STUDIES ON RAT KIDNEY GAMMA-GLUTAMYLCYSTEINE SYNTHETASE AND EFFECT OF DIETARY NUCLEOTIDES ON THE IMMUNE FUNCTION OF MICE

An improved purification scheme for rat kidney (gamma)-glutamylcysteinesynthetase ((gamma)GCSase) was developed. Such a preparation yields only onemajor band and one minor band on polyacrylamide gel electrophoresis.Further, the preparation is devoid of contaminating adenylate kinaseactivity. The K(,eq) for the following reaction was estimated to be 400 atpH 8.2: Glu + (alpha)AB ('M('2+)) (gamma)Glu(alpha)AB + ADP + P(,i) The reaction mechanism of (gamma)GCSase was studied using isotope exchange at equilibrium techniques and inhibition studies. Of the four isotope exchanges measured ((alpha)AB()(gamma)Glu(alpha)AB, Glu()(gamma)Glu(alpha)AB, ATP()ADP and P(,i)()ATP) only the ATP()ADP exchange did not show a saturable effect when substrate concentrations were elevated to three times their Km values. Inhibition studies using L-methionine-S-sulfoximine (MSOX) yielded results which showed MSOX was competitive with respect to Glu, noncompetitive with respect to (alpha)AB and uncompetitive with respect to ATP. Results from both experiments are consistent with a random BC-random RQ reaction mechanism. Only ATP and ADP bind oligatorally to free enzyme. The immune function of mice was studied using two inbred strains of mice maintained on one of three diets: rodent chow, nucleotide-free (NF) diet or nucleotide-free + RNA (NFRNA) diet. Immune response towards allogeneic challenges was measured in vivo using either free allografting of caridao tissue or allogeneic spleen cell injections (popliteal lymph node weight gain assay). In both allogeneic model systems, mice maintained on the NF diet exhibited diminished immune response to the challenges relative to the responses shown by the two control groups (chow and NFRNA). A syngeneic model was also tested. A lymphoid leukemia was injected subcutaneously into the flanks of mice, and tumor growth and recipient mouse mortality were monitored. Mice maintained on a NF diet survived longer than mice in the two control groups. Tumor growth in mice fed a NF diet was slower than tumor growth in mice fed either control diet

Current methodology to assess bioequivalence of levothyroxine sodium products is inadequate

Levothyroxine sodium is a drug with a narrow therapeutic index for which an individual patient must have his or her dose carefully titrated to achieve the necessary therapeutic effect. In addition, exogenous levothyroxine cannot be distinguished from the endogenously produced hormone. Since 2004, generic formulations have been approved for the most frequently prescribed brands of levothyroxine sodium. This review examines the methodology and statistical acceptance criteria and summarizes findings of a previously published relative bioavailability study that brings into question the use of standard criteria to assess bioequivalence of levothyroxine sodium. The key findings reviewed were the following: (1) in the absence of baseline correction for endogenous T4 levels, products that differed by as much as 25% to 33% would be declared bioequivalent; (2) the use of baseline correction reduced the likelihood of declaring products bioequivalent when they actually differed by 25% to 33%; (3) even with baseline correction, products that differed by 12.5% would be declared bioequivalent; and (4) there was evidence of significant carryover from one dosing period to the next even with washout periods of up to 53 days. In conclusion, the current recommended methodology in the United States to assess bioequivalence for levothyroxine sodium products is inadequate to differentiate products that differ by 12.5%, a clinically relevant difference. Recommendations are made for modifications to the criteria that could improve the likelihood that products that differ by a clinically significant amount in their bioavailability would not be accepted as bioequivalent

Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF- I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 ± 3,669 binding sites/cell) with a dissociation constant of 0.36 ± 0.10 nM. Two different β-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphetyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-γ1, and phosphatidylinositol 3-kinase. IGF.I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50 values for the stimulation of ERK1 and ERK2 by

Effects of sibutramine treatment in obese adolescents: a randomized trial

Increased prevalence of adolescent obesity requires effective treatment options beyond behavior therapy. To see whether sibutramine reduced weight more than placebo in obese adolescents who were receiving a behavior therapy program. 12-month, 3:1 randomized, double-blind trial conducted from July 2000 to February 2002. 33 U.S. outpatient clinics. 498 participants 12 to 16 years of age with a body mass index (BMI) that was at least 2 units more than the U.S. weighted mean of the 95th percentile based on age and sex, to the upper limit of 44 kg/m2. Site-specific behavior therapy plus 10 mg of sibutramine or placebo. Blinded study medication dose was uptitrated to 15 mg or placebo at month 6 if initial BMI was not reduced by 10%. Body mass index, waist circumference, body weight, fasting lipid and glycemic variables, safety, and tolerability. Seventy-six percent of patients in the sibutramine group and 62% of patients in the placebo group completed the study. The estimated mean treatment group difference at month 12 (linear mixed-effects model) favored sibutramine for change from baseline in BMI (-2.9 kg/m2 [95% CI, -3.5 to -2.2 kg/m2]) and body weight (-8.4 kg [CI, -9.7 to -7.2 kg]) (P < 0.001 for both). The sibutramine group had greater improvements in triglyceride levels, high-density lipoprotein cholesterol levels, insulin levels, and insulin sensitivity (P < or = 0.001 for all). The rate of tachycardia was greater with sibutramine vs. placebo (12.5% vs. 6.2%; difference, 6.3 percentage points [CI, 1.0 to 11.7 percentage points]) but did not lead to increased withdrawal (2.4% vs. 1.5%; difference, 0.9 percentage point [CI, -1.7 to 3.5 percentage points]). The 1-year study duration precluded assessment of long-term weight maintenance and putative health benefits and harms, and 24% and 38% of the sibutramine and placebo groups, respectively, did not complete follow-up. Sibutramine added to a behavior therapy program reduced BMI and body weight more than placebo and improved the profile of several metabolic risk factors in obese adolescents