Search PubMed⌕ Search

Biomedical subjects

J M Little

Publications and source records attributed to J M Little.

At least 37 records · Page 2Linked to original sources
PubMed1997

Long-term duct-occluded segmental pancreatic autografts: absence of microvascular diabetic complications.

BACKGROUND: Current insulin therapies for control of glucose metabolism in patients with type I diabetes mellitus prevent major metabolic consequences of insulin deficiency, but none prevents or arrests long-term complications. In experimental models of canine diabetes, retinopathy, neuropathy, and nephropathy have been shown to develop within 5 years. The aim of this study was to determine in a canine model whether glucose control provided by segmental duct-occluded pancreas autografts could prevent the long-term complications of diabetes. METHODS: Thirty-five outbred mongrel dogs underwent segmental pancreas autotransplantation with residual pancreatectomy. Follow-up over 5 years included endocrine, retinal fundus photography, fluorescein angiography, and nerve conduction studies. At endpoint, analysis of organ specific changes was undertaken. RESULTS: Long-term survival was achieved in 14 dogs for 4 to 5 years and in 3 dogs for 3 to 5 years. Glycosylated hemoglobin levels remained within normal limits, although response to glucose challenge was suboptimal. Fundus photography and fluorescein angiography demonstrated the absence of retinal vascular aneurisms, capillary leakage, and obliteration. Retinal digest showed no vascular changes and normal endothelial/pericyte ratios. Nerve conduction was normal, and histology of nerves revealed normal density of myelinated fibers and absence of intrafascicular vessels and glycogen deposits, with no change in spectrum of fiber diameters and ovoids. Renal histology revealed no evidence of nephropathy with normal glomerular basement membranes. CONCLUSIONS: We have demonstrated that duct-occluded segmental pancreatic autografts are capable of providing satisfactory metabolic control for up to 5 years, thereby preventing development of the long-term microvascular complications of diabetes.

Animals↗
PubMed1997

Application of photoaffinity labeling with [11,12-3H]all-trans-retinoic acid to characterization of rat liver microsomal UDP-glucuronosyltransferase(s) with activity toward retinoic acid.

[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 microM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H] all-trans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes.

Affinity Labels↗
PubMed1997

Glucuronidation of all-trans-retinoic acid and 5,6-epoxy-all-trans-retinoic acid. Activation of rat liver microsomal UDP-glucuronosyltransferase activity by alamethicin.

The effects of detergent, alamethicin (a channel-forming peptide), and the inducers phenobarbital and 3-methylcholanthrene on glucuronidation of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been investigated using liver microsomes from Sprague-Dawley and Fischer 344 rats. Conditions for enzymatic glucuronidation were optimized for substrate concentration, protein, and time by using atRA and Sprague-Dawley microsomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epoxy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs. 116 pmol/mg/min for 5,6-epoxy-atRA and atRA, respectively). The product of incubation of microsomes with atRA and UDP-glucuronic acid was identified as a glucuronide by beta-glucuronidase hydrolysis and by HPLC analysis. Alamethicin was shown to be a highly effective activator of glucuronidation activity; atRA and 5,6-epoxy-atRA glucuronidation rates were increased 2- and 3-fold, respectively, compared with detergent activation. Alamethicin (but not detergent) significantly increased retinoid glucuronidation by microsomes from Fischer 344 rats treated with phenobarbital and 3-methylcholanthrene, compared with untreated controls. The two compounds were equally effective inducers of activity, although 5,6-epoxy-atRA was again the better substrate. The same control and induced Fischer rat microsomes were photolabeled with [32P]5-azido-UDP-glucuronic acid in the absence or presence of detergent, two concentrations of alamethicin, and a 10-fold molar excess of unlabeled UDP-glucuronic acid. Photoincorporation into microsomal proteins from detergent-disrupted induced microsomes was 2-3 times greater than that of controls. Alamethicin increased photoincorporation of the probe into UDP-glucuronosyltransferase proteins an additional 1.5-2-fold in control and induced microsomes, compared with the respective detergent-activated samples.

Affinity Labels↗
PubMed1997

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT).

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).

Affinity Labels↗
PubMed1996

Photoaffinity labeling of human recombinant sulfotransferases with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate.

Photoaffinity labeling with 2-azidoadenosine 3', 5'-[5'-32P]bisphosphate was used to identify and characterize adenosine 3',5'-bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co-incubation with unlabeled adenosine 3',5'-bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 microM. To verify that the 31 35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3',5'-[5'-32P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3',5'-bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites.

