Characterization of a Negative cis-Acting DNA Element Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

The region -160 to -127 nt of the upstream of CYP-2B1/B2 gene has been found to function as a negative cis-acting element on the basis of DNase-I footprint and gel mobility shift assays as well as cell-free transcriptional assays using Bal-31 mutants. A reciprocal relationship in the interaction of the negative and the recently characterized positive elements with their respective protein factors has been found under repressed and induced conditions of the gene. The negative element also harbors the core glucocorticoid responsive sequence, TGTCCT. It is concluded that the negative element mediates the repressed state of the gene under the uninduced condition and also mediates the repressive effect of dexamethasone, when given along with the inducer phenobarbitone in rats. Dexamethasone is able to antagonize the effects of phenobarbitone at as low a concentration as 100 mu g/kg body wt in these animals. (C) 1995 Academic Press,Inc.

Interaction of sheep liver apo-serine hydroxymethyltransferase with pyridoxal-5'-phosphate: A physicochemical, kinetic, and thermodynamic study

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer of M(r) 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofactor, Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 +/- 2 and 58 +/- 2 degrees C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme, However, sedimentation analysis of the apoenzyme revealed the presence of two peaks of S-20,S-w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (M(r) 200,000 +/- 15,000) while the second peak had a M(r) of 130,000 +/- 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis. (C) 1996 Academic Press, Inc.

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.

Antioxidant and antihepatotoxic effect of Spirulina laxissima against carbon tetrachloride induced hepatotoxicity in rats

The vast biodiversity of nature provides bioactive compounds that may be useful in the fight against chronic diseases. This study was designed to investigate the protective effects of the ethanol extract of Spirulina laxissima West (Pseudanabaenaceae) (EESL) against carbon tetrachloride (CCl4) induced hepatotoxicities in rats. Male albino rats of Sprague-Dawley strain were treated orally with the ethanol extract of S. laxissima (50, 100 mg kg(-1) body wt.) 1 h before each CCl4 administration. The ethanol extract of S. laxissima showed the maximum antioxidant property in vitro. There were statistically significant losses in the activities of antioxidant enzymes and an increase in TBARS and liver function marker enzymes in the serum of the CCl4-treated group compared with the control group. However, all the tested groups were able to counteract these effects. The antioxidant activity of the extracts might be attributable to its proton-donating ability, as evidenced by DPPH. In the present study, the decline in the level of antioxidant observed in CCl4-treated rats is a clear manifestation of excessive formation of radicals and activation of the lipid peroxidation system resulting in tissue damage. The significant increases in the concentration of antioxidant enzymes in tissues of animals treated with CCl4 + EESL indicate the antioxidant effect of EESL. This study suggests that EESL can protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its antioxidant and radical-scavenging effects.

Graphics Processing Units for the Real-time Linear Elastostatic Simulation of Liver

Biomedical engineering solutions like surgical simulators need High Performance Computing (HPC) to achieve real-time performance. Graphics Processing Units (GPUs) offer HPC capabilities at low cost and low power consumption. In this work, it is demonstrated that a liver which is discretized by about 2500 finite element nodes, can be graphically simulated in realtime, by making use of a GPU. Present work takes into consideration the time needed for the data transfer from CPU to GPU and back from GPU to CPU. Although behaviour of liver is very complicated, present computer simulation assumes linear elastostatics. One needs to use the commercial software ANSYS to obtain the global stiffness matrix of the liver. Results show that GPUs are useful for the real-time graphical simulation of liver, which in turn is needed in simulators that are used for training surgeons in laparoscopic surgery. Although the computer simulation should involve rendering also, neither rendering, nor the time needed for rendering and displaying the liver on a screen, is considered in the present work. The present work is just a demonstration of a concept; the concept is not really implemented and validated. Future work is to develop software which can accomplish real-time and very realistic graphical simulation of liver, with rendered image of liver on the screen changing in real-time according to the position of the surgical tool tip approximated as the mouse cursor in 3D.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

The role of lysine-256 in the structure and function of sheep liver recombinant serine hydroxymethyltransferase

The active site lysine residue, K256, involved in Schiffs base linkage with pyridoxal-5'-phosphate (PEP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H(4)folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H(4)folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.