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.

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tissues within 1 h whereas in vitro only 19 per cent of the activity was transported by the mid-gut of silkworm (unpublished work).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.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Variation of Snowline and Mass Balance of Glaciers of Warwan and Bhut Basins of Western Himalaya Using Remote Sensing Technique

Glaciers are natural reservoirs of fresh water in frozen state and sensitive indicators of climate change. Among all the mountainous glaciated regions, glaciers of Himalayas form one of the largest concentrations of ice outside the Polar Regions. Almost all the major rivers of northern India originate from these glaciers and sustain perennial flow. Therefore, in view of the importance and role of the glaciers in sustaining the life on the Earth, monitoring the health of glaciers is necessary. Glacier's health is monitored in two ways (i) by mapping the change in extent of glaciers (ii) by finding variation in the annual mass balance. This paper has been discussed the later approach for monitoring the health of glaciers of Warwan and Bhut basins. Mass balance of glaciers of these two basins was determined based on the extraction of snow line at the end of ablation season. A series of satellite images of AWiFS sensor were analysed for extraction of snowline on the glaciers for the period of 2005, 2006 and 2007. The snow line at the end of ablation season is used to compute accumulation area ratio (AAR = Accumulation area/Glacier area) for each glacier of basins. An approach based on relationship of AAR to specific mass balance (computed in field) for glaciers of Basapa basin was employed in the present study. Mean of specific mass balance of individual glacier for the year 2005, 2006 and 2007 of Warwan basin was found to be -ve 0.19 m, -ve 0.27 m and -ve 0.2 m respectively. It is 0.05 m, -ve 0.11 m and -ve 0.19 m for Bhut basin. The analysis suggests a loss of 4.3 and 0.83 kmA(3) of glacier in the monitoring period of 3 years for Warwan and Bhut basins respectively. The overall results suggest that the glaciers of Warwan basin and Bhut basins have suffered more loss of ice than gain in the monitoring period of 3 years.

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4'-imidazolidine]-2',5'-dione (DFH) and 8-(3,4-dichlorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4'-imidazolidine]-2',5'-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8ES. Incorporation of tritiated thymidine ([H-3) thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy. (C) 2008 Elsevier Inc. All rights reserved.

The high temperature mechanical characteristics of superplastic 3 mol% yttria stabilized zirconia

A detailed study was undertaken to characterize the deformation behavior of a superplastic 3 mol% yttria-stabilized tetragonal zirconia (3YTZ) over a wide range of strain rates, temperatures and grain sizes. The experimental data were analyzed in terms of the following equation for high temperature deformation: Image Full-size image ∞ σn d−pexp(−Q/RT), where Image Full-size image is the strain rate, σ is the flow stress, d is the grain size, Q is the activation energy, R is the gas constant, T is the absolute temperature, and n and p are constants termed the stress exponent and the inverse grain size exponent, respectively. The experimental data over a wide range of stresses revealed a transition in stress exponent. Deformation in the low and high stress regions was associated with n not, vert, similar 3 and p not, vert, similar 1, and n not, vert, similar 2 and p not, vert, similar 3, respectively. The transition stress between the two regions decreased with increasing grain size. The activation energy was similar for both regions with a value of not, vert, similar 550 kJ mol−1. Microstructural measurements revealed that grains remained essentially equiaxed after the accumulation of large strains, and very limited concurrent grain growths occurred in most experiments. Assessment of possible rate controlling creep mechanisms and comparison with previous studied indicate that in the n not, vert, similar 2 region, deformation occurs by a grain boundary sliding process whose rate is independent of impurity content. Deformation in the n not, vert, similar 3 region is controlled by an interface reaction that is highly sensitive to impurity content. It is concluded that an increase in impurity content increases yttrium segregation to grain boundaries, which enhances the rate of the interface reaction, thereby decreasing the apparent transition stress between the n not, vert, similar 2 and n not, vert, similar 3 regions. This unified approach incorporating two sequential mechanisms can rationalize many of the apparently dissimilar results that have been reported previously for deformation of 3YTZ.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli

We have designed a novel coupled transcriptional construct wherein Escherichia coil uracil DNA glycosylase (UDC:) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (P-trc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide cor responding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.

