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.

Dielectric properties of Poly(vinylidene fluoride)/CaCu3Ti4O12 composites

The possibility of obtaining relatively high dielectric constant polymer-ceramic composite by incorporating the giant dielectric constant material, CaCu3Ti4O12 (CCTO) in a Poly(vinylidene fluoride) (PVDF) polymer matrix by melt mixing and hot pressing process was demonstrated. The structure, morphology and dielectric properties of the composites were characterized using X-ray diffraction, Thermal analysis. scanning electron microscope, and impedance analyzer. The effective dielectric constant a(epsilon(eff)) of the composite increased with increase in the volume fraction of CCTO at all the frequencies(100 Hz-1 MHz) under study. The dielectric loss did not show any variation up to 40% loading of CCTO, but showed an increasing trend beyond 40%. The room temperature dielectric constant as high as 95 at 100 Hz has been realized for the composite with 55 vol.% of CCTO, which has increased to about 190 at 150 degrees C. Theoretical models like Maxwell's, Clausius-Mossotti, Effective medium theory, logarithmic law and Yamada were employed to rationalize the dielectric behaviour of the composite and discussed. (C) 2010 Published by Elsevier Ltd.

Tuning the beta-turn segment in designed peptide beta-hairpins: Construction of a stable type I ' beta-turn nucleus and hairpin-helix transition promoting segments

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu- Val-Val-OMe ((D)Xxx = (D)Ala, 3a; (D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD3OH and DMSO-d(6). However, in CDCl3 evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl3 and a significant population of both helices and hairpins in CD3OH and DMSO-d(6). The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central beta-turn permits generation of models for robust beta-hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions. (c) 2006 Wiley Periodicals, Inc.

Electronic structure and bonding of beta-rhombohedral boron using cluster fragment approach

Theoretical studies using density functional theory are carried out to understand the electronic structure and bonding and electronic properties of elemental beta-rhombohedral boron. The calculated band structure of ideal beta-rhombohedral boron (B-105) shows valence electron deficiency and depicts metallic behavior. This is in contrast to the experimental result that it is a semiconductor. To understand this ambiguity we discuss the electronic structure and bonding of this allotrope with cluster fragment approach using our recently proposed mno rule. This helps us to comprehend in greater detail the structure of B-105 and materials which are closely related to beta-rhombohedral boron. The molecular structures B12H12-2, B28H21+1, BeB27H21, LiB27H21-1, CB27H21+2, B57H36+3, Be3B54H36, and Li2CB54H36, and corresponding solids Li8Be3B102 and Li10CB102 are arrived at using these ideas and studied using first principles density functional theory calculations.

Hybrid peptide hairpins containing alpha- and omega-amino acids: Conformational analysis of decapeptides with unsubstituted beta-, gamma-, and delta-residues at positions 3 and 8

The effects of inserting unsubstituted omega-amino acids into the strand segments of model beta-hairpin peptides was investigated by using four synthetic decapeptides, Boc-Lcu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val- OMe: pepticle 1 (Xxx=Gly), pepticle 2 (Xxx=beta Gly=beta hGly=homoglycine, beta-glycine), pepticle 3 (Xxx=gamma Abu=gamma-aminobutyric acid), pepticle 4 (Xxx= delta Ava=delta-aminovaleric acid). H-1 NMR studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for P-hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a beta-hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the gamma Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II' beta-turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 rim, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

Studies on follicular atresia: role of gonadotropins and gonadal steroids in regulating cathepsin-D activity of preovulatory follicles in the rat

Preovulatory follicular atresia was studied using pregnant mare serum gonadotropin (PMSG)-primed rats (15 IU/rat) which were deprived of hormonal support either by allowing the metabolic clearance of the PMSG or by injecting a specific PMSG antiserum (PMSG a/s). Atresia was monitored by an increase in lysosomal cathepsin-D activity and a decrease in the receptor activity of the granulosa cells (GC) isolated from the preovulatory follicles. It was shown that the increase in lysosomal activity and the decrease in receptor activity seen at 96 h after PMSG (or PMSG plus PMSG a/s) could be arrested both by follicle stimulating hormone (FSH) and luteinizing hormone (LH). Injection of cyanoketone or clomiphene citrate together with FSH/LH prevented this 'rescue' suggesting a role for estrogens in the regulation of atresia. Although the administration of estradiol-17 beta (20 micrograms/rat) together with PMSG a/s could show a 'rescue effect' in terms of reduction in cathepsin-D activity the gonadotropin receptor activities of these granulosa cells were not restored. The injection of dihydrotestosterone (DHT) to 48 h PMSG-primed rats induced atresia as noted by an increase in cathepsin-D activity. However, the exogenous administration of FSH along with DHT prevented this atretic effect suggesting that DHT is not having a direct effect on atresia. Determination of androgen: estrogen content of the granulosa cells and an analysis of the individual profile of androgen and estrogen revealed that the increase in cathepsin-D activity could be correlated only with the decrease in GC estrogen content. This along with the observation that GC showed a loss of estrogen synthesis well before the increase in cathepsin-D activity strongly points out that the lack of estrogen rather than an increase in androgen is the principle factor responsible for the atresia of preovulatory follicles in the rat.

Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.

The structure of 3[beta],20[alpha]-bis(dimethylamino)pregn-5-en-18-ol

Abstract. C25H44N20 , M r= 388.6, orthorhombic, P21212 I, a = 6.185 (2), b = 18.123 (2), c = 20.852 (2) A, U= 2337.2 A 3, Z = 4, D x = 1.104 Mg m -a, 2(Cu Ka) = 1.5418 A,/~ = 0.47 mm -~, F(000) = 864, T= 293 K. Final R - 0.038 for 1791 reflections with I >_ 3a(I). Rings A and C are in chair conformation. Ring B is in an 8fl,9a-half-chair conformation. Ring D adopts a conformation in between 13fl,14a-half-chair and 13t-envelope. There is a quasitrans fusion of rings A and B, whilst ring systems B/C and C/D are trans fused about the bonds C(8)-C(9)and C(13)-C(14).

X-ray studies on crystalline complexes involving amino acids and peptides. XV. Crystal structures of L-lysine D-glutamate and L-lysine D-aspartate monohydrate and the effect of chirality on molecular aggregation

L-Lysine D-glutamate crystallizes in the monoclinic space group P2(1) with a = 4.902, b = 30.719, c = 9.679 A, beta = 90 degrees and Z = 4. The crystals of L-lysine D-aspartate monohydrate belong to the orthorhombic space group P2(1)2(1)2(1) with a = 5.458, b = 7.152, c = 36.022 A and Z = 4. The structures were solved by the direct methods and refined to R values of 0.125 and 0.040 respectively for 1412 and 1503 observed reflections. The glutamate complex is highly pseudosymmetric. The lysine molecules in it assume a conformation with the side chain staggered between the alpha-amino and the alpha-carboxylate groups. The interactions of the side chain amino groups of lysine in the two complexes are such that they form infinite sequences containing alternating amino and carboxylate groups. The molecular aggregation in the glutamate complex is very similar to that observed in L-arginine D-aspartate and L-arginine D-glutamate trihydrate, with the formation of double layers consisting of both types of molecules. In contrast to the situation in the other three LD complexes, the unlike molecules in L-lysine D-aspartate monohydrate aggregate into alternating layers as in the case of most LL complexes. The arrangement of molecules in the lysine layer is nearly the same as in L-lysine L-aspartate, with head-to-tail sequences as the central feature. The arrangement of aspartate ions in the layers containing them is, however, somewhat unusual. Thus the comparison between the LL and the LD complexes analyzed so far indicates that the reversal of chirality of one of the components in a complex leads to profound changes in molecular aggregation, but these changes could be of more than one type.

Negative differential resistance in doped poly(3-methylthiophene) devices

The current density-voltage (J-V) characteristics of poly(3-methylthiophene) devices show a negative differential resistance (NDR) at room temperature with a large peak to valley current ratio (similar to 507). This NDR can be tuned by two orders of magnitude by controlling the carrier density due to the variation of the space-charge region in the device. The temperature and scan rate dependent J-V measurements infer that the NDR is mainly driven by the trapping and de-trapping of carriers. The photo-generation of carriers is observed to reduce the NDR effect.

The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase

Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.

Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L.

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Conformational studies on beta-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C(gamma)-endo and C(gamma)-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.

Effect of Zirconium on the Processing MAPS For Hot-Working of Alpha-Beta Brass

The effect of zirconium on the hot working characteristics of alpha and alpha-beta brass was studied in the temperature range of 500 to 850-degrees-C and the strain rate range of 0.001 to 100 s-1. On the basis of the flow stress data, processing maps showing the variation of the efficiency of power dissipation (given by [2m/(m+1)] where m is the strain rate sensitivity) with temperature and strain rate were obtained. The addition of zirconium to alpha brass decreased the maximum efficiency of power dissipation from 53 to 39%, increased the strain rate for dynamic recrystallization (DRX) from 0.001 to 0.1 s-1 and improved the hot workability. Alpha-beta brasses with and without zirconium exhibit a domain in the temperature range from 550 to 750-degrees-C and at strain rates lower than 1 s-1 with a maximum efficiency of power dissipation of nearly 50 % occurring in the temperature range of 700 to 750-degrees-C and a strain rate of 0.001 s-1. In the domain, the alpha phase undergoes DRX and controls the hot deformation of the alloy whereas the beta phase deforms superplastically. The addition of zirconium to alpha-beta brass has not affected the processing maps as it gets partitioned to the beta phase and does not alter the constitutive behavior of the alpha phase