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.

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.

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.

In vitro plant regeneration from different seedling explants of blackgram [Vigna mungo (L.) Hepper] via organogenesis

Callus induction and morphogenesis from different blackgram explants were tested on MS basal medium supplemented with B5 vitamins, IAA, NAA, IBA, KIN and BAP individually and in combinations. The explants were hypocotyl, epicotyl, axillary bud, cotyledonary node and immature leaf. The optimal levels of the frequency of callus induction was 22.8 mu M of IAA or 16.1 mu M NAA and in combination with 2.2 mu M of BAP. Among the seedling explants, hypocotyl was found to be more efficient in producing callus. Shoots mere induced from callus cultures of hypocotyls, epicotyls, axillary bud, cotyledonary node and immature leaf with varying frequencies in the medium containing KIN (2.3-9.3 mu M) or BAP (2.2-8.8 mu M) and in combination with IAA (2.8 mu M) or NAA (2.6 mu M). Multiple shoots were obtained using cotyledonary node segments. The regenerated shoots rooted best on MS basal medium containing 9.8 mu M IBA. Seventy three per cent of the shoots produced roots, and 80-85% of the plantlets survived under greenhouse condition.

In vitro regeneration from immature cotyledon explant and protein profile changes during organogenesis in groundnut (Arachis hypogaea L.)

Complete plants were regenerated from in vitro cultured immature cotyledon segments of groundnut (Arachis hypogaea L. cv. TMV-7) by organogenesis. Callus cultures were best Initiated from immature cotyledon segments on MS (Murashige and Skoog) salts containing B5 vitamins supplemented with indole-3-acetic acid (IAA) and alpha -naphthalene acetic acid (NAA; 4.0 mg L-1) and kinetin (KIN; 0.5 L-1). Calluses were transferred to a medium containing KIN (2.0 mg L-1) and IAA and NAA (0.5 mg L-1) for shoot Initiation. The regenerated shoots were transferred to a medium containing Indole-3-butyric acid (IBA; 2.0 mg L-1) and KIN (0.2 mg L-1) for developing roots. In vitro produced plantlets developed sucessfully, matured, and set seed. The protein profiles [sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)] of callus, callus with shoot, and callus with shoot and root showed differences.

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.

A comparative analysis of regeneration in natural forests and joint forest management plantations in Uttara Kannada district, Western Ghats

Five villages undertaking joint forest management (JFM) were chosen in Uttara Kannada district, Karnataka for assessing regeneration in plantations and nearby natural forests of the village. Species number, stem density, diversity index, similarity in species composition in less disturbed and disturbed forests and plantations in the village were compared. Stem density was low in all the disturbed forests; however, the species number was low in disturbed forests of three villages and high in two villages. Plantations showed lower diversity values compared to the adjacent natural forests. Regeneration in all less disturbed forests was better compared to the disturbed counterparts. Villages were ranked based on number of landless families, per, capita forest available and number of cut stems. Assessment of village forests using ranks indicates that parameters such as per capita availability, cut stems in the forests may determine the success of JFM.