Biotransformation of cholesterol to diosgenin by freely suspended and immobilised cells of Dioscorea bulbifera L.

The cell suspension cultures, established from the friable callus which was risen from the nodal segments of Dioscorea bulbifera L. in Murashige-Skoog (MS) medium supplemented with indole-3-butryic acid (20 mg L- 1), was examined for cell growth in MS medium fed with cholesterol (50 mg L- 1 and 100 mg L- 1) after 8, 10, 12, 14, 16, and 18 days of culture. The growth index of the cell suspension culture on the 8th day was 1.2 and gradually inclined to 1.9 on the 16th day and remained the same at the 18th day. There is no marked difference in the cell growth of cholesterol-treated and control cell suspension culture. The maximum accumulation of diosgenin was noticed on the 14th day in control and cholesterol-treated cell suspension culture and immobilised cell cultures. The highest concentration of diosgenin, 2.94% and 2.14% dry weight, was obtained in immobilised cell culture and cell suspension culture treated with 100 mg L- 1 cholesterol, respectively.

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N-7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

Expression of hyoscyamine 6 beta-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L.

Hyoscyamine 60-hydroxylase (H6H: EC 1.14.11.11), a key enzyme at the terminal step of tropane alkaloid biosynthesis, converts hyoscyamine to scopolamine. The accumulation of scopolamine in different organs, in particular the aerial parts for storage, is subject to the expression of hyoscyamine 6-phydroxylase as well as its transport from the site of synthesis. To understand the molecular basis of this regulation, we have analyzed, in parallel, the relative levels of hyoscyamine and scopolamine, and the accumulation of H6H (both protein and transcript) in leaves, stems and roots of D. metel. The root, stem and leaf tissues all contain about 0.51-0.65 mg g(-1) dry weight of scopolamine. Hyoscyamine content was extremely low in leaf and stem tissues and was about 0.28 mg g(-1) dry weight in the root tissue. H6H protein and its transcript were found only in roots but not in the aerial parts viz. stems and leaves. The immunolocalization studies performed on leaf, stem, root as well as hairy root tissues showed that H6H was present only in the pericycle cells of young lateral and hairy roots. These studies suggest that the conversion of hyoscyamine to scopolamine takes place in the root pericycle cells, and the alkaloid biosynthesized in the roots gets translocated to the aerial parts in D. metel. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the conversion of l-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Cα proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5'-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5'-phosphate. However, there was an alteration in the λmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Cα and Cβ of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Cα proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Cα proton abstraction by SHMT is proposed.

Conformational restrictions of peptides via backbone modification: Solution and crystal-state analysis of Boc-L-Pro-DZPhe-Gly-NH2

An N-alpha-protected model tripeptide amide containing, in the central position, an alpha,beta-dehydrophenylalanine (Z-configurational isomer), Boc-L-Pro-DELTA-Z-Phe-Gly-NH2 (Boc, tert-butyloxycarbonyl), has been synthesized by solution methods and fully characterized. IR absorption and H-1 NMR studies provided evidence for the occurrence of a significant population of a conformer containing two consecutive, intramolecularly H-bonded (type II-III') beta-bends in solution. However, an X-ray diffraction analysis clearly indicates that only the type-II beta-bend structure survives in the crystal state.

L-p Wiener-Tauberian theorems for M(2)

We prove two sided and one sided analogues of the Wiener-Tauberian theorem for the Euclidean motion group, M(2).

Accumulation of glycine in the fat body of the silkworm, bombyx mori L. [31]

INVESTIGATIONS of intestinal transport of amino-acids in the locust1,2 and silkworm3,4 have shown no evidence for active accumulation in a transport from the insect gut of amino-acids. When glycine-2-14C was administered in vivo to fifth instar larvae of the silkworm, 96 per cent of the radioactivity was incorporated into various tissues within 1 h whereas in vitro only 19 per cent of the activity was transported by the mid-gut of silkworm (unpublished work). These results suggested that continued absorption of glycine by the intestine could be aided by a facilitated diffusion mechanism in which amino-acids are rapidly removed from the site of absorption either by accumulation into other tissues or by degradation. Although the insect fat body has been assigned both accumulatory and dissimilatory roles5, the mechanism of accumulation of amino-acids has not been investigated. Our present experiments show that the silkworm fat body possesses an efficient mechanism for accumulating glycine and that both the accumulation and the release of glycine are metabolically controlled.

Studies on the biosynthesis of β-n-oxalyl-l-α,β-diaminopropionic acid, the Lathyrus sativus neurotoxin

The biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP), HOOC· CO·NH·CH2·CH(NH2·COOH is of interest, since this neurotoxin has been isolated from the seeds of Lathyrus sativus, the consumption of which causes the disease neurolathyrism in humans. The concentration of this non-protein amino acid in the seeds increases on germination. When the seeds are germinated in the presence of [14C2]- oxalic acid, the isolated ODAP is labelled exclusively in the oxalyl moiety. An oxalyl- CoA synthetase requiring the obligatory presence of ATP, CoA and Mg2+ can be demonstrated in crude extracts of the seedlings. When l-α,β-diaminopropionic acid is incubated with the enzyme in the presence of the components for oxalyl activation, net formation of ODAP can be shown. The enzymic reaction is specific to the β-amino group of l-α,β-diaminopropionic acidm and the higher homologues like α,γ-diaminobutyric acid, ornithine and lysine are inactive in this system. ODAP is not formed with α,β-diaminopropionic acid when the enzyme extract is prepared from Pisum sativum although oxalyl-CoA formation can be demonstrated.

Trace element nutrition of the silkwormBombyx mori L. I. Effect of trace elements

Trace elements zinc, copper, manganese, molybdenum and cobalt have been shown to have varying effects on growth and trace element composition of the silkworm. Results indicate the important role of manganese in the normal metabolism of the insect. Cobalt has been shown to exert a very favourable effect on growth and silk yield.

Intestinal transport in the silkworm, Bombyx mori L., of amino acids from mixtures

The rate of absorption of amino acids from mixtures has been studied in the silkworm midgut by using an in vitro perfusion technique. The rates differ for individual amino acids. A characteristic absorption pattern is observed which is independent of the amino acid composition of the mixture used. The metabolic inhibitors dinitrophenol and cyanide have no effect on the amino acid transport from mixtures. Based on these results an energy-independent, carrier-mediated transport is postulated.

Trace element nutrition of the silkwormBombyx mori L. II. Relationship between cobalt, vitamin B12, benzimidazole and purines

Benzimidazole is toxic to silkworms due to its antagonistic action to purines. Cobalt and copper possess the capacity to reverse its toxicity because of their ability to form complexes with benzimidazole. For reasons unknown cobalt exerts a favourable effect in its free form rather than in the vitamin form on the insect growth.

Biochemical Characterization Of Β-N-Oxalyl-L-Α,Β-Diaminopropionic Acid, The Lathyrus Sativus Neurotoxin As An Excitant Amino Acid

Abstract—β-N-Oxalyl-l-α,β-diaminopropionic acid (ODAP), the toxin isolated from the seeds of Luthyrus sativus produces head retraction, tremors and convulsions when injected into a variety of experimental animals. In 12-day-old rats, it has been found that the convulsive behaviour is accompanied by profound biochemical changes in the brain. The brain homogenates prepared from ODAP injected animals show a higher rate of respiration. There is a decrease in the brain glucose, glycogen, ATP, phosphocreatine and acetylcholine levels of the convulsing animals. The inorganic phosphate, lactic acid and acetylcholineesterase levels increase. These results establish that ODAP is a typical convulsant.