Hussain M, Daignault S, Agarwal N, Grivas PD, Siefker-Radtke AO, et al.
Cancer •
Epidermal growth factor receptor overexpression is associated with poor outcomes in urothelial carcinoma (UC). Cetuximab (CTX) exhibited an antitumor effect in in vivo UC models. The efficacy of gemcitabine/cisplatin (GC) with or without CTX in patients with advanced UC was evaluated. Patients with advanced UC, measurable disease, and adequate organ function were randomized 1:2 to cisplatin (70 mg/m(2) ) on day 1 plus gemcitabine (1000 mg/m(2) ) on days 1, 8, and 15 (arm A) or GC plus CTX (500 mg/m(2) ) on days 1 and 15 (arm B). The primary endpoint was the overall response rate. The secondary endpoints were the response duration, safety, progression-free survival, overall survival, determination of whether or not CTX sensitized nonresponders to GC, and exploratory biomarker analysis. The accrual targets were 27 and 54 patients for the 2 arms, respectively. The overall response rate was reported by arm with binomial confidence intervals (CIs). Kaplan-Meier methods were used for time-to-event endpoints. Eighty-eight eligible patients were randomized; 87 were toxicity-evaluable, and 85 were response-evaluable. The overall response rates were 57.1% for arm A (95% CI = 37%-76%) and 61.4% for arm B (95% CI = 48%-74%). The median progression-free survival times were 8.5 months for arm A (95% CI = 5.7-10.4 months) and 7.6 months for arm B (95% CI = 6.1-8.7 months). The median overall survival times were 17.4 months for arm A (95% CI = 12.8 months to unreached) and 14.3 months for arm B (95% CI = 11.6-22.2 months). The most common grade 3/grade 4 adverse events in both arms were myelosuppression and nausea. Thromboembolism, acneiform rash, fatigue, pain, hypersensitivity reactions, elevated transaminases, hyponatremia, and hypomagnesemia were more common in arm B; 3 grade 5 adverse events occurred in arm B. The presence of primary disease significantly correlated with thromboembolism. An increased soluble E-cadherin level after cycle 2 correlated with a higher risk of death. GC plus CTX was feasible but was associated with more adverse events and no improvements in outcomes.
American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists •
A possible case of levofloxacin-induced thrombocytopenia is reported. A 73-year-old Caucasian woman with stage IV squamous cell cancer of the oral cavity arrived at the hospital with a 6-day history of epistaxis; petechiae over her arms, legs, and abdomen; and bruises over her forearms. Her comorbidities included hypertension, type 2 diabetes mellitus, and coronary artery disease. Two weeks before arrival at the hospital, the patient had been admitted to the hospital with community-acquired pneumonia (CAP) and given a 10-day course of levofloxacin 500 mg daily, which she completed 4 days before this admission. Her platelet count was 7,000 cells/mm(3) on admission. Her home medications included aspirin 325 mg daily, ranitidine 150 mg twice daily, alprazolam 0.25 mg daily, and methadone 10 mg twice daily. She last received cetuximab six weeks before this hospital admission. No other new medications were recently introduced. She had no known drug allergies and no recent heparin exposure. The patient was given a platelet transfusion and treated empirically with prednisone for possible immune thrombocytopenic purpura, though drug-induced thrombocytopenia (DIT) was also suspected. She was restarted on her home medications except for aspirin. She was discharged with a 7-day course of oral corticosteroids. At discharge, her platelet count was 38,000 cells/mm(3). Corticosteroids were discontinued when DIT was established to be the most likely diagnosis. A 73-year-old woman with stage IV squamous cell cancer of the oral cavity developed a possible case of levofloxacin-induced thrombocytopenia after receiving the drug for 10 days for treatment of CAP.
A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine alpha-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P. pastoris strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole P. pastoris cells were used to synthesize alpha-galactosyl (alpha-Gal) trisaccharide (Galalpha1,3Galbeta1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM alpha-Gal was accumulated in a 200-ml reaction. The Pichia system described here is simple and flexible. This work demonstrates that recombinant P. pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides.
The interaction of alpha-galacosyl antigen and its binding protein (anti-Gal antibody) has been studied by quartz crystal microbalance through a SAM immobilization technique which overcomes the nonspecific interactions and molecule trapping for current piezoelectric biosensors.
Chen Y, Janczuk A, Chen X, Wang J, Ksebati M, et al.
Carbohydrate research •
The syntheses of two carbohydrate-linked cisplatin analogs, namely lacto- and alpha-Gal-cisplatin, are described. A fusion enzymatic approach was used to create the diazido-alpha-Gal trisaccharide intermediate in the synthesis of alpha-Gal-cisplatin.
Chembiochem : a European journal of chemical biology •
A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galalpha l,3Lac through galactose metabolism has been developed. The plasmid contains a lambda promoter, a c1857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine - Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galalpha 1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 gL(-1)) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5'-diphosphoglucose. This work demonstrates that large-scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.
UDP-galactose:beta-galactosyl-alpha1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of galactosyl-alpha-1,3-beta-galactosyl structures in mammalian glycoconjugates. In humans the gene for alpha3GT is inactivated, and its product, the alpha-Gal epitope, is the target of a large fraction of natural antibodies. alpha3GT is a member of a family of metal-dependent-retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn(2+) activates the catalytic domain of alpha3GT (alpha3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn(2+) binding to two sites. At physiological metal ion concentrations, Zn(2+) gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was perturbed by substituting Co(2+) and Zn(2+) for Mn(2+) and by mutagenesis of a conserved D(149)VD(151) sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were found to be essential for activity, and the kinetic properties of a Val(150) to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage.
Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.
Synthesis of polymerizable beta-lactosyl, Galalpha1-->3Gal and alpha-mannosyl acrylamide derivatives with either a hydrophobic aromatic spacer or a hydrophilic biocompatible oligoethoxyl spacer was accomplished. Radical terpolymerizations of beta-lactosyl monomer. alpha-mannosyl monomer, and acrylamide were conducted in aqueous media with ammonium persulfate and N,N,N',N'-tetramethylethylenediamine as initiators. The resulting water soluble glycopolymers were further transformed efficiently by a recombinant alpha1-->3 galactosyltransferase to afford mediators bearing Galalpha1-->3Gal termini as xenoactive antigens and alpha-mannosyl termini as specific ligands for bacterial cells. The binding of the resulting multivalent glycopolymer to bacteria was tested by its ability to inhibit agglutination of yeast to E. coli. The binding of human natural anti-Gal antibodies to the alpha-Gal containing glycopolymers and a monovalent alpha-Gal-Man glycoconjugate was demonstrated by an ELISA inhibition assay.
