• Title/Summary/Keyword: translation in vitro

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Effects of Gut Extract Protein and Insulin on Glucose Uptake and GLUT 1 Expression in HC 11 Mouse Mammary Epithelial Cells

  • Myung, K.H.;Ahn, B.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.8
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    • pp.1210-1214
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    • 2002
  • The large and rapid changes of glucose utilization in lactating mammary tissue in response to changes in nutritional state must be largely related by external signal of insulin. This also must be related with the quantity and composition of the diet in vivo. To characterize the mode of gut extract protein with insulin, in vitro experiment was conducted with HC11 cells. The gut extract protein has not only the same effect as insulin alone but also the synergistic effect with insulin in 2-Deoxy[3H] glucose uptake. Although the gut extract did not modulates glucose uptake via increasing the rate of translation of the GLUT1 protein, northern blot analysis indicated that the gut extract protein increased the expression of GLUT1 mRNA by a threefold and also there was a dose-dependent increase in the expression of GLUT1 mRNA. The gut extract protein is therefore shown to be capable of modulating glucose uptake by transcription level with insulin in HC 11 cells.

Flower-Inducing Activity in the Phloem Exudata and Gene Expression Specific to Photoperiodic Floral Induction in Pharbitis Cotyledons

  • Kim, Kang-Chang;Lee, Jin-Hwan;Her, Yoon-Kang;Maeng, Jue-Son
    • Journal of Plant Biology
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    • v.39 no.4
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    • pp.257-263
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    • 1996
  • Flower-inducing activity in the phloem exudata of Pharbitis cotyledons was investigated using the bioassay of Pharbitis and Lemna. By SDS-PAGE and 2-D gel electrophoresis of the phloem exudate, two polypeptides of 11 kDa and of approximately 32 kDa (pI 6.9) showing qualitative changes during the flower induction were detected. A polypeptide of approximately 20 kDa (pI 4.8) specifically labeled in vivo with [35S]methionine was found during the inductive dark period in Pharbitis cotyledon tissues. The polypeptide of the equivalent molecular mass and with the identicl pI value was also detected by in vitro translation assay. Thus, it is assumed that the 20 kDa polypeptide plays a role in the process of flower induction in Pharbitis cotyledons.

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Transfer RNA Acceptor Stem Determinants for Specific Aminoacylation by Class II Aminoacyl-tRNA Synthetases

  • Musier, Karin
    • BMB Reports
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    • v.31 no.6
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    • pp.525-535
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    • 1998
  • A critical step in the faithful translation of genetic information is specific tRNA recognition by aminoacyl-tRNA synthetases. These enzymes catalyze the covalent attachment of particular amino acids to the terminal adenosine of cognate tRNA substrates. In general, there is one synthetase for each of the twenty amino acids and each enzyme must discriminate against all of the cellular tRNAs that are specific for the nineteen noncognate amino acids. Primary sequence information combined with structural data have resulted in the division of the twenty synthetases into two classes. In recent years, several high-resolution co-crystal structures along with biochemical data have led to an increased understanding of tRNA recognition by synthetases of both classes. The anticodon sequence and the amino acid acceptor stem are the most common locations for critical recognition elements. This review will focus on acceptor stem discrimination by class II synthetases. In particular, the results of in vitro aminoacylation assays and site-directed and atomic group mutagenesis studies will be discussed. These studies have revealed that even subtle atomic determinants can provide signals for specific tRNA aminoacylation.

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miR-421, miR-155 and miR-650: Emerging Trends of Regulation of Cancer and Apoptosis

  • Farooqi, Ammad Ahmad;Qureshi, Muhammad Zahid;Coskunpinar, Ender;Naqvi, Syed Kamran-Ul-Hassan;Yaylim, Ilhan;Ismail, Muhammad
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.5
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    • pp.1909-1912
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    • 2014
  • It is becoming progressively more understandable that between transcription and translation there lies another versatile regulator that quantitatively controls the expression of mRNAs. Identification of miRNAs as key regulators of wide ranging signaling cascades and modulators of different cell-type and context dependent activities attracted basic and clinical scientists to study modes and mechanisms in details. In line with this approach overwhelmingly increasing in vivo and in vitro studies are deepening our understanding regarding miR-421, mir-155 and miR-650 mediated regulation of cellular activities. We also attempt to provide an overview of long non coding RNAs.

Genetic Organization of the Recombinant Bacillus pasteurii Urease Genes Expressed in Escherichia coli

  • Kim, Sang-Dal;Hausinger, Robert P.
    • Journal of Microbiology and Biotechnology
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    • v.4 no.2
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    • pp.108-112
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    • 1994
  • The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment. A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major ($M_r$ 67,000) and minor ($M_r$ 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.

