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D Irwin

Publications and source records attributed to D Irwin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Divergent regulation of human and rat proglucagon gene promoters in vivo.

A single mammalian proglucagon gene is expressed in the brain, islets, and intestinal enteroendocrine cells, which gives rise to a unique profile of proglucagon-derived peptides (PGDPs) in each tissue. The biological importance of glucagon, glucagon-like peptide (GLP)-1, and GLP-2 has engendered considerable interest in the factors regulating the synthesis and secretion of the PGDPs in vivo. Although rat proglucagon gene transcription has been extensively studied, the factors important for control of human proglucagon gene expression have not been examined. We now report that, despite conservation of proximal promoter G1-G4 enhancer-like elements, human proglucagon reporter plasmids containing these elements are transcriptionally inactive in islet cell lines. Remarkably, larger human proglucagon promoter fragments, such as the 1604 hGLU-Luc, are expressed in GLUTag enteroendocrine cells but not in islet cell lines. A total of 5775 bases of human proglucagon promoter were required for expression in islet cell lines. Analysis of human proglucagon promoter expression in transgenic mice demonstrated that approximately 1.6 kb of human proglucagon gene sequences directs expression of a human growth hormone reporter gene to the brain and intestinal enteroendocrine cells but not islet cells in vivo. These findings provide the first evidence demonstrating divergence in the mechanisms utilized for tissue-specific regulation of the human and rodent proglucagon genes.

Animals↗
PubMed1998

Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis.

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

Amino Acid Sequence↗
PubMed1997

Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca.

Cellulase E4 from Thermomonospora fusca is unusual in that it has characteristics of both exo- and endo-cellulases. Here we report the crystal structure of a 68K M(r) fragment of E4 (E4-68) at 1.9 A resolution. E4-68 contains both a family 9 catalytic domain, exhibiting an (alpha/alpha)6 barrel fold, and a family III cellulose binding domain, having an antiparallel beta-sandwich fold. While neither of these folds is novel, E4-68 provides the first cellulase structure having interacting catalytic and cellulose binding domains. The complexes of E4-68 with cellopentaose, cellotriose and cellobiose reveal conformational changes associated with ligand binding and allow us to propose a catalytic mechanism for family 9 enzymes. We also provide evidence that E4 has two novel characteristics: first it combines exo- and endo-activities and second, when it functions as an exo-cellulase, it cleaves off cellotetraose units.

Actinomycetales↗
PubMed1995

Hepatitis B transmission from contaminated cryopreservation tank.

Over a 25-month period, six multiply transfused patients undergoing cytotoxic treatment for haematological or other malignant disorders developed icteric acute hepatitis B virus (HBV) infection. Bone marrow or peripheral-blood stem cells had been harvested from all six patients and stored in the same cryopreservation tank for possible future transplantation. Human DNA, HBsAg, and HBV DNA with sequences identical to those from four patients with related infections were subsequently found in the liquid nitrogen. Leakage of the cryopreservation bags used to store bone marrow harvested from the first patient when acutely infected with HBV led to contamination of the tank and its contents with HBV and subsequent transmission to patients after transplantation. This incident emphasises the continuing need to screen donors of tissue to be cryopreserved for bloodborne virus infections. It also reinforces the requirement for primary containers used to cryopreserve human tissue to be sealed in a way which prevents exchange of material between the specimen and the liquid nitrogen.

Acute Disease↗
PubMed1994

Characterization and sequence of a Thermomonospora fusca xylanase.

TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. The enzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed by plasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after 18 h at 75 degrees C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The Vmax values for TfxA and the fragment are 600 and 540 mumol/min/mg, respectively, while the Kms are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequence of 12 other xylanases from seven different organisms and belongs to family G.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetales↗
PubMed1993

Severity of sickness at admission to hospital in Colchester 1985 and 1990.

In 1991 concern was expressed by hospital physicians and general practitioners about hospital services in Colchester, Essex, following a period of declining real resources. One focus of complaints was that over a period of about five years it had become increasingly difficult to admit acutely ill medical patients. We wished to obtain some objective data to corroborate or refute this anecdotal evidence. We therefore studied the severity of illness at admission in a random sample of emergency admissions for acute chest problems in 1985 and 1990. The main finding was that APACHEII scores were significantly higher (P = 0.002) in the 1990 sample than in the 1985 sample. These objective data contributed to a decision to allocate pounds 100,000 extra funding to the acute medical service in 1991-92.

Aged↗
PubMed1993

Clinical aspects of HIV-related lymphoma.

