Purification and characterization of UDP-N-acetylglucosamine:alpha-D-mannoside beta(1,6)N-acetylglucosaminyltransferase

Over the past twenty years, evidence has accumulated indicating that transformation with tumor viruses, carcinogens, or transfection with oncogenes alters the glycosylation of cell surface glycolipids and glycoproteins. It has been demonstrated that BHK cells transformed with either Rous sarcoma or polyoma virus differ in their N-linked oligosaccharides from non-transformed parental cells. The significant difference in glycosylation of these transformed cells is an increase in the structure (GlcNAc$\beta$(1,6)Man$\alpha$(1,6)Man), known as the $\beta$(1,6) branch. The increase in the appearance of this branch correlates with a 5-fold increase in the specific activity of the Golgi enzyme GlcNAc-T V (EC 2.4.1.155), the enzyme directly responsible for synthesis of the $\beta$(1,6) branch. Significantly, increased expression of the $\beta$(1,6) branch and GlcNAc-T V has been reported for many human breast tumor biopsies. By contrast, studies have documented in several cell types a correlation between decreased expression of this branch, decreased activity of GlcNAc-T V, and decreased metastatic potential. To determine the molecular mechanism of the regulation of GlcNAc-T V expression after neoplastic transformation, we have focused on isolating a cDNA encoding the enzyme. In pursuit of this goal we have purified GlcNAc-T V from a Triton X-100 extract of rat kidney acetone powder. GlcNAc-T V was purified by sequential affinity chromatography on a UDP-hexanolamine Sepharose column and on a synthetic oligosaccharide inhibitor Sepharose column. Silver-stained SDS-PAGE of the purified enzyme revealed two major bands at apparent molecular weights of 69 and 75 kDa. The enzyme was recovered in a 26% yield with about a 400,000-fold increase in specific activity. The optimal ranges of pH and Triton X-100 concentrations for enzyme activity were 6.5-7.0 and 1.0-1.5%, respectively. Mn\sp{+2}, Ca\sp{+2}, and Mg\sp{+2} were each found to have a negligible ($\u3c$10%) effect on activity. Moreover, the enzyme was fully active in the presence of 20 mM EDTA. Enzyme activity was stabilized and enhanced by the addition of 20% glycerol, 0.5 mg/ml IgG, and 0.2 M NaCl. The K\sb{\rm m} and v\sb{\rm max} of the purified enzyme towards a synthetic trisaccharide acceptor was 87 $\mu$M and 18.8 $\mu$mol/(mg*min), respectively. This K\sb{\rm m} value is similar to that previously reported for this acceptor using the enzyme from crude extracts of BHK cells

Case Series Regarding Parastomal Variceal Bleeding: Presentation and Management

Parastomal variceal bleeding (PVB) is a serious complication occurring in up to 27% of patients with an ostomy and concurrent cirrhosis and portal hypertension. The management of PVB is difficult and there are no clear guidelines on this matter. Transjugular intrahepatic portosystemic shunt (TIPS), sclerotherapy, and /or coil embolization are all therapies that have been shown to successfully manage PVB. We present a case series with five different patients who had a PVB at our institution. The aim of this case series is to report our experience on the management of this infrequently reported but serious condition. We also conducted a systemic literature review focusing on the treatment modalities of 163 patients with parastomal variceal bleeds. In our series, patient 1 had embolization and sclerotherapy without control of bleed and expired on the day of intervention due to hemorrhagic shock. Patient 2 had TIPS in conjunction with embolization and sclerotherapy and had no instance of rebleed 441 days after therapy. Patient 3 did not undergo any intervention due to high risk for morbidity and mortality, the bleed self-resolved and there was no further rebleed, this same patient died of sepsis 73 days later. Patient 4 had embolization and sclerotherapy and had no instance of rebleed 290 days after therapy. Patient 5 had TIPS procedure and was discharged five days post procedure without rebleed, patient has since been lost to follow-up