Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.     

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.     

GNL3L Is a Nucleo-Cytoplasmic Shuttling Protein: Role in Cell Cycle Regulation

GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501–582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the ‘G2/M’ phase of cell cycle whereas depletion of endogenous GNL3L results in ‘G2/M’ arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L^∆NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L^∆NES results in faster ‘S’ phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote ‘S’ phase progression during cell proliferation.     

Transbuccal delivery of 5-Aza-2′-deoxycytidine: Effects of drug concentration,buffer solution,and bile salts on permeation

Delivery of 5-aza-2′-deoxycytidine (decitabine) across porcine buccal mucosa was evaluated as an alternative to the complex intravenous infusion regimen currently used to administer the drug. A reproducible high-performance liquid chromatography method was developed and optimized for the quantitative determination of this drug. Decitabine showed a concentration-dependent passive diffusion process across porcine buccal mucosa. An increase in the ionic strength of the phosphate buffer from 100 to 400 mM decreased the flux from 3.57±0.65 to 1.89±0.61 μg/h/cm². Trihydroxy bile salts significantly enhanced the flux of decitabine at a 100 mM concentration (P>.05). The steady-state flux of decitabine in the presence of 100 mM of sodium taurocholate and sodium glycocholate was 52.65±9.48 and 85.22±7.61 μg/cm²/h, respectively. Two dihydroxy bile salts, sodium deoxytaurocholate and sodium deoxyglycocholate, showed better enhancement effect than did trihydroxy bile salts. A 38-fold enhancement in flux was achieved with 10 mM of sodium deoxyglycocholate. Published: July 13, 2007     

Physiological and biochemical responses to acute ozone-induced oxidative stress in Medicago truncatula.

Oxidative signaling mediated by reactive oxygen species (ROS) is a central component of biotic and abiotic stresses in plants. Acute ozone (O(3)) fumigation is a useful non-invasive treatment for eliciting endogenous ROS in planta. In this study, 38 different accessions of the model legume, Medicago truncatula, from various geographical regions were fumigated with 300 nmol mol(-1) of O(3) for a period of six hours. Phenotypic symptoms were evaluated 24 and 48 h after the end of treatment. A majority of the accessions showed distinct visible damage. Eight accessions showing varying sensitivities to ozone were subjected to biochemical analysis to evaluate correlations between ozone damage and levels of ROS, antioxidants, and lipid peroxidation. Two-way analysis of variance indicated highly significant interactions between O(3) damage and levels of ROS, ascorbate, glutathione and lipid peroxidation. There were significant differences among the accessions for these traits before and after the end of O(3) fumigation, as indicated by equal variance Student's t-test. This study suggests that multiple physiological and biochemical mechanisms may govern O(3) tolerance or sensitivity. Surveying a large collection of germplasm led to identification of multiple resistant and sensitive lines for investigating molecular basis of O(3) phytotoxicity. The most resistant JE154 accession also showed enhanced tolerance to chronic O(3) and dehydration stress, suggesting germplasm with increased tolerance to acute O(3) can be a useful resource for improving resistance to multiple abiotic stressors.     

The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist

Meta III is formed during the decay of rhodopsin's active receptor state at neutral to alkaline pH by thermal isomerization of the retinal Schiff base C15=N bond, converting the ligand from all-trans 15-anti to all-trans 15-syn. The thereby induced change of ligand geometry switches the receptor to an inactive conformation, such that the decay pathway to Meta III contributes to the deactivation of the signaling state at higher pH values. We have examined the conformation of Meta III over a wider pH range and found that Meta III exists in a pH-dependent conformational equilibrium between this inactive conformation at neutral to alkaline pH and an active conformation similar to that of Meta II, which, however, is assumed at very acidic pH only. The apparent pKa of this transition is around 5.1 and thus several units lower than that of the Meta I/Meta II photoproduct equilibrium with its all-trans 15-anti ligand, but still about 1 unit higher than that of the opsin conformational equilibrium in the absence of ligand. The all-trans-15-syn-retinal chromophore is therefore not an inverse agonist like 11-cis- or 9-cis-retinal, which lock the receptor in an inactive conformation, but a classical partial agonist, which is capable of activating the receptor, yet with an efficiency considerably lower than the full agonist all-trans 15-anti. As the Meta III chromophore differs structurally from this full agonist only in the isomeric state of the C15=N bond, this ligand represents an excellent model system to study principal mechanisms of partial agonism which are helpful to understand the partial agonist behavior of other ligands.     

Molecular basis for RASSF10/NPM/RNF2 feedback cascade–mediated regulation of gastric cancer cell proliferation

Ras-association domain family (RASSF) proteins are encoded by numerous tumor suppressor genes that frequently become silenced in human cancers. RASSF10 is downregulated by promoter hypermethylation in cancers and has been shown to inhibit cell proliferation; however, the molecular mechanism(s) remains poorly understood. Here, we demonstrate for the first time that RASSF10 inhibits Cdk1/cyclin-B kinase complex formation to maintain stable levels of cyclin-B for inducing mitotic arrest during cell cycle. Using LC-MS/MS, live cell imaging, and biochemical approaches, we identify Nucleophosmin (NPM) as a novel functional target of RASSF10 and revealed that RASSF10 expression promoted the nuclear accumulation of GADD45a and knockdown of either NPM or GADD45a, resulting in impairment of RASSF10-mediated G2/M phase arrest. Furthermore, we demonstrate that RASSF10 is a substrate for the E3 ligase ring finger protein 2 (RNF2) and show that an NPM-dependent downregulation of RNF2 expression is critical to maintain stable RASSF10 levels in cells for efficient mitotic arrest. Interestingly, the Kaplan–Meier plot analysis shows a positive correlation of RASSF10 and NPM expression with greater gastric cancer patient survival and the reverse with expression of RNF2, suggesting that they may have a role in cancer progression. Finally, our findings provide insights into the mode of action of the RASSF10/NPM/RNF2 signaling cascade on controlling cell proliferation and may represent a novel therapeutic avenue for the prevention of gastric cancer metastasis.