Nitrogen Economy of the Main Shoot of Field-Grown Barley (Hordeum vulgare L.): I. DRY WEIGHT, DURATION, AND NITRATE CONTENT IN DIFFERENT ORGANS

The main shoot of field-grown Jyoti barley (Hordeum vulgareL.) grown at 40 kg ha^–1 was separated into different organsat various stages of growth and development. Changes in freshand dry weights and duration for which the parts remained metabolicallyactive (green) were recorded. Weight duration (gram x day),a factor contributing to total NO^–₃ reduction of eachorgan, was calculated. The dry matter percentage increased inthe successively formed laminae and sheaths. The weight durationof the different components viz. internodes, spike (excludingthe grains), laminae, and sheaths, respectively were 40.2, 10.5,21.8, and 17.8. Nitrate content (µmol g^–1 dry wt.)was high in the initially formed organs and declined in thesuccessively formed ones. In the laminae, there was a sharpfall after achieving maximal values except for the flag laminawhere the changes in NO^–₃ content were marginal over afairly long period. The ear components had low NO₃^– concentration.Total NO^–₃ content (µmol) in the main shoot showedpeaks at 49, 84, and 118 d after sowing.     

Fine structure of the organ of attachment of the teleost, Garra gotyla gotyla (Ham)

We describe the morphology of the attachment organ (AO) of the teleost, Garra gotyla gotyla (Cyprinidae). It is located ventrally around the mouth opening and used by the species for attachment to submerged rocks in sub-Himalayan streams and rivers where it lives. The AO consists of three crescentic parts and a central callus part. Scanning electron microscopy (SEM) shows the former to possess numerous tubercles, each of which bears about 23–27 curved spines. Light microscopy shows the epidermis of the tuberculated parts to possess one type of cell arranged into 7–8 rows. Transmission electron microscopy (TEM) reveals these cells to contain abundant tonofilaments (hence called the filament cells). The epidermis of the callus part possesses the filament cells and additionally mucous cells, which are absent in the tuberculated parts. The superficial epidermis is apparently keratinized (thickness: 5–8 μm), and a part of the cells of the outer row is modified into spines. These cells show a thick plasma membrane envelope and possess mucous granules (diameter: 0.1–0.3 μm) and bundles of tonofilaments. The cells of the inner two to four rows possess similar organelles and additionally, prominent Golgi bodies and rough endoplasmic reticulum. Immunohistochemically, the cells of the outer row and the spines stain positively for cytokeratin. The cells of the innermost rows (five to eight) possess few tonofilaments and no mucous granules. It is evident that the filament cells of the mid- to upper epidermis are specialized for the production of mucous granules and tonofilaments, which is unique for the teleost epidermis concerned. It appears that the tuberculated parts with spines assist in anchorage and interlocking with the substratum, while the central callus part probably utilizes both suction and frictional mechanisms, and mucous secretion protects the spines from damage during anchorage and abrasion.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Synergistic regulation of endothelial tight junctions by antioxidant (Se) and polyunsaturated lipid (GLA) via Claudin-5 modulation

Tight junctions (TJs) in endothelial cells act as cell-cell adhesion structures, governing paracellular permeability (PCP). Disruption can lead to leaky vascular bed and potentially to oedema and swelling of tissues, the aetiology of mastalgia. These changes may also cause vascular spread of cancer cells. This study aimed to determine whether the function of TJs in endothelial cells can be strengthened by gamma linolenic acid (GLA), selenium (Se) and iodine (I) in the presence of 17beta-estradiol (17beta-estradiol), which causes leakage of endothelial cells by disruption of TJs in endothelium. GLA, I, and Se individually increased transendothelial resistance. The combination of all three agents also had a significant effect on TER. Addition of GLA/Se/I reduced PCP of the endothelial cells. Treatment with GLA/Se/I reversed the effect of 17beta-estradiol in reducing TER and increasing PCP. Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h there was increasing relocation to endothelial cell-cell junctions of the TJ proteins Claudin-5, Occludin, and ZO-1. Interestingly, this relocation was particularly evident with treatments containing I when probing with Claudin-5 and those containing Se for Occludin. There was a small increase in overall protein levels when examined by Western blotting after treatment with GLA/Se/I when probed with Claudin-5 and Occludin. We report that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells, by way of regulating the distribution of Claudin-5, Occludin, and ZO-1. Interestingly, this combination was also able to completely reverse the effect of 17beta-estradiol in these cells.     