Adenosine Diphosphate↗
PubMed1996

Logic, hermeneutics, and informed consent.

A belief in the validity of informed consent is one of the most important consequences of the doctrine of autonomy in medical ethics. Truly informed consent requires full disclosure of all relevant information by the doctor, competence of the patient to appreciate what the information signifies, understanding of the facts and issues by the patient, a voluntary choice by the patient and an autonomous authorisation for treatment or entry into a trial. Each of these conditions is hard to fulfil. In particular, full autonomy cannot exist in illness, and this is acknowledged by the act of consultation. The issue is further complicated by the stochastic nature of biological systems and the responses to illness and treatment. Disclosure of likely outcomes is, of necessity, unsatisfactory when a patient seeks surety when entering the clinical process with a serious and life threatening disease. Neither strict logic nor the law provide answers to this problem. The legal and moral issues have become confused. This is unfortunate, because the legal concern often centres on avoidance of actions in law rather than on the more fundamental issue of benefit to the patient. There is a need to teach doctors that discussion is a necessary part of the doctor-patient relationship, that fully informed consent is seldom-if ever-possible, and that skill in understanding what the patient is seeking is more important than the development of rigid and legally "complete" consent forms.

Communication↗
PubMed1995

Frantz's tumour: papillary and cystic carcinoma of the pancreas.

Frantz's tumour (papillary and cystic tumour) of the pancreas is a rare neoplasm usually seen in young women. It is of low grade malignancy and deserves special note among pancreatic malignancies as it is frequently amenable to local resection and has a good long-term survival rate after excision. Three such cases have been treated at Westmead Hospital, one young male and two females. In two the disease was confined to the pancreas. In one, local invasion outside the pancreas and trans-coelomic spread to the ovaries was present at the time of diagnosis. Complete surgical removal of macroscopic disease was achieved in all three and all remain disease free between 2 and 4 years post-surgery. All have good exocrine and endocrine pancreatic function. These cases are discussed in detail. The need to be aware of this uncommon variant of pancreatic cancer is stressed. Investigation and treatment options are reviewed. The role of cytology studies in diagnosis and the potential for long-term surgical control of this tumour are highlighted on the basis of our limited experience and that presented in recent surgical literature.

Adolescent↗
PubMed1995

Including smoking status as a new vital sign: it works!

BACKGROUND: Despite the adverse health consequences of smoking, many physicians still neglect to counsel smokers to quit. This study evaluated the effect of including smoking status as a vital sign on the frequency of physician discussions with patients about smoking and physician advice to quit smoking. METHODS: A consecutive sample of adult ambulatory patients in our metropolitan family practice residency program completed exit surveys on physician and nurse counseling about smoking. Control group data were collected for 1 month before the change was made to include smoking status as a vital sign on patient charts. Charts were then marked with a stamp as a chart prompt in the vital signs section. Data were collected for 2 months after smoking status was added to the stamp. RESULTS: There were 637 individuals surveyed, of whom 179 were current smokers; 95 in the "prestamp" group and 84 in the "poststamp" group. The percentage of patient-physician encounters during which smoking was discussed increased from 47% to 86% (P < .001). Physician advice to quit increased from 50% to 80% (P < .001). Physician discussion of smoking with patients increased across all of the five stages of change but most dramatically (53% to 95%) in the "preparation" stage. Physicians were much less likely to counsel patients in the "precontemplation" stage to quit smoking. CONCLUSIONS: Including smoking as a new vital sign significantly increased the likelihood of smoking-related discussions between patients and their physicians. The stamp is inexpensive and easy to use, and because it is a one-time office system change, it is more likely to be implemented and maintained in busy practices.

Adult↗
PubMed1995

Characterization of human liver microsomal UDP-glycosyltransferases using photoaffinity analogs.

