Nuclear translocation of insulin receptor substrate-1 by oncogenes and Igf-I. Effect on ribosomal RNA synthesis

The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally localized in the cytosol/membrane fraction of the cell. We show here that a substantial fraction of IRS-1 is translocated to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1 occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized role in rRNA synthesis and/or processing.     

NF-kappa B mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments

Fibronectin fragments (FN-f) that bind to the alpha(5)beta(1) integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene beta (GRO-beta). Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1beta expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-kappaB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.     

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze]

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clonedr1 (droughtresponsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clonedr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clonedr3, responded similarly to clonedr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters     

A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites

The superfamily of plant and bacterial type III polyketide synthases (PKSs) produces diverse metabolites with distinct biological functions. PKS18, a type III PKS from Mycobacterium tuberculosis, displays an unusual broad specificity for aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20)) to produce tri- and tetraketide pyrones. The crystal structure of PKS18 reveals a 20 A substrate binding tunnel, hitherto unidentified in this superfamily of enzymes. This remarkable tunnel extends from the active site to the surface of the protein and is primarily generated by subtle changes of backbone dihedral angles in the core of the protein. Mutagenic studies combined with structure determination provide molecular insights into the structural elements that contribute to the chain length specificity of the enzyme. This first bacterial type III PKS structure underlines a fascinating example of the way in which subtle changes in protein architecture can generate metabolite diversity in nature.     

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.     

EpiMix Based Novel Vaccine Candidate for Shigella: Evidence of Prophylactic Immunity in Balb/c Mice

International Journal of Peptide Research and Therapeutics - Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent...     

New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit micromolar EC₅₀ values and against T. gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.     

Targeted novel surface-modified nanoparticles for interferon delivery for the treatment of hepatitis B

The purpose of the present work was to develop hepatitis B surface antigen (HBsAg) surface-adsorbed cationic poly (d,l-lactic-co-glycolic acid) PLGA nanoparticles for interferon alpha (IFNα) delivery targeted to hepatocytes. Cationic PLGA nanoparticles loaded with IFNα were prepared using the double emulsification technique. Delipidated HBsAg was passively adsorbed on the surface of nanoparticles by using the simple dipping and drying method. Surface morphology and size distribution of nanoparticles were analyzed by scanning electron microscopy and dynamic light-scattering method, respectively. The biodistribution behavior of plain and HBsAg-coated (99m)Tc-tagged PLGA nanoparticles was also examined followed by intravenous injection. The results revealed that ～75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly 3-fold greater in magnitude than the plain PLGA nanoparticles. These data demonstrated that the novel formulation of nanoparticles has potential application in hepatic-targeted drug delivery.     

Oxytocin influences the production of glycyrrhizin from cell cultures of Abrus precatorius Linn.

Oxytocin, a peptide animal hormone, was used as a growth regulator to test its effect on biomass accumulation and production of secondary plant constituent glycyrrhizin in the cell cultures of Abrus precatorius. Glycyrrhizin is an important phytoconstituent of liquorice which is widely used in the pharmaceutical and food industries. Cell suspension cultures of A. precatorius were developed from leaf explant of in vitro germinated plant in Murashige and Skoog medium supplemented with 30 g/l sucrose, 1 mg/l naphthalene acetic acid and 1 mg/l kinetin. The influence of oxytocin on biomass accumulation as well as on the production of glycyrrhizin was observed in the cell cultures of A. precatorius. Treatment of A. precatorius cell cultures with 100 μg/l oxytocin, improved glycyrrhizin production up to 34.27 mg/l on the dry cell weight basis third day after oxytocin treatment, which is over four times that of the control cultures, simultaneously nearly two fold increase in the biomass 2 days after the oxytocin treatment was recorded over the control cultures.