Xylan-Degrading Enzymes from the Thermophilic Fungus Humicola Zanuginosa (Griffon and Maublanc) Bunce: Action Pattern of Xylanase and beta-Glucosidase on Xylans, Xylooligomers and Arabinoxylooligomers

The mode of action of xylanase and beta-glucosidase purified from the culture filtrate of Humicola lanuginosa (Griffon and Maublanc) Bunce on the xylan extracted from sugarcane bagasse and on two commercially available larchwood and oat spelt xylans, on xylooligomers and on arabinoxylooligomers was studied. While larchwood and oat spelt xylans were hydrolyzed to the same extent in 24 h, sugarcane bagasse xylan was hydrolyzed to a lesser extent in the same period. It was found that the rate of hydrolysis of xylooligomers by xylanase increased with increase in chain length, while beta-glucosidase acted rather slowly on all the oligomers tested. Xylanase exhibited predominant ''endo'' action on xylooligomers attacking the xylan chain at random while beta-glucosidase had ''exo'' action, releasing one xylose residue at a time. On arabinoxylooligomers, however, xylanase exhibited ''exo'' action. Thus, it appears that the presence of the arabinose substituent has, in some way, rendered the terminal xylose-xylose linkage more susceptible to xylanase action. It was also observed that even after extensive hydrolysis with both the enzymes, substantial amounts of the parent arabinoxylooligomer remained unhydrolyzed together with the accumulation of arabinoxylobiose. It can therefore be concluded that the presence of the arabinose substituent in the xylan chain results in linkages that offer resistance to both xylanase and beta-glucosidase action.

Phyllosphere of Cotton as a Habitat for Diazotrophic Microorganisms

Positive nitrogenase activities ranging from 0.18 to 0.78 nmol of C2H4 cm−2 h−1 were detected on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants. Beijerinckia sp. was observed to be the predominant nitrogen-fixing microorganism in the phyllosphere of these varieties. A higher level of phyllosphere nitrogen-fixing activity was recorded in the variety Varalaxmi despite a low C/N ratio in the leaf leachates. Leaf surfaces of the above variety possessed the largest number of hairy outgrowths (trichomes) which entrapped a majority of microbes. Immersion of plant roots in nutrient medium containing 32Pi led to the accumulation of label in the trichome-borne microorganisms, thereby indicating a possible transfer of nutrients from leaf to microbes via trichomes. Extrapolation of acetylene reduction values suggested that 1.6 to 3.2 kg of N ha−1 might be contributed by diazotrophs in the phyllosphere of the variety Varalaxmi during the entire growth period.

The effect of bottom reflectivity on the performance of a solar pond

The reflectivity of the bottom of a solar pond increases on account of the accumulation of dirt or the presence of undissolved salt. The effect of the reflection of the solar radiation at the bottom of the pond on the seasonal performance of the pond has been studied using a three zone model. The spectral reflectivity of dirt and common salt were measured in the laboratory and used in the analysis. The results obtained from the analysis show that the presence of dirt at the bottom of the pond does not affect the performance of the pond substantially. On the other hand, the presence of undissolved salt at the bottom of the pond results in substantial deterioration of the pond performance.

Induction of riboflavin-carrier protein in the immature male rat by estrogen: kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.

Growth inhibition of human promonocytic leukaemic U937 cells by interferon gamma is irreversible and not cell cycle phase-specific.

Growth of human promonocytic leukaemic U937 cells was found arrested within 24 h upon exposure to interferon gamma (IFN-gamma). Removal of the interferon did not result in the resumption of growth, as is evident from the absence of doubling of viable cell count and(3)H-thymidine incorporation. 5-Bromo-2'-deoxyuridine-based flow cytometric analysis of the growth-arrested cells, 24 h subsequent to the removal of IFN-gamma, showed absence of DNA synthesis, confirming the irreversible nature of the growth inhibition. Propidium iodide-based flow cytometric analysis of the growth-arrested cells showed a distribution which is typical of a growth inhibition without resulting in the accumulation of cells in any specific phase of the cell cycle. These results indicated that IFN-gamma arrested growth of U937 cells in an irreversible and cell cycle phase-independent manner. These observations were in contrast to our earlier report on the reversible and cell cycle phase-specific growth inhibition of human amniotic (fetal epithelial) WISH cells by the interferon. Copyright 1999 Academic Press.

The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn(2+) and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg(2+) alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg(2+), PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp(122), Asp(222), and Glu(220)) together with immunoprecipitation assays provided compelling evidence to link both the Mg(2+)- and Mn(2+) and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.