal murine tissues and tumor cell lines. However, in two experimental models of metastasis, where changes in branching of oligosaccharides on P2B/LAMP-1 have been shown to occur, comparable levels of P2B/LAMP-1 mRNA were found in both metastatic and nonmetastatic cell lines.

Amino Acid Sequence↗
PubMed1989

Thiol proteases released in vitro by Fasciola hepatica.

Immature Fasciola hepatica release 11 distinct proteases when cultured in vitro for 16 h as revealed by gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six of these proteases are active in the pH range 4.5 to 8.0. Five are acid proteases, being most active in the pH range 3.0 to 4.5. The majority of proteases released in vitro by immature flukes are also released by mature flukes; however, a 40-kDa protease released by immature flukes is a very minor protease released by mature flukes. The activity of all proteases is inhibited by leupeptin, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, phenylmethylsulfonyl fluoride and iodoacetamide and enhanced or stabilized by the reducing agents cysteine and dithiothreitol. Therefore, all F. hepatica in vitro-released proteases identified by gelatin-substrate SDS-PAGE are thiol proteases.

Animals↗
PubMed1982

Pilot study on use of PMPs.

Patient management problems (PMPs) have been used for many years in an attempt to design tests capable of measuring clinical problem-solving ability. Problems have arisen particularly related to criterion validity, though some studies have suggested that content and construct validity are satisfactory. One of the major problems has been that a common definition of problem-solving ability has not been developed yet. In addition, various formats have been used with considerable variety in construction, possibly influencing outcomes, and scoring procedures have not been standardized. A serious charge has been that PMPs are content specific and therefore are unreliable and not valid for measuring any general characteristic such as problem-solving ability (assuming this exists).

Australia↗
PubMed1979

A shortcut to rational prescribing. Matching the bug and the drug.

The rational use of antibacterial chemotherapy in the clinical setting is a necessary objective, but a difficult task. This article presents a particular interpretation of history that links bacteria, conditions and chemotherapy in a prescribing rule that applies to much of the bacterial infection seen in clinical practice. Whilst retaining a rational basis, use of the rule simplifies day-to-day prescribing of antibacterial chemotherapy.

Adult↗
PubMed1977

What drug information does the general practitioner need?

There is a great need for imporved drug information services within the Australian medical community. The need has been only partly assessed and should be more objectively surveyed as part of any development of drug information services. Whatever service is established, it should be easily accessible, pay particular attention to the language used in communication, and have a significant ongoing educational component.

Australia↗
PubMed1976

Promoting discovery as a teaching technique.

What one discovers or learns for oneself tends to be retained for long periods and used effectively in later problem solving. This article examines the reasons for this, suggesting methods and illustrating how one may teach in the clinical environment to achieve such discovery learning.

Education, Medical↗
PubMedDate not supplied

Effects of exposure temperature on brown adipose tissue uncoupling protein mRNA levels.

The effect of temperature on the amount of uncoupling protein mRNA in rat brown adipose tissue was examined after 1 and 14 days of exposure to cold. The relative amounts after 1 day, compared with rats kept at a thermoneutral temperature of 28 degrees C, were 3.2 at 19 degrees C, 3.3 at 11 degrees C, and 2.1 at 3 degrees C. This suggests that in warm-acclimated rats, a maximal response to a cold stimulus in brown adipose tissue is reached by 19 degrees C. In contrast to these results, the relative amounts of uncoupling protein mRNA after 14 days of cold exposure, compared with rats left at 28 degrees C, were 1.2 at 19 degrees C, 1.9 at 11 degrees C, and 2.1 at 3 degrees C. Since it is known that the amount of uncoupling protein in cold-acclimated rats increases continuously with decrease in temperature, the amount of protein reflects the mRNA levels during later times but not the initial time of exposure to cold.

Adipose Tissue, Brown↗