The number of cases HIV-associated non-Hodgkin's lymphoma continues to increase as the AIDS epidemic grows. Approximately 3% of AIDS-defining illnesses are non-Hodgkin's lymphoma. The number of non-Hodgkin's lymphoma cases may actually be higher because many cases go unreported. There is also evidence that increasing numbers of patients who are surviving longer on antiretroviral therapy are developing non-Hodgkin's lymphoma. A majority of HIV-related lymphomas are large cell, either high-grade immunoblastic or aggressive intermediate grade, diffuse cleaved, or small noncleaved (Burkitt's-like). HIV-related non-Hodgkin's lymphomas behave aggressively. They are predominantly extranodal and often show unusual patterns of organ involvement. They are typically stage III or IV at the time of diagnosis. Current treatment strategies involve the use of combination chemotherapy regimens with or without antiretroviral therapy. Current studies are evaluating the efficacy of low-dose chemotherapy regimens versus standard-dose regimens with granulocyte-macrophage colony-stimulating factor support. New strategies for treating AIDS-associated non-Hodgkin's lymphoma will incorporate our current knowledge of AIDS-related lymphoma pathogenesis. Factors that reflect a patient's state of immunodeficiency seem to be the most important prognostic features determining clinical outcome after treatment. Patients with good prognostic features may benefit the most from aggressive treatment regimens. AIDS-related primary central nervous system lymphomas continue to comprise approximately 15% of AIDS-related non-Hodgkin's lymphoma cases. Treatment is limited. Although whole-brain radiation therapy can result in an improved neurologic status, the median survival remains 3 to 4 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Neoplasms↗
PubMed1993

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.

Actinomycetales↗
PubMed1991

Human T-lymphocytes synthesize and secrete a protease resistant proteoglycan in a delayed, serum-dependent response to concanavalin A.

In the presence of radioactive precursors, the addition of Concanavalin A (Con A) to cultures of mononuclear cells (MC) from human blood resulted in marked accumulation in the medium of a protease-resistant proteoglycan (PG) with chondroitin sulfate (CS) chains of an average molecular weight of 28 KDa. This PG was shown to be elaborated primarily by T-cells. The addition of serum was necessary for maximal stimulation with Con A and under these conditions PG accumulation in the medium rose slowly during the first few days of culture and much more rapidly between days 6-8.

Blood↗
PubMed1989

The effects of "matrigenin" activity from bovine bone on the glycosaminoglycans of bovine articular cartilage in culture. A model for cartilage repair by bone derived factors.

A bovine articular cartilage longterm culture system was used to study proteoglycan synthesis by a pulse labeling technique. A brief preincubation of the cartilage slices with bacterial collagenase was found to be an effective method of depleting proteoglycan. The addition of a fraction from bovine bone that contained partially purified "matrigenin" activity to the cultures resulted in the stimulation of incorporation of radioactive precursors into cartilage chondroitin sulfate, suggesting increased proteoglycan synthesis. The stimulatory effect of matrigenin activity was observed earlier and was more sustained if the cartilage slices were preincubated with collagenase. The system appears to be useful for testing the hypothesis that bone derived factors stimulate the repair of damaged cartilage.

Animals↗
PubMed1987

The effect of "matrigenin" activity from bone on glycosaminoglycan and proteoglycan synthesis by cultured cells from articular tissues.

An activity that stimulates glycosaminoglycan and proteoglycan synthesis by confluent connective tissue cells in culture ("matrigenin" activity) has been partially purified from bovine bone. This activity is expressed on fibroblastic cells from several types of connective tissues, including synovium, and on cartilage tissue, but not on cells of unrelated origin. For synovial fibroblastic cells, matrigenin activity stimulated both hyaluronic acid and proteoglycan synthesis. The major proteoglycan whose synthesis was stimulated, contained primarily chondroitin-6-sulfate chains and eluted in the range of 400-600 Kdaltons. It is suggested that matrigenin activity in bone matrix has the potential to modulate remodelling (repair) in bone and in the synovial and cartilagenous regions abutting on bone.

Animals↗
PubMed1985

Lipoprotein composition as a component in the lipoprotein clearance defect in experimental diabetes.

The hypertriglyceridemia associated with streptozotocin-induced diabetes in rats is largely reflected in the plasma lipoproteins of density less than 1.006 g/ml. Analysis of the plasma apolipoproteins of these rats indicated marked alterations in both the total levels and in the lipoprotein distribution of the major apolipoproteins. In whole plasma, diabetes was associated with significant increases in apolipoprotein (apo)-AIV, apo-AI, and apo-B (mainly in the intestinally derived apo-B240) and a marked decrease in apo-E. In the d less than 1.006 g/ml lipoprotein fraction (very-low-density lipoproteins (VLDL], there were significant increases in apo-B240, apo-AI, and apo-AIV and decreased levels of apo-E and the C apolipoproteins. The decrease in apo-C was primarily due to lower levels of apo-CII, and the ratio of the lipoprotein lipase inhibitor, apo-CIII, to the lipoprotein lipase activator, apo CII, was significantly increased over that in controls. The comparative clearance of triglycerides of VLDL particles from control and diabetic rat plasma was tested in recirculating heart perfusion in vitro. During 45-min perfusions of hearts from control donor rats, lipolysis of triglycerides of VLDL from diabetic rats was only 63-64% of that using plasma VLDL from control rats. Perfusion of hearts from diabetic rats with VLDL from control rats gave lipolysis values of only 53% of that obtained with normal hearts. Where both the VLDL and hearts were obtained from diabetic rats, lipolysis was 23% of that observed when both the lipoprotein and the organ were from control rats. The data suggest that in addition to depressed lipoprotein lipase activity in the tissue from diabetic rats, there are also major compositional changes in circulating lipoproteins which may contribute to defective triglyceride clearance from the circulation.

Animals↗
PubMed1985

Phosphorylation of specific rat plasma membrane proteins during promotion of gamma-glutamyl transpeptidase-positive hepatic foci and inhibition by di(2-ethylhexyl)phthalate.

The protein kinase activity of isolated plasma membranes from the livers of rats treated with three promoting regimens was examined using both exogenous proteins and endogenous plasma membrane proteins as substrates. Male rats first received either an initiating dose (30 mg/kg) of the hepatocarcinogen diethylnitrosamine or the 0.9% NaCl solution vehicle by i.p. injection at 18 h following partial hepatectomy. Ten days later, the three promoting regimens were begun. These consisted of 10 weeks of treatment with either (a) a choline-deficient (CD) diet, (b) a choline-supplemented (CS) diet containing 0.06% phenobarbital (PHB) (CS plus PHB), or (c) a CD diet containing 0.06% PHB (CD plus PHB). In addition, two other groups of rats received either (a) a CS diet containing 2% di(2-ethylhexyl)phthalate (DEHP) (CS plus DEHP) or (b) a CD diet containing 2% DEHP (CD plus DEHP). DEHP is a widely used plasticizer and environmental contaminant which we have shown previously inhibits the development of putative preneoplastic gamma-glutamyl transpeptidase (GGT) positive foci in rat liver. Total liver plasma membrane protein kinase activity using both protamine sulfate and histone was cyclic adenosine 3':5'-monophosphate independent and did not appear to be a marker of promotion. Its activity was increased by both DEHP which suppresses the development of GGT positive foci and a CD diet which promotes the appearance of GGT positive foci. The CD, CS plus PHB, and CD plus PHB dietary regimens, which promote the appearance of GGT positive foci, induced the phosphorylation of a Mr 40,000 plasma membrane protein in vitro by endogenous protein kinases. Plasma membranes from DEHP-treated rats did not demonstrate phosphorylation of this Mr 40,000 protein. DEHP dietary treatment also blocked the ability of epidermal growth factor to enhance the phosphorylation of its Mr 175,000 receptor protein in isolated liver plasma membranes. These results suggest that the phosphorylation of a Mr 40,000 plasma membrane protein may be important to the early promotional phase of liver carcinogenesis, and that one mechanism by which DEHP inhibits the emergence of GGT positive foci may be by blocking the response of initiated cells to stimulation by epidermal growth factor.

Animals↗
PubMed1985

The effect of human bone matrix extracts on the biosynthesis of macromolecules by human mononuclear cells in culture.

In chronic rheumatoid arthritis, mononuclear cells (MC) accumulate in the subchondral bone and form a prominent part of both destructive lesions and repair reactions. A fraction from human bone matrix extracts (BME) stimulated glycosaminoglycan (gag) and glycoprotein synthesis by fibroblastic cells but its effects on MC metabolism had not been studied. A method was established for the study of incorporation of radioactive precursors into total protein, IgG and gag synthesized and secreted by peripheral blood MC cultured in microwells in the presence or absence of Concanavalin A (ConA). Relatively low concentrations of BME suppressed spontaneous synthesis of radioactive IgG (protein A bound) and TCA precipitable protein but had little effect on gag synthesis. In general, stimulation of the cultures with ConA overcame the inhibitory effects on protein synthesis by the BME. A large stimulation of gag synthesis induced by ConA was not affected by BME. The interactions between the BME and the stimulatory effect of ConA on DNA synthesis were studied in detail and were found to be complex, not immunologically specific and appeared to be due to binding of lectin by the carbohydrate moieties of the glycoproteins in the BME. On the basis of a model of the lectin-BME interaction, the hypothesis is postulated that the carbohydrate moieties of subchondral bone glycoproteins may have the capacity to act as a solid state "trap" for certain circulating antigens which may then also interact with surface glycoproteins of the MC accumulating in the subchondral bone. The physiological role of gag synthesis by MC is not known.

Arthritis, Rheumatoid↗