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Structural Studies on IRES 4-2 Domain of Foot-and-mouth Disease Virus

  • Kim, Young-Mee;Yoo, Jun-Seok;Cheong, Hae-Kap;Lee, Chul-Hyun;Cheong, Chae-Joon
    • Journal of the Korean Magnetic Resonance Society
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    • v.7 no.2
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    • pp.89-97
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    • 2003
  • Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus within the picornavirus which has a single copy of a positive sense RNA. The translation initiation process of FMDV occurs by a cap-independent mechanism directed by a highly structured element (∼435 nt) termed an internal ribosome entry site (IRES). We have designed and prepared FMDV 4-2 RNA (28nt) by in vitro transcription. The 2D NMR data revealed that FMDV 4-2 IRES domain RNA has a flexible loop and bulge conformation. In further study, we need to make an isotope labeled RNA sample and conduct 3D NMR experiments to completely determine the 3D structure. This study may establish a new drug design strategy to treat foot-and mouth disease.

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Influence of Coronoid Fracture on Elbow Stability: A Kinematic Study Based on New Clinical Relevant Fracture Classification

  • Jeon, In-Ho;Joaquin, Sanchez-Sotelo;Steinmann, Scott;Zhao, Kristin;An, Kai-Nan;Morrey, Bernard F.
    • The Academic Congress of Korean Shoulder and Elbow Society
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    • 2009.03a
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    • pp.128-129
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    • 2009
  • This study suggests isolated Type IV-MO or Type IV-LO fractures could be treated with nonsurgical treatment because they do not interfere with normal elbow kinematics. Valgus and external rotation instability were influenced by total articular surface, however, posterior and proximal translation were influenced by isolated articular surface involvement of coronoid. Further clinical studies are warranted to validate these in vitro findings.

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Cytochrome C methylation: Current Knowledge of its Biological Significance

  • Park, Kwang-Sook;Frost, Blaise F.;Lee, Hyang-Woo;Kim, Sang-Duk;Paik, Woon-Ki
    • Archives of Pharmacal Research
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    • v.11 no.1
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    • pp.7-13
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    • 1988
  • The yeast cytochrome c gene has been recloned, and the resulting cytochrome c mRNA has been translated in rabbit reticulocyte lysate translation system. The newly synthesized apocytochrome c could be methylated by exogenously added cytochrome c-lysine N-methyltransferase. Enzymatic methylation of in vitro synthesized apocytochrome c was found to facilitate specifically its import into mitochondria of yeast, but not of rat liver.

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Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130

  • Seo, In-Ae;Lee, Hyun-Kyoung;Shin, Yoon-Kyung;Lee, Sang-Hwa;Seo, Su-Yeong;Park, Ji-Wook;Park, Hwan-Tae
    • The Korean Journal of Physiology and Pharmacology
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    • v.13 no.2
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    • pp.131-138
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    • 2009
  • The binding of interleukin-6 (IL-6) cytokine family ligands to the gp130 receptor complex activates the Janus kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3) signal transduction pathway, where STA T3 plays an important role in cell survival and tumorigenesis. Constitutive activation of STAT3 has been frequently observed in many cancer tissues, and thus, blocking of the gp130 signaling pathway, at the JAK level, might be a useful therapeutic approach for the suppression of STAT3 activity, as anticancer therapy. AG490 is a tyrphostin tyrosine kinase inhibitor that has been extensively used for inhibiting JAK2 in vitro and in vivo. In this study, we demonstrate a novel mechanism associated with AG490 that inhibits the JAK/STAT3 pathway. AG490 induced downregulation of gp130, a common receptor for the IL-6 cytokine family compounds, but not JAK2 or STAT3, within three hours of exposure. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. It most likely occurred by translation inhibition of gp130 in association with phosphorylation of the eukaryotic initiation factor-2 a. The inhibition of protein synthesis of gp130 by AG490 led to immediate loss of mature gp130 in cell membranes, due to its short half-life, thereby resulting in reduction in the STAT3 response to IL-6. Taken together, these results suggest that AG490 blocks the STAT3 activation pathway via a novel pathway.

Export of Human Proinsulin in E. coli : High Export of Proinsulin Fusion Protein but not of Proinsulin Itself (대장균에서 인체 프로인슐린의 분비 발현 : 프로인슐린 융합체의 고분비 발현과 프로인슐린의 저분비 발현)

  • Yup Kang
    • KSBB Journal
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    • v.11 no.2
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    • pp.165-172
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    • 1996
  • To obtain a correctly folded human proinsulin, export of proinsulin using Staphylococcal protein A signal sequence-mediated secretion pathway has been attempted in E.coli. A secretion operon for proinsulin was constructed by consecutively connecting T7 promoter, SPA ribosome binding site, SPA signal sequence gene, and human proinsulin gene. Little immunoreactive proinsulin was detected in the periplasmic space and. culture medium, and not even in cytoplasmic space. The qualitative analysis of transcribed proinsulin mRNA and the in vitro transcription/translation experiment suggests that the negligible level of proinsulin export appears to be due to intracellular degradation of proinsulin, rather than due to the blockage during translocation. However, expression of proinsulin fusion protein such as MBP-proinsulin could dramatically increase export of proinsulin in E.coli.

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