Journal articles: 'Heterologe Expression'

1

Hahn, T., L. Kilian, K. Muffler, S. Lang, and R. Ulber. "Erstmalige heterologe Expression einer Fucosidase." Chemie Ingenieur Technik 82, no. 9 (August 27, 2010): 1488. http://dx.doi.org/10.1002/cite.201050112.

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Lutz-Wahl, S., M. Wälz, M. L. Magri, K. Liebeton, J. Eck, and L. Fischer. "Heterologe Expression und Charakterisierung einer stereoselektiven Nitril-Hydratase." Chemie Ingenieur Technik 78, no. 9 (September 2006): 1399–400. http://dx.doi.org/10.1002/cite.200650178.

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Crüsemann, Max, Raphael Reher, Isabella Schamari, Alexander O. Brachmann, Tsubasa Ohbayashi, Markus Kuschak, Davide Malfacini, et al. "Heterologe Expression, Biosynthese und ökologische Funktion des selektiven Gq‐Signaltransduktionsinhibitors FR900359." Angewandte Chemie 130, no. 3 (December 20, 2017): 844–49. http://dx.doi.org/10.1002/ange.201707996.

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Richter, A., M. Müller, H. Kiesecker, and H. J. Jacobsen. "Expressionsstabilität in transgenen Erbsen – Heterologe Expression von Polygalakturonase-inhibierenden Proteinen in transgenen Erbsen." Journal für Verbraucherschutz und Lebensmittelsicherheit 1, S1 (November 2006): 132. http://dx.doi.org/10.1007/s00003-006-0090-6.

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Yan, Fu, David Auerbach, Yi Chai, Lena Keller, Qiang Tu, Stephan Hüttel, Amelie Glemser, et al. "Biosynthese und heterologe Expression der Vioprolide: rationale gentechnische Eingriffe in die Biosynthese und 4-Methylazetidincarbonsäure-Bildung." Angewandte Chemie 130, no. 28 (June 8, 2018): 8890–95. http://dx.doi.org/10.1002/ange.201802479.

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Szankowski, I., H. Li, H. Flachowsky, T. Fischer, V. Hanke, G. Forkmann, D. Treutter, T. Hoffmann, and W. Schwab. "Modifikation der Flavonoid-Biosynthese in Apfel (Malus domestica Borkh.) durch heterologe Expression des Lc-Gens aus Mais." Journal für Verbraucherschutz und Lebensmittelsicherheit 2, S1 (December 2007): 106. http://dx.doi.org/10.1007/s00003-007-0269-5.

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Elliott, Beth, Christine Richardson, Jamie Winderbaum, Jac A. Nickoloff, and Maria Jasin. "Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells." Molecular and Cellular Biology 18, no. 1 (January 1, 1998): 93–101. http://dx.doi.org/10.1128/mcb.18.1.93.

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ABSTRACT Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology. Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence. Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs. Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs. We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination. Gene conversion tract lengths were examined in 80 recombinants. Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less. These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.

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Hidayani, Andi Aliah, Odang Carman, and Alimuddin. "Effectiveness of B-actin promoter on driving target gene expression in common carp transgenesis." Jurnal Akuakultur Indonesia 10, no. 1 (January 1, 2011): 16. http://dx.doi.org/10.19027/jai.10.16-23.

