Two-stage affinity purification for inducibly phosphorylated membrane proteins

Characterisation of tyrosine phosphorylations induced in immune cells in response to inflammatory stimuli may help elucidate the molecular bases of the diversity of immune responses. We have used anti-phosphotyrosine antibodies in combination with cell surface biotinylation in a two-step affinity purification procedure to recover pervanadate-induced tyrosine phosphorylated proteins from sub-cellular compartments, including the cell surface, of murine T cells and macrophages prior to separation by solution-phase isoelectric focussing and one-dimensional gel electrophoresis and identification by tandem mass spectrometry.     

Documentation of immune profile of microglia through cell surface marker study in glioma model primed by a novel cell surface glycopeptide T11TS/SLFA-3

STATEMENT OF THE PROBLEM: The sheep erythrocyte membrane glycoprotein T11TS/SLFA-3 can form a ligand-receptor complex with CD2 present on immunocyte and exert stimuli for activation and proliferation. Regression of brain tumor with the application of T11TS indicates the probable role of microglia, the chief immunomodulatory cell within the brain compartment. In the present study microglial activation and immunophenotypic modulation were assessed in T11TS treated brain tumor-bearing animal models. Rat glioma models induced by chemical carcinogen ENU were treated with three consecutive doses of T11TS. Microglial cells from brain were isolated and assessed through E-rosette formation, SEM and FACS for CD2, MHC class II, CD25, and CD4. The preliminary indication of presence of CD2 on microglia through E-rosette formation was confirmed by SEM and FACS. MHC class II and CD2 single and double positive subpopulations exist, and their expression is also modulated in different doses of T11TS. A general trend of highest receptor saturation and microglial activation, measured through the activation marker CD25 and CD4 expression, was observed in 2nd dose of T11TS administration, which was then dampened via a complex immune feedback mechanism in the 3rd dose.     

Differential responses of UV-B irradiation on the viability and intracellular calcium influx in goat hepatocytes–in vitro effect

Ultraviolet-B (UV-B) irradiation in the range of 280-320nm has shown to be a promising immunomodulatory tool in xenogenic hepatocyte transplantation. Most of the studies documenting the effect(s) of UV-B irradiation on hepatic transplantation have been carried out in small model systems with very little information available in larger animals. The aim of the present investigation was to study in vitro the effect(s) of UV-B irradiation (302 nm) at 0, 250, 500, 1250 and 2500 J/m2 on the viability and cellular responses in the isolated goat hepatocytes. The results showed that the cells irradiated at 0, 250, 500, 1250 and 2500 J/m2 demonstrated a viability of 90-95%. However, intracellular [Ca2+]i influx as quantitated by Flu 3-acetete showed a significant increase with irradiation as observed in confocal microscope. The intracellular pH (quantitated by the flourescence of BCCEF) although tend to show an increase with UV-B irradiation was not statistically significant. The present observations suggest that there is a modulation in the intracellular [Ca2+]i concentration within the hepatocytes at higher dose of UV-B irradiation without altering the viability of hepatocytes. These observations are significant for the xenotransplantation of cells.     

Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.     

Spherical scatters in the lens of Acrossocheilus hexagonolepis as revealed by scanning electron microscopy and UV-visible spectroscopy

Scanning electron microscopy revealed the presence of spherical granules 0.25-0.75 micron in diameter in the lens fibres of the hill stream fish Acrossocheilus hexagonolepis. The density of the granules was approximately 8,500 per mm2. The size of the particles and their distribution pattern suggested they functioned in spherical scattering of the light. The absorbance and transmittance of light at different wavelengths obtained from the spectroscopic analysis of the lens indicated that a small percentage of light was neither absorbed nor transmitted. Since reflection usually does not take place from the ocular refractive structure, it may be that this proportion of the light was scattered from the lens.     

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.     

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6–14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R₆) and nona-arginine (R₉)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6–14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.