Assessment of genetic fidelity of micropropagated <Emphasis Type="Italic">Swertia chirayita</Emphasis> plantlets by ISSR marker assay

Inter simple sequence repeat (ISSR) marker assay was employed to validate the genetic fidelity of Swertia chirayita plantlets multiplied in vitro by axillary multiplication upto forty-two passages. Sixteen ISSR primers generated a total of 102 amplicons among the tissue-cultured plants. Forty-eight amplicons were amplified in the outlier (a Swertia species). The outlier (negative control) was employed to rule out the possibility that the invariant fingerprint was due to chance alone and that the ISSR technique employed was not discriminatory enough to detect the off-types. A homogenous amplification profile was observed for all the micropropagated plants. The results confirmed the clonal fidelity of the tissue culture-raised S. chirayita plantlets and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

Micropropagation of Dendrocalamus asper {Schult. & Schult. F.} Backer ex k. Heyne): an exotic edible bamboo

Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February?CApril) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS?+?15???M BAP. For shoot multiplication, MS medium supplemented with 10???M BAP and 75???M Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ? strength MS medium supplemented with 5???M each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6?months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.     

Mass spectrometric measurement of end-tidal xenon concentration for clinical stable xenon/computerized tomography cerebral blood flow studies

We have demonstrated the feasibility of using a compact dedicated mass spectrometer to monitor end-tidal xenon concentration in human subjects during stable xenon computerized tomography measurements of regional cerebral blood flow. End-tidal carbon dioxide concentration is monitored simultaneously and noninvasively without degrading the dynamic response to xenon. For clinical regional cerebral blood flow studies we employed a Nuclide 3-60-G Sectorr mass spectrometer with a 3 in radius, 60 degrees magnetic sector and a variable (0-5000 V) ion accelerating potential. The required high vacuum (10(-7) Torr) was achieved and maintained by means of a turbomolecular pump. A needlemetering valve was incorporated into an anesthesia mask connector, and exhaled gases were transported to the mass spectrometer via a 6 ft length of Teflon tubing (1/16 in i.d.). Molecular flow conditions between the sample and analysis chambers were provided by use of a gold foil leak (0.0005 in. hole). At an inlet pressure of 400 m Torr (achieved by means of the needle valve), the inlet system was characterized by a gas transport lag-time of 1.3 s and a rise-time constant of 85 ms. Xenon (doubly charged ion: m/z 68) and carbon dioxide (doubly charged ion: m/z 22) were monitored alternately at 75 ms intervals. Our experience with mass spectrometry has demonstrated the feasibility of using a compact dedicated instrument for accurately and non-invasively monitoring end-tidal xenon concentration in a clinical setting.     

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.     

Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

Effect of picroliv on low density lipoprotein receptor binding of rat hepatocytes in hepatic damage induced by paracetamol.

Picroliv from root and rhizome of Picrorhiza kurroa showed reversal of low density lipoprotein (LDL) binding to paracetamol-induced damaged hepatocytes of rats. Changes in levels of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase, conjugated dienes and lipids of hepatocytes were significantly prevented by picroliv at different doses. The effect of picroliv on enzyme levels, LDL receptor binding and lipids in damaged hepatocytes was found to be comparable to silymarin, a known hepatoprotective agent.     

Glutamine Metabolism Regulates Proliferation and Lineage Allocation in Skeletal Stem Cells

1. Download high-res image (128KB)
2. Download full-size image

     

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^?/? mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.