Regeneration of plantlets from mature embryo calli of Western Ghats land race cultivar of rice, Oryza sativa L

The Malnad region located in the Western Ghats of Karnataka is known for the cultivation of indigenous rain fed land race cultivar of rice. The present study was to investigate the callogenic and caulogenic potentialities of the two indigenous rice cultivar namely Karimundaga and Kanadatumba using dehusked mature embryo explants. For callus and shoot bud differentiation, the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D (1-3 mg/L), IAA (1-2 mg/L), Kn (1-4 mg/L) and BAP (1-4 mg/L). The morphogenic potentialities of the two rice cultivar differed in texture of callus. In both the cultivar callogenic frequency was optimized at 1 mg/L 2,4-D concentration, it was 94% in Karimundaga and 58% in Kanadatumba. Supplementation of IAA either alone (1-2 mg/L) or in combination with Kn or BAP at 1 to 4 mg/L concentration of each induces shoot bud differentiation from the calli. In the cultivar Karimundaga caulogenic frequency was highest (10.60 +/- 2.55) at 1.0 mg/L IAA and 4.0 mg/L BAP concentration. While in the cultivar Kanadatumba highest number of shoot buds (7.90 +/- 2.69) was differentiated at 1.0 mg/L IAA and 4.0 mg/L Kn concentration. The calli derived regenerants were successfully acclimatized in the greenhouse and agro-morphological variations were evaluated. The growth characteristics and yield related parameters exhibited by in vitro plants were lower than the in vivo plants.     

Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio

White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM) generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1) administration. Myocytes were treated with ET-1 (10 nM) for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK) activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin–dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

Rat heart is a site of leptin production and action

Leptin, the 16-kDa peptide hormone product of the ob gene, is produced primarily by adipocytes and was initially thought to exert its effects exclusively through actions on the hypothalamus via distinct leptin receptors termed OB-R. However, recent data show that leptin is produced elsewhere and that receptors are present in many other tissues. Using real-time PCR, we determined whether leptin and its receptors are present in the rat heart and demonstrated regional distribution patterns and gender differences as well as the effect of ischemia and reperfusion. Gene expression of leptin and its receptors (OB-Ra, OB-Rb, and OB-Re) was identified in myocytes and whole heart homogenates from all regions of the heart of male and female rats, with the highest abundance in left and right atria of male and female rats, respectively. No differences in regional distribution of OB-R were evident in male rat hearts. In female rats, expression was highest in right atria for all three isoforms and was significantly greater than in male rats. Ischemia and reperfusion significantly downregulated leptin and OB-R expression, although this was more pronounced in male rat hearts. Leptin release in the coronary effluent was also detected using ELISA, although this was generally unaffected by global ischemia and reperfusion. Our results demonstrate for the first time the presence of the leptin system, including the peptide and its receptors, in all regions of the rat heart. In view of emerging evidence for cardiac effects of leptin, it is proposed that the heart is a target for leptin action and that the peptide modulates function through a paracrine- or autocrine-dependent manner.     

Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.     

rugose (rg), a Drosophila A kinase anchor protein,is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.     

Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation

A novel method for selection of transgenic plants utilizing the sweet pepper ( Capsicum annuum L.) ferredoxin-like protein ( pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (beta-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.     

Autoregulation of regulatory proteins is key for dynamic operation of GAL switch in Saccharomyces cerevisiae

Autoregulation and nucleocytoplasmic shuttling play important roles in the operation of the GAL regulatory system. However, the significance of these mechanisms in the overall operation of the switch is unclear. In this work, we develop a dynamic model for the GAL system and further validate the same using steady-state and dynamic experimental expression data. Next, the model is used to delineate the relevance of shuttling and autoregulation in response to inducing, repressing, and non-inducing-non-repressing media. The analysis indicates that autoregulation of the repressor, Gal80p, is key in obtaining three distinct steady states in response to the three media. In particular, the analysis rationalizes the intuitively paradoxical observation that the concentration of repressor, Gal80p, actually increases in response to an increase in the inducer concentration. On the other hand, although nucleocytoplasmic shuttling does not affect the dynamics of the system, it plays a dominant role in obtaining a sensitive response to galactose. The dynamic model was also used to obtain insights on the preculturing effect on the system behavior.     

Evaluating two process scale chromatography column header designs using CFD

Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale‐up. Computational Fluid Dynamics (CFD) provides a cost‐effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:837–844, 2014