Role of lipid peroxidation in impairment of mitochondrial function at complex I by methyl isocyanate treatment of rats in vivo

The subcutaneous administration of methyl isocyanate (MIC) in 1.0 LD50 dose in rats caused a significant effect on hepatic mitochondrial function only at complex I region of the respiratory chain. MIC administration at 1.0 LD50 dose also resulted in significant increases in malondialdehyde and ferrous ion concentration in liver mitochondria. It is suggested that the augmented lipid peroxidation in hepatic mitochondria, catalyzed by iron, possibly mobilized from intracellular stores leads to the inhibition of enzymes of mitochondrial respiration at complex I region, in vivo, in rats receiving a lethal dose of MIC subcutaneously.

5-Fluorouracil induces structural changes in rat liver tRNA

5-fluorouracil (FUra) has been shown to modulate the aminoacylation function of rat liver tRNA. The present study was aimed at studying the structure-function relationship of FUra-substituted tRNA. Male Wistar rats (2-3 month old) were given a single i.p. injection of FUra at 50, 250, or 500 mg/kg body wt. and FUra-substituted total liver tRNA, i.e. tRNA(FUra50, 250, and 500, respectively, were isolated 3 h later. Normal tRNA (tRNA(N)) was isolated from saline-treated control rats. Thermal denaturation studies showed higher melting temperatures for tRNA(FUra) compared to tRNA(N). Heat denaturation followed by renaturation of total tRNA did not affect the activity of tRNA(N) and tRNA(FUra50), where as tRNA(FUra250 and 500) lost 35% and 72% of activity, respectively, compared to the corresponding group of non-denatured tRNA. Antibodies specific to rat liver tRNA recognized normal and FUra-substituted tRNA in the order of tRNA(N) > tRNA(FUra50) > or = tRNA(FUra250) > tRNA(FUra500) in an avidin-biotin micro-enzyme linked immunosorbant assay. tRNA(N) or tRNA(FUra50) preincubated with tRNA antiserum showed 74% and 59% of aminoacylation activity, respectively, compared to that of corresponding tRNA preincubated with normal rabbit IgG. However, activities of similarly treated tRNA(FUra250 and 500) were not affected. The observations of possible changes in the secondary structure of rat liver tRNA upon incorporation of FUra are discussed.

Functions Of Cytochrome-C In Regulation Of Electron-Transfer And Protein Folding

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

Effects of menthofuran, a monoterpene furan on rat liver microsomal enzymes, in vivo

Oral administration (250 mg/kg) of menthofuran, a monoterpene furan, to rats once daily for 3 days caused hepatotoxicity as judged by a significant increase in serum glutamate pyruvate transaminase (SGPT) and decreases in glucose-6-phosphatase and aminopyrine N-demethylase activities. Administration of menthofuran also resulted in a decrease in the levels of liver microsomal cytochrome P-450, whereas cytochrome b(5) and NAD(P)H-cytochrome c reductase activities were not affected. These effects of menthofuran were both dose- and time-dependent. Pretreatment of rats with phenobarbital (PB) prior to menthofuran treatment potentiated hepatotoxicity suggesting that a PB-induced cytochrome P-450 catalyzed the formation of reactive metabolite(s) responsible for the hepatotoxicity.

Rapid purification of tRNA(Lys) from rat liver

Fast protein liquid chromatography (FPLC) system using Mono Q (HR 5/5) anion-exchange column chromatography followed by highly cross-linked urea-polyacrylamide gel electrophoresis (urea-PAGE) was used for the purification of lysine-specific tRNA (tRNA(Lys)) from rat liver. Crude tRNA from rat liver was fractionated with a linear gradient of NaCl (0.3-0.8 M) in triethanolamine-HCl buffer, pH 4.5, and the activity of tRNA(Lys) was found to elute between 0.51 and 0.57 M NaCl. Using this concentration range of NaCl, tRNA(Lys) was refractionated on the same column with a shallow gradient, where a single peak of tRNA(Lys) activity was obtained. tRNA(Lys)-rich fractions recovered from the second run were electrophoretically separated on 16% polyacrylamide-7 M urea gel into one major band and three minor bands. The major band showed a specific activity of 997 pmols/A260 U for tRNALys with a 43-fold purification and approximately 17% recovery. The minor bands displayed negligible or no activity for lysine. tRNA(Lys) obtained by this method was found to be homogeneous by competitive aminoacylation. The advantages of FPLC followed by urea-PAGE in the purification of an amino acid-specific tRNA over conventional column chromatography are discussed.

The primary structure of sheep liver cytosolic serine hydroxymethyltransferase and an analysis of the evolutionary relationships among serine hydroxymethyltransferases

The complete amino-acid sequence of sheep liver cytosolic serine hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver serine hydroxymethyltransferase consisted of 483 amino-acid residues. A comparison of this sequence with 8 other serine hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the serine hydroxymethyltransferases belong to the alpha/beta category of proteins. The predicted secondary structure of sheep liver serine hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5'-phosphate containing enzyme, suggesting that sheep liver serine hydroxymethyltransferase might have a similar pyridoxal 5'-phosphate binding domain. In addition, a conserved glycine rich region, G L Q G G P, was identified in all the serine hydroxymethyltransferases and could be important in pyridoxal 5'-phosphate binding. A comparison of the cytosolic serine hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that serine hydroxymethyltransferase was a highly conserved protein. It was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5'-phosphate against modification with [C-14]iodoacetic acid, while C247 and C261 were buried in the native serine hydroxymethyltransferase. However, the cysteines are not conserved among the various serine hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by serine hydroxymethyltransferase remains to be elucidated.

CDNA Cloning, Overexpression in Escherichia coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The K-m values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.

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.

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).