Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli

Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.     

Experimental pathogenicity evaluation of Mycoplasma canadense from bovine mastitis in vitro and in vivo laboratory models

Mycoplasma canadense, a clinical isolate from milk of a mastitic buffalo, was experimentally tested for its pathogenic potential in hamster tracheal ring and rabbit fallopian tube explant organ cultures (in vitro) and rat and rabbit mammary gland (in vivo) models. The activity percentage reduction in M. canadense infected hamster tracheal rings was 99.1% in comparison to 16.4% in control rings. Mycoplasma canadense, also induced complete ciliostasis at 11-day post-infection in rabbit fallopian tube explants. Histopathological lesions in these infected organ cultures were loss of cilia, desquamation or denudation of epithelium, infiltration of inflammatory cells and proliferation of macrophages as well as oedema in lamina propria. At the end of the experiments, M. canadense organisms were reisolated in pure colonies from the infected but not the control organ cultures. In the rat and rabbit mammary glands, M. canadense organisms persisted upto 6-day and 7-day postinfection, respectively and caused histopathological changes suggestive of subacute to chronic mastitis during the experimental period. The results indicate that the tested M. canadense clinical isolate was virulent.     

Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.     

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF^Skp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser⁶⁴ and Ser⁷², we have identified Thr⁴¹⁷ as a unique Pim-1 phosphorylation target. Phosphorylation of Thr⁴¹⁷ controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.     

Comparative effect of Ocimum sanctum, Commiphora mukul, folic acid and ramipril on lipid peroxidation in experimentally-induced hyperlipidemia

Treatment with C. mukul and O. sanctum, showed a significant decrease in cholesterol and triglyceride levels respectively. O. sanctum also significantly increased serum HDL-cholesterol compared to control. Serum MDA levels were significantly reduced in all the treated groups compared to control suggesting that each of the drugs under study were effective in their free radical scavenging action. Erythrocyte SOD activity was increased in all the treatment groups with C. mukul showing the maximum effect followed by O. sanctum, folic acid and ramipril. The erythrocyte CAT activity was significantly increased in all the drug treated groups with maximum increase seen in O. sanctum and ramipril treated groups, whereas lesser effects were observed with C. mukul and folic acid groups. Thus, the indigenous drugs, C. mukul and O. sanctum had beneficial effect on hypercholesterolemic rabbit model, both in terms of lipid profile as well as antioxidant potential. Ocimum sanctum was found to be the most promising of all the drugs. Moreover, it could be hypothesized that these plant products along with folic acid and ramipril can be explored for synergistic effect for treatment for hypercholesterolemic conditions.     

Protein disulfide isomerase assisted protein folding in malaria parasites

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.     

Conformation and assembly of polypeptide scaffolds in templating the synthesis of silica: an example of a polylysine macromolecular "switch"

Although the role of polycationic macromolecules in catalyzing the synthesis of silica structures is well established, detailed understanding of the mechanisms behind the production of silica structures of controlled morphologies remains unclear. In this study, we have used both poly-L-lysine (PLL) and/or poly-D-lysine (PDL) for silica synthesis to investigate mechanisms controlling inorganic morphologies. The formation of both spherical silica particles and hexagonal plates was observed. The formation of hexagonal plates was suggested, via circular dichroic spectroscopy (CD), to result from the assembly of helical polylysine molecules. We confirm that the formation of PLL helices is a prerequisite to the hexagonal silica synthesis. In addition, we present for the first time that the handedness of the helicity of the macromolecule does not affect the formation of hexagonal silica. We also show, by using two different silica precursors, that the precursor does not have a direct effect on the formation of hexagonal silica plates. Furthermore, when polylysine helices were converted to beta-sheet structure, only silica particles were obtained, thus suggesting that the adoption of a helical conformation by PLL is required for the formation of hexagonally organized silica. These results demonstrate that the change in polylysine conformation can act as a "switch" in silica structure formation and suggest the potential for controlling morphologies and structures of inorganic materials via control of the conformation of soft macromolecular templates.     

PI3K-independent AKT activation in cancers: a treasure trove for novel therapeutics

AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.     

Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid

The Asian citrus psyllid, Diaphorina citri Kuwayama, is a worldwide pest of citrus, which vectors the putative causal pathogen of huanglongbing. Current management practices warrant continuous monitoring of field populations for insecticide resistance. Baseline activities of acetylcholinesterase (AChE), general esterase, and glutathione S-transferase as well as sensitivity of AChE to selected organophosphate and carbamate insecticides were established for a susceptible laboratory strain (Lab) and compared with several field populations of D. citri from Florida. The specific activity of AChE in various D. citri populations ranged from 0.77 to 1.29 microM min(-1) mg of protein(-1); the Lab strain was characterized by the highest activity. Although reduced AChE sensitivity was observed in the Lab strain compared with field populations, overlap of 95% confidence intervals of I50 values (concentration required for 50% AChE activity inhibition) suggests no significant difference in AChE sensitivity among all populations tested for a given insecticide. There was no significant evidence of target site insensitivity in field populations that were exposed to the selected organophosphate and carbamate insecticides tested. The specific activity of general esterase and glutathione S-transferase was lowest in the Lab strain and was generally comparable to that of the field populations evaluated. The current data provide a mode-of-action specific baseline for future monitoring of resistance to organophosphate and carbamate insecticides in populations of D. citri.