The photoaffinity analogs [beta-32P]5-azido-UDP-glucuronic acid ([32P]5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose ([32P]5N3UDP-Glc) were used to characterize UDP-glycosyltransferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to 56-kdalton range, known to contain UDP-glucuronosyl transferases (UGTs), was concentration dependent, and photolabeled proteins were susceptible to trypsin digestion only in the presence of detergent. The latter was demonstrated by the appearance on Western blots of the trypsin-treated, detergent-disrupted microsomes of a protein band of slightly lower molecular mass than, and presumably derived from, the UGTs. However, a labeled cleavage product was found only in samples photolabeled with [32P]5N3UDP-GlcUA and not in those labeled with [32P]5N3UDP-Glc. In detergent-treated microsomes, all of the nucleotide sugars that were tested protected better against photoinsertion of [32P]5N3UDP-GlcUA than of [32P]5N3UDP-Glc, with UDP-glucose being the most effective, followed by UDP-GlcUA and UDP-galactose. The pattern of inhibition of a series of uridinyl analogs toward photolabeling by the two probes was quite different: one inhibitor that was ineffective in blocking photoincorporation of [32P]5N3UDP-GlcUA (L-DPASiU) was one of the most potent inhibitors of photolabeling with [32P]5N3UDP-Glc. A similar dichotomy was seen with several inhibitors in enzymatic assays measuring hyodeoxycholic acid 6-O glucuronidation and glucosidation activities; the most potent inhibitors of HDCA glucosidation were not as effective against glucuronidation. The results indicate a lumenal orientation for human microsomal UGTs and provide substantial evidence that two distinct enzyme systems are involved in 6-O glucuronidation and 6-O glucosidation of HDCA.

Affinity Labels↗
PubMed1994

Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs.

The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Hepatocellular carcinoma in western Sydney. Aetiology, changes in incidence, and opportunities for better outcomes.

OBJECTIVES: To examine the incidence of hepatocellular carcinoma (HCC) in western Sydney over the last 14 years, to assess risk factors for the disease among ethnic groups of Australian residents, and to consider the opportunities for improving its usually poor outcome. DESIGN AND SUBJECTS: Retrospective case-record review of clinical features in all (122) patients discharged from a 900-bed tertiary-referral teaching hospital with a diagnosis of HCC from January 1979 to March 1993. MAIN OUTCOME MEASURES: Annual number of new cases; risk factors according to birthplace; surgical resectability of tumours. RESULTS: New cases admitted each year at least doubled between 1979-1985 and 1986-1992. This apparent increase involved individuals born in Australia (50% of all patients) as well as immigrants. Cirrhosis was found in 93% at liver biopsy or autopsy. Excessive alcohol intake was an associated risk factor for 46% of Australian-born patients and for 13% of those born overseas. Among the latter, HCC was associated with markers of hepatitis B virus infection in 64%. Since hepatitis C virus (HCV) tests became available in 1990, five of nine patients tested were anti-HCV positive. Surveillance screening of patients known to have cirrhosis detected eight cases of early HCC. Seven of these had surgical resection and all are alive. CONCLUSIONS: New diagnoses of HCC have increased recently, irrespective of country of birth. In Australian-born patients alcoholic liver disease remains a major aetiological factor but the role of HCV requires further evaluation. Among immigrants, cirrhosis from chronic viral hepatitis accounts for most cases. We propose that prevention of cirrhosis caused by chronic viral hepatitis should have the greatest long-term impact on prevention of HCC in Australia. The role of surveillance of people with cirrhosis to detect small and potentially resectable tumours should be explored.

Adult↗
PubMed1994

Bile acid glucuronidation by rat liver microsomes and cDNA-expressed UDP-glucuronosyltransferases.

Four rat UDP-glucuronosyltransferases (UGTs), UGT2B1, UGT2B2, UGT2B3 and UGT2B6, synthesized in COS-7 cells from appropriate cDNA clones were screened for activity towards a range of bile acids, neutral steroids and retinoic acid. For comparison, as well as optimization of enzymatic assays and product identification, rat liver microsomal preparations from Sprague-Dawley, Fischer 344 and phenobarbital-induced Fischer 344 male rats were also used as enzyme sources. Only two of the expressed proteins, UGT2B1 and UGT2B2, were active in bile acid glucuronidation. UGT2B1 exhibited a high substrate specificity for the carboxyl function of bile acids, whereas UGT2B2 demonstrated less specificity, accepting both hydroxyl and carboxyl functions of bile acids. The preferred substrates for both cloned enzymes were mono-hydroxylated bile acids, followed by di-hydroxylated 6-OH compounds. The levels of UGT activity were sufficient to allow for the identification of the biosynthesized products. The data presented here demonstrate that bile acid glucuronidation is carried out, at least in part, by members of the UGT2B subfamily. Similar results have been obtained previously for neutral steroid glucuronidation. UGT2B3 and UGT2B6 was not involved in BA glucuronidation; none of the cloned enzymes was active toward retinoic acid.

Affinity Labels↗