Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.     

Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis (Hemiptera: Aleyrodidae) adults using 16S rDNA amplicon pyrosequencing and FISH

Dialeurolonga malleswaramensis Sundararaj (Hemiptera: Aleyrodidae) is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene (mtCO1) helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswaramensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization (FISH) in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.     

Modeling the binding sites of anti-hen egg white lysozyme antibodies HyHEL-8 and HyHEL-26: an insight into the molecular basis of antibody cross-reactivity and specificity

Three antibodies, HyHEL-8 (HH8), HyHEL-10 (HH10), and HyHEL-26 (HH26) are specific for the same epitope on hen egg white lysozyme (HEL), and share >90% sequence homology. Their affinities vary by several orders of magnitude, and among the three antibodies, HH8 is the most cross-reactive with kinetics of binding that are relatively invariable compared to HH26, which is highly specific and has quite variable kinetics. To investigate structural correlates of these functional variations, the Fv regions of HH8 and HH26 were homology-modeled using the x-ray structure of the well-characterized HH10-HEL complex as template. The binding site of HH26 is most charged, least hydrophobic, and has the greatest number of intramolecular salt bridges, whereas that of HH8 is the least charged, most hydrophobic and has the fewest intramolecular salt bridges. The modeled HH26-HEL structure predicts the recently determined x-ray structure of HH26, (Li et al., 2003, Nat. Struct. Biol. 10:482-488) with a root-mean-square deviation of 1.03 A. It is likely that the binding site of HH26 is rendered rigid by a network of intramolecular salt bridges whereas that of HH8 is flexible due to their absence. HH26 also has the most intermolecular contacts with the antigen whereas HH8 has the least. HH10 has these properties intermediate to HH8 and HH26. The structurally rigid binding site with numerous specific contacts bestows specificity on HH26 whereas the flexible binding site with correspondingly fewer contacts enables HH8 to be cross-reactive. Results suggest that affinity maturation may select for high affinity antibodies with either "lock-and-key" preconfigured binding sites, or "preconfigured flexibility" by modulating combining site flexibility.     

Protein structure to function via dynamics

Protein folding, binding, catalytic activity and molecular recognition all involve molecular movements, with varying extents. The molecular movements are brought upon via flexible regions. Stemming from sequence, a fine tuning of electrostatic and hydrophobic properties of the protein fold determine flexible and rigid regions. Studies show flexible regions usually lack electrostatic interactions, such as salt-bridges and hydrogen-bonds, while the rigid regions often have larger number of such electrostatic interactions. Protein flexible regions are not simply an outcome of looser packing or instability, rather they are evolutionally selected. In this review article we highlight the significance of protein flexibilities in folding, binding and function, and their structural and thermodynamic determinants. Our electrostatic calculations and molecular dynamic simulations on an antibody-antigen complex further illustrate the importance of protein flexibilities in binding and function.     

Spectrum of <Emphasis Type="Italic">CREBBP</Emphasis> mutations in Indian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RSTS), a developmental disorder comprising abnormalities that include mental retardation, an unusual facial appearance, broad thumbs and big toes is frequently associated with molecular lesions in the CREB-binding protein gene, CREBBP. The objective of the present study was to identify and analyse CREBBP mutations in Indian RSTS patients on which there are no data. Direct sequencing of CREBBP performed in 13 RSTS patients identified the three zinc fingers (CH1, CH2, CH3) and HAT domain as mutational hotspots in which ten novel pathogenic mutations were localized. Functional analysis revealed that three of these mutations affecting amino acids Glu1459, Leu1668 and Glu1724 were critical for histone acetyltransferase activity. Twenty-eight novel CREBBP single-nucleotide polymorphisms (SNPs) were also identified in the Indian population. Linkage disequilibrium studies revealed associations between (i) SNP (rs129974/c.3836-206G>C) and mutation (p.Asp1340Ala); (ii) (rs130002) with mutation (p.Asn435Lys) and (iii) SNPs rs129974, rs130002 and SNP (c.3836-206G>C) signifying a disease affection status. In conclusion, the present study reports the highest detection rate of CREBBP mutations (76.9%) in RSTS patients to date, of which ten are predicted to be pathogenic and three critical for histone acetyltransferase activity. Moreover, identification of the association of CREBBP polymorphisms with disease susceptibility could be an important risk factor for the pathogenesis of RSTS.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence