Decreased expression of the pancreatic secretory trypsin inhibitor II gene during aging of the rat liver

The genetic constitution and differential gene expression of an organism play important roles in controlling the species-specific rate of aging and the maximum life span potential. We utilized a differential-display polymerase chain reaction technique to identify the age-dependent expression of genes in the rat liver. We demonstrate in this report, for the first time, that expression of the pancreatic secretory trypsin inhibitor II (PSTI-II) gene declines drastically during aging. We confirmed this decrease by Northern blot analysis. Low PSTI-II levels in aged animals might result in a lack of protection from prematurely activated trypsin-like proteases, which would thus enhance inflammation.Supported by funds from the Department of Biotechnology and by Research Fellowships (to H.P. and G.Z.) from the Council of Scientific and Industrial Research (Government of India).     

Differential pulse anodic voltammetric determination of pantoprazole in pharmaceutical dosage forms and human plasma using glassy carbon electrode

Pantoprazole is used as an anti-ulcer drug through inhibition of H(+), K(+)-adenosine 5(')-triphosphatase in gastric parietal cells. It reduces the gastric acid secretion regardless of the nature of stimulation. The use of differential pulse voltammetry for the determination of pantoprazole in pharmaceutical dosage forms and human plasma using a glassy carbon electrode has been examined. The best voltammetric response was reached for a glassy carbon electrode in Britton-Robinson buffer solution of pH 5.0 submitted to a scan rate of 20.0 mVs(-1) and a pulse amplitude of 50.0 mV. This electroanalytical procedure was able to determine pantoprazole in the concentration range 6.0 x 10(-6)-8.0 x 10(-4)M. Precision and accuracy of the developed method was checked with recovery studies. The limit of detection and limit of quantitation were found to be 4.0 x 10(-7) and 9.0 x 10(-7)M, respectively. Rapidity, precision, and good selectivity were also found for the determination of pantoprazole in pharmaceutical dosage forms and human plasma. For comparative purposes high-performance liquid chromatography with a diode array and UV/VIS detection at 290.0 nm determination also was developed.     

Physicochemical characterization of silicon-containing glycolipids by DSC,FT-Raman spectroscopy and X-ray diffraction

Derivatives of dimethylalkylchlorosilanes are novel substances which may be used in formulations for drug targeting. In order to design their properties it is essential to perform physicochemical characterization. For this purpose, a combination of differential scanning calorimetry (DSC), FT-Raman spectroscopy and X-ray diffraction is well suited. For the starting material dimethyloctadecylchlorosilane (DMOC), the assignment of Raman bands is discussed. The influence of sugar-containing head groups on the structures of the hydrocarbon chains of 1-O-(dimethyldodecylsilyl)-[2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside] and 1-O-(dimethyloctadecylsilyl)-[2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside] was investigated using the band position of the symmetric methylene mode. The temperature dependence of conformationally sensitive bands in the CH(2)-stretching region (2800-2900 cm(-1)), C-C-stretching region (1000-1150 cm(-1)) and CH(3)-rocking region (830-900 cm(-1)) was studied to characterize the state of order of the alkyl chains. Using X-ray diffraction, the repeating distances of layered structures was determined. The phase transitions occurring were found to be completely reversible. The subcell of DMOC shows an orthorhombic perpendicular packing structure in the crystalline state.     

Interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers-the effect of counterions

The interaction of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) with two types of phospholipid vesicles (MLV and SUV) was investigated using DSC and 1H NMR. It was suggested that the benzyl group like the micellisation process (J. Colloid Interface Sci. 218 (1999) 529) changes its position when interacting with phosphatidylcholine bilayers and incorporates into the bilayer. In order to enhance counterion-water interactions, the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). It follows from the obtained results that for both types of liposomes and both manners in which the surfactant was added, the interaction of DBeAX with liposomes and consequent changes in the phospholipid bilayer organisation depend on the kind of counterion. Results are discussed in terms of counterion ability to modify water structure.     

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best fit" membrane permeability parameters at 5 and 10 degrees C/min for canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg)[cpa]=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp)[cpa]=46.4-56.0kJ/mol (11.1-13.4kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.     

Ectopic expression of MHC class II genes (RT1.B(I) beta/alpha) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16 alpha-carbonitrile and dexamethasone

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.     

If space is provided,bulky modification on the rim of azurin's beta-barrel results in folded protein

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.