Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Fallibility of plasma urea and creatinine as indices of renal function in diarrhoeic calves treated with conventional or nutritional oral rehydration solutions

The prevalence and severity of gingival overgrowth in organ-transplant patients medicated with cyclosporin are greater in patients concomitantly medicated with nifedipine; however, no relationship between the gingival overgrowth and any of the nifedipine pharmacological variables has been demonstrated. The study examined the effect of five nifedipine pharmacological variables (nifedipine dosage, plasma concentration and gingival crevicular fluid concentration, M1 metabolite plasma concentration and the nifedipine: M1 ratio). The effect of the nifedipine variables on the gingival overgrowth score were examined using univariate and multivariate regression analysis. Adjustment for the effect of other risk factors was made by adding the distribution of each of the nifedipine variables in turn to a stepwise regression model containing previously identified risk factors for this condition. Despite the high levels of nifedipine sequestered in the GCF, only the plasma concentration of nifedipine was identified as a risk factor for the severity of gingival overgrowth in these patients (P = 0.01) once adjusted for other known risk factors (R2 for the model = 55%).     

Prostanoid biosynthesis by blood monocytes of children with hyperprostaglandin E syndrome

Hyperprostaglandin E syndrome (HPS), the prenatal variant of Bartter's syndrome, is characterized by a marked and selective stimulation of prostaglandin E (PGE2) synthesis. In the study group HPS patients showed increased urinary levels of PGE2, an index of renal, and of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M), an index of systemic PGE2 synthesis of 470% and of 570%, respectively. In addition, plasma concentration of PGE-M was also elevated 6.3-fold when compared with a control group. The urinary levels of other prostanoids were unaltered. During indomethacin treatment in both groups prostanoid excretion rates were suppressed to similar levels. To investigate the origin of stimulated prostanoid biosynthesis in HPS patients CD14+ monocytes were isolated from plasma samples, and the prostanoid synthesis was analyzed. The pattern and amounts of metabolites synthesized from endogenous arachidonic acid pools did not vary significantly between monocytes of the HPS and the control group. Thromboxane A2 (TXA2) was formed as the major prostanoid product. Using PGH2 as an exogenous substrate, again no difference in PGE2 biosynthesis was observed, indicating no difference in PGE-synthetic activity between both groups. Additionally, mRNA expression analysis of CD14+ monocytes via RT-PCR delineated the constitutive expression of cyclooxygenase-1, cyclooxygenase-2, and thromboxane synthase mRNA in cells from HPS patients and controls without statistical differences between these two groups. In conclusion, our data show that monocytes are not the source for the increased PGE2 biosynthesis in children with HPS, and a genetic defect in PGE synthesis can be excluded as the primary event in the pathogenesis in HPS.     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

Solution structure of the heparin-binding domain of vascular endothelial growth factor

Tricorn protease was previously described as the core enzyme of a modular proteolytic system displaying multicatalytic activity. Here we elucidate the mode of cooperation between Tricorn and its interacting factors, and we identify two additional factors, F2 and F3, closely related aminopeptidases of 89 kDa. In conjunction with these three factors, Tricorn degrades oligopeptides in a sequential manner, yielding free amino acids. We have been able to reconstitute a proteolytic pathway comprising the proteasome, Tricorn, and its interacting factors, F1, F2, and F3, which converts proteins efficiently into amino acids. Therefore, it is quite likely that Tricorn also acts in vivo downstream of the proteasome and, in cooperation with its interacting factors, completes protein catabolic pathways.     

Swyer James syndrome following Mycoplasma pneumoniae pneumonia: report of one case

Systemic vasculopathy in patients with neurofibromatosis type 1 is rare. We describe a case of unusual cerebral and renal involvement in a young, 29-year-old patient, who died of a cerebral ischemic attack one year after our observation.     

Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).