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Promoter in transgene construct plays an important role on regulating of transgene expression level in transgenic fish. In fish transgenesis, researcher convinced that use all-fish gene construct is safety and prospective. This study was performed to compare effectiveness b-actin promoter, - the promoter which has ubiquitous, constitutive, housekeeping characteristics, from common carp (homologous) and from tilapia and medaka b-actin promoters (heterologous) in driving of green fluorescent protein (GFP) expression as a model of target gene on common carp transgenesis. These gene constructs were separately microinjected into cytoplasm of 60 one-cell-stage common carp embryos. The results suggested that 70% survival rate at embryo stage and 45% hatching rate values showed that the microinjection was performed successfully. Percentage of embryos expressing GFP gene were slightly higher when injected using common carp and medaka promoters than those of using tilapia promoter. Percentage of larvae expressing GFP using common carp promoter was similar with medaka promoter. Furthermore, GFP expression using common carp b-actin promoter could be detected at one-week-old larvae, while GFP expressing using medaka b-actin promoter was lasted at 2-day-old larvae. The results demonstrated that homologous promoter more effective in driving of a target gene expression than that of heterologous promoter. Key words: homologous promoter, GFP, transgenesis, common carp ABSTRAK Promoter dalam konstruksi transgen berperan penting dalam pengaturan tingkat ekspresi transgen pada ikan transgenik. Dalam transgenesis ikan, peneliti meyakini bahwa penggunaan konstruksi gen "all-fish" adalah aman dan prospektif. Penelitian ini dilakukan untuk membandingkan efektivitas promoter β-aktin, - promoter yang memiliki ciri ubiquitous, constitutive, dan housekeeping, dari ikan dari ikan mas (homolog) dan ikan nila dan ikan medaka (heterolog) dalam mengendalikan ekspresi gen GFP sebagai model gen pada transgenesis ikan mas. Setiap konstruksi gen tersebut diinjeksikan secara terpisah ke sitoplasma embrio ikan mas fase 1 sel sebanyak 60 embrio. Hasil penelitian dengan kelangsungan hidup embrio 70% dan derajat penetasan 45% menunjukkan bahwa kegiatan mikroinjeksi berhasil dengan baik. Persentase embrio mengekspresikan gen GFP yang diinjeksi konstruksi gen dengan promoter β-aktin ikan mas dan ikan medaka sedikit lebih tinggi dibandingkan dengan yang menggunakan promoter β-aktin ikan nila. Selanjutnya, ekspresi gen GFP yang dikendalikan oleh promoter β-aktin ikan mas dapat dideteksi pada larva berumur 1 minggu, sedangkan ekspresi GFP dengan promoter β-aktin ikan medaka hanya bisa terdeteksi hingga larva berumur 2 hari. Hasil penelitian menunjukkan bahwa promoter homolog adalah lebih efektif dalam mengatur ekspresi gen target dibandingkan dengan promoter heterolog. Kata kunci: promoter homolog, GFP, transgenesis, ikan mas

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Mérida-Floriano, Angela, Will P. M. Rowe, and Josep Casadesús. "Genome-Wide Identification and Expression Analysis of SOS Response Genes in Salmonella enterica Serovar Typhimurium." Cells 10, no. 4 (April 19, 2021): 943. http://dx.doi.org/10.3390/cells10040943.

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A bioinformatic search for LexA boxes, combined with transcriptomic detection of loci responsive to DNA damage, identified 48 members of the SOS regulon in the genome of Salmonella enterica serovar Typhimurium. Single cell analysis using fluorescent fusions revealed that heterogeneous expression is a common trait of SOS response genes, with formation of SOSOFF and SOSON subpopulations. Phenotypic cell variants formed in the absence of external DNA damage show gene expression patterns that are mainly determined by the position and the heterology index of the LexA box. SOS induction upon DNA damage produces SOSOFF and SOSON subpopulations that contain live and dead cells. The nature and concentration of the DNA damaging agent and the time of exposure are major factors that influence the population structure upon SOS induction. An analogy can thus be drawn between the SOS response and other bacterial stress responses that produce phenotypic cell variants.

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Deng, Yong-Sheng, Fan-Ying Kong, Bin Zhou, Song Zhang, Meng-Meng Yue, and Qing-Wei Meng. "Heterology expression of the tomato LeLhcb2 gene confers elevated tolerance to chilling stress in transgenic tobacco." Plant Physiology and Biochemistry 80 (July 2014): 318–27. http://dx.doi.org/10.1016/j.plaphy.2014.04.017.

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11

Schytter, Helene. "HAVNEN OG DET HETEROGENE BYRUM - NORDHAVN BETRAGTET GENNEM GEORGES BATAILLES HETEROLOGI." K&K - Kultur og Klasse 38, no. 109 (July 1, 2010): 85–102. http://dx.doi.org/10.7146/kok.v38i109.15793.

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THE HARBOUR AND THE HETEROGENEOUS URBAN. NORDHAVN READ ON THE BASIS OF BATAILLES HETEROLOGYThis article deals with the revitalisation of post-industrial waterfronts on the basis of the French philosopher Georges Bataille’s concept of the heterogeneous. Using the northern harbour area in Copenhagen (Nordhavn) as its analysis site, the article investigates the way in which a heterologically based position can be introduced into urban space-making, pointing to a different urban ideal and the possibility of including something formless and wholly other in the conventional development process. Based on Bataille’s heterology, Nordhavn can predominantly be considered a radically different urban space on a spatial, reflexive and socio-cultural level. The harbour area can be characterised on the basis of four spatial types (ruin, shed, accumulation monument and terrain vague), each expressing aheterogeneous aesthetic identity as something more or less excluded, non-ideal, and entropy- and waste-like. The heterogeneous typology thus establishes the experience of a progressive spatial formlessness, as well as emphasising the aesthetic and cultural potential in urban wastelands. In this sense, the heterogeneous urban environment underlines the importance of the procedural, non-planned, unexpected, other and hybrid in relation to the current discussions about how to create a vibrant urban space. In the age of gentrification, wall-to-wall city design and homogenised waterfronts, a heterogeneously inspired urbandevelopment may thus generate an alternative aesthetic strategy leading towards a more value-pluralistic city.

