ABSTRACT
Objective To construct the ICAT gene interference lentivirus expression vector targeting and to establish stable transfected cell line HL60 .Methods The interference sequence targeted at human ICAT gene was designed and synthesized ,after annealing ,which was connected to PGLV3 interference vector and with PG‐p1‐VSVG ,PG‐p1‐REV ,PG‐p1‐RRE were co‐transfected into 293T cells The lentivirus particles were packaged and generated .The virus titer was detected .HL60 cells were transfected for establishing the stable cell line ;RT‐PCR and Western Blot techniques were used to detect ICAT gene and protein expression in sta‐ble HL60 cells ,then the results were compared with those in the control group .Results The lentivirus expression vector targeted at ICAT was successfully constructed and the virus titer was 2 × 108 U/mL .Stable transfected HL60 cell line was established .The effective interference verification revealed that shICAT could significantly reduce the mRNA and protein level of ICAT ( P<0 .001) .Conclusion The shRNA lentiviral expression vector of ICAT gene is successfully constructed and the HL 60 cell line stably interfering ICAT expression is established .
ABSTRACT
An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other ‘omics’ approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.
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Aim To analyze differential expression and interaction of β-catenin/ICAT proteins in HL60 cells when they were induced into monocytic differentiation, and to figure out the mechanism of NSC67657 in cellu-lar induction. Methods HL60 cells were treated by 10 μmol · L-1 NSC67657 , and cellular differentiation could be observed by cytochemical staining and flow cytometry. Then, RT-PCR and Western blot were em-ployed to determine the differential expression of β-catenin/ICAT genes and proteins. Co-immunoprecipi-tation assay was used to confirm the interaction of β-catenin/ICAT proteins, and laser co-focus light mi-croscopy technology was used to co-indentify proteins differential expression and intracellular location. Re-sults HL60 cells could be induced into monocytic dif-ferentiation after 5 days treatment using 10μM NSC67657 . The CD14 ( +)% cells could be up to o-ver 90%, and cytochemical staining reports were con-sistent with this result. The expressions of ICAT gene and protein were up-regulated significantly ( P <0. 01 ) , but the expressions ofβ-catenin gene and pro-tein, on the contrary, were down-regulated(P<0. 05) when HL60 cells were induced into monocytic differen-tiation. From co-immunoprecipitation assay findings, ICAT protein interacted with β-catenin protein, and the absorbance of protein electrophoresis bands in-creased in differentiated cells. From laser co-focus light microscopy assay findings, the fluorescence of ICAT and β-catenin protein could be both observed in cytoplasm and nucleus. In drug treated HL60 cells, the fluorescence of ICAT protein was enhanced both in cytoplasm and nucleus, however, the fluorescence ofβ-catenin protein, which looked like transferring into different organelles, decreased significantly in nucleus, but increased in cytoplasm. Conclusions HL60 cells could be induced into monocytic differentiation by NSC67657 and β-catenin/ICAT proteins differentially expressed during cellular differentiation. The enhanced interaction of β-catenin/ICAT proteins and β-catenin protein transferring from nucleus into cytoplasm indi-cates that NSC67657 probably induces HL60 cells into monocytic differentiation through down-regulating β-catenin protein and blockingβ-catenin protein from nu-cleus.
ABSTRACT
The inflammatory lesions that affect the paranasal sinuses receive the generic denomination sinusitis; the maxillary sinus is the most commonly affected. This inflammation can have various origins, including the tooth. We describe a case of maxillary sinusitis in a 56-year-old patient who experienced pain on the left-side maxilla, referred to atooth and performed a partial review of the literature.
Las lesiones inflamatorias que afectan los senos paranasales reciben la denominación genérica de sinusitis, siendo el seno maxilar el más comúnmente afectado. Esta inflamación puede tener diversos orígenes, entre ellos el dentario. Se describe un caso de sinusitis del seno maxilar de origen dentario de un paciente de 56 años que consultó por dolor en la zona maxilar del lado izquierdo referido a una pieza dentaria y se realiza una revisión parcial de la literatura.
Subject(s)
Humans , Male , Middle Aged , Tooth Diseases/complications , Maxillary Sinusitis/etiology , Maxillary Sinusitis , Cone-Beam Computed TomographyABSTRACT
Quantitative proteomics is a novel subject of proteomics research. There are several new techniques employed for the protein quantitative study. Isobaric tags for relative and absolute quantitation (iTRAQ) technology for protein quantitation using mass spectrometry is a recent powerful means of determining relative and absolute protein levels in up to four samples simultaneously. The iTRAQ reagent produced high quality, reproducible result in enriched complexes, organelles, and whole cell lysates. The status of the recent promising techniques and their possible future evolution were reviewed.
ABSTRACT
Objective:To Clone the cDNA of human ICAT,and express it in tumor cell,to investigate its anti-tumor effect.Method:the ICAT cDNA was amplified by RT-PCR,the ukaryotic expression plasmid pcDNA3.1-ICAT was constructed,MCF-7 and MDA-MB231 breast cancer cells were transfected by pcDNA3.1-ICAT using liposome and the cancer cell proliferation was estimated by MTT.Result:The product of RT-PCR was about 260bp,its sequence was the same as that reported,the transfection efficiency for MCF-7 and MDA-MB-23 cells was about 26%,and inhibiting efficiencies on the proliferation of the cancer cell transfected were 18.1% and 16.2% respectively.Conlusion:The cDNA of ICAT was cloned,and its expression in MCF-7 and MDA-MB-23 cells may inhibit the proliferation of these cancer cells.