The dazzling array of basal branches in the mtDNA macrohaplogroup M from India as inferred from complete genomes

Many efforts based on complete mitochondrial DNA (mtDNA) genomeshave been made to depict the global mtDNA landscape, but thephylogeny of Indian macrohaplogroup M has not yet been resolvedin detail. To fill this lacuna, we took the same strategy asin our recent analysis of Indian mtDNA macrohaplogroup N andselected 56 mtDNAs from over 1,200 samples across India forcomplete sequencing, with the intention to cover all Indianautochthonous M lineages. As a result, the phylogenetic statusof previously identified haplogroups based on control-regionand/or partial coding-region information, such as M2, M3, M4,M5, M6, M30, and M33, was solidified or redefined here. Moreover,seven novel basal M haplogroups (viz., M34–M40) were identified,and yet another five singular branches of the M phylogeny werediscovered in the present study. The comparison of matrilinealcomponents among India, East Asia, Southeast Asia, and Oceaniaat the deepest level yielded a star-like and nonoverlappingpattern, reflecting a rapid mode of modern human dispersal alongthe Asian coast after the initial "out-of-Africa" event.     

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Development of high affinity camptothecin-bombesin conjugates that have targeted cytotoxicity for bombesin receptor-containing tumor cells

Mammalian bombesin (BN) receptors are among those most frequently overexpressed by a number of common tumors including prostate, breast, lung, and colon cancers. The aim of this study was to develop a camptothecin-bombesin (CPT-BN) conjugate that interacts with all classes of BN receptors and possibly functions as a prodrug via a labile linker with site-specific cytotoxicity for cancer cells bearing these receptors. CPT was coupled to analogs of [D-Tyr6,beta-Ala11,Phe13,Nle14]BN-(6-14) (BA0) using carbamate linkers (L1 and L2) with built-in nucleophile-assisted releasing groups for intracellular cleavage of free cytotoxic agents. One conjugate, CPT-L2-BA3, bound to all three BN receptor classes with high affinity and functioned as a full agonist at each. 125I-CPT-L2-BA3 was rapidly internalized by cells expressing each BN receptor class and, using fluorescent imaging, was found to co-localize with BN receptors initially and later to be internalized in cytoplasmic compartments. HPLC analysis of internalized ligand showed that 40% was intact, 25% was metabolized by releasing free CPT, and 35% was metabolized to other breakdown products. CPT-L2-BA3 inhibited the growth of NCI-H1299 non-small cell lung cancer cells in 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) and clonal growth assays. CPT-L2-BA3 was cytotoxic in an MTT assay for cells transfected with each class of BN receptor; however, it had significantly less effect in cells lacking BN receptors. These results indicate that CTP-L2-BA3 is a potent agonist that is cytotoxic for cells overexpressing any of the three BN receptor classes and functions as a prodrug for receptor-mediated cytoxicity. It therefore should be a useful prototype to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism.     

Carnitine,carnitine acetyltransferase,and glutathione in Alzheimer brain

Glutathione and total carnitine (i.e., free carnitine plus acid-soluble carnitine esters) were measured in an affected (superior frontal gyrus; SFG) and unaffected (cerebellum: CBL) region of Alzheimer disease (AD) and control brains. Average glutathione content in AD SFG (n=13) and AD CBL (n=7) (7.9±2.1 and 11.9±4.0 nmol/mg protein, respectively (mean ±S.D.)) was similar to that in control SFG (n=13) and CBL (n=6) (7.7±2.0 and 11.6±2.6 nmol/mg protein, respectively). However, glutathione increased significantly with age in AD brain (p=0.003) but not in control brain. Average total carnitine in AD SFG (84±47 pmol/mg protein; n=10) and AD CBL (108±86 pmol/mg protein; n=7) was not significantly different from that in the corresponding regions of control brain (148±97 (n=10) and 144±107 (n=6) pmol/mg protein, respectively). However, a significant decline of total carnitine with age in both regions was noted for AD brain, but not for control brain. Carnitine acetyltransferase activity in the AD SFG (n=13) was not significantly different from that of control SFG (n=13) (1.83±1.05 and 2.04±0.82 nmol/min/mg protein, respectively). However, carnitine acetyltransferase activity of AD CBL (n=7) was significantly lower than that of control CBL (n=6) (1.33±0.88 versus 2.26±0.66 nmol/min/mg protein; p=0.05).     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.