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Su, Tao, Bing Han, Chong Zhang, Xin Li, and Xin-Hui Xing. "Heterologus expression of particulate methane mooxygenase gene in different host bacterial cells for biocatalysis." Journal of Biotechnology 136 (October 2008): S303. http://dx.doi.org/10.1016/j.jbiotec.2008.07.1888.

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13

Yang, Sha, Xian-Feng Tang, Na-Na Ma, Li-Yan Wang, and Qing-Wei Meng. "Heterology expression of the sweet pepper CBF3 gene confers elevated tolerance to chilling stress in transgenic tobacco." Journal of Plant Physiology 168, no. 15 (October 2011): 1804–12. http://dx.doi.org/10.1016/j.jplph.2011.05.017.

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14

Hsieh, Tsai-Hung, Jent-Turn Lee, Pei-Tzu Yang, Li-Hui Chiu, Yee-yung Charng, Yu-Chie Wang, and Ming-Tsair Chan. "Heterology Expression of the ArabidopsisC-Repeat/Dehydration Response Element Binding Factor 1 Gene Confers Elevated Tolerance to Chilling and Oxidative Stresses in Transgenic Tomato." Plant Physiology 129, no. 3 (June 14, 2002): 1086–94. http://dx.doi.org/10.1104/pp.003442.

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15

Enerbäck, S., B. G. Ohlsson, L. Samuelsson, and G. Bjursell. "Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis." Molecular and Cellular Biology 12, no. 10 (October 1992): 4622–33. http://dx.doi.org/10.1128/mcb.12.10.4622.

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When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.

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Enerbäck, S., B. G. Ohlsson, L. Samuelsson, and G. Bjursell. "Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis." Molecular and Cellular Biology 12, no. 10 (October 1992): 4622–33. http://dx.doi.org/10.1128/mcb.12.10.4622-4633.1992.

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When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.

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Finzel, J., V. Florian, H. Schoepe, G. Woitow, H. J. Selbitz, and S. Springer. "Vorkommen und Bekämpfung des Clostridium-perfringens-Typ-A-assoziierten Durchfalls der Saugferkel unter besonderer Berücksichtigung der Immunprophylaxe." Tierärztliche Praxis Ausgabe G: Großtiere / Nutztiere 40, no. 06 (2012): 375–82. http://dx.doi.org/10.1055/s-0038-1623143.

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Zusammenfassung Gegenstand und Ziel: Clostridium perfringens Typ A wird häufig in Verbindung mit Durchfällen von Saugferkeln isoliert. Die Bedeutung des Alpha-(α-) und Beta(β)2-Toxins für die Pathogenese der Erkrankung ist nicht abschließend geklärt. Zur Bekämpfung der Erkrankung wurden in der Vergangenheit insbesondere stallspezifische Impfstoffe eingesetzt. Ziel der Untersuchungen war die Typisierung und quantitative Bestimmung des α- und β2-Toxins von C.-perfringens-Stämmen, die im Rahmen der Herstellung bestandsspezifischer Impfstoffe isoliert wurden. Mithilfe eines Intoxikationsmodells wurde weiterhin die Wirksamkeit eines kommerziell erhältlichen Impfstoffs gegen den C.-perfringens-Typ-A-assoziierten Durchfall der Saugferkel geprüft. Material und Methoden: Im ersten Teil der Untersuchungen wurden 1434 C.-perfringens-Stämme, die von an Durchfall erkrankten Ferkeln stammten, mithilfe einer Multiplex-PCR typisiert. Gleichzeitig wurde das α- und β2-Toxin-Bildungsvermögen der Stämme mittels ELISA-Tests untersucht. Zur Prüfung der α- und β2-toxoidhaltigen C.-perfringens-Typ-A-Vakzine für Schweine (Clostriporc A, IDT Biologika GmbH) wurden im zweiten Teil der Untersuchungen 18 Jungsauen im letzten Drittel der Trächtigkeit zweimal im Abstand von 3 Wochen immunisiert. Ergebnisse: 87,9% der Stämme gehörten zur Toxovar A (cpa, cpb2), 6,3% zur Toxovar A (cpa) und 5,8% zur Toxovar C (cpa, cpb, cpb2). Bei der quantitativen Untersuchung des Toxinbildungsvermögens der C.-perfringens-Typ-A-Stämme (cpa, cpb2) zeichneten sich Stämme, die geringe oder mittlere Gehalte des α-Toxins bildeten, häufig durch eine starke Expression des β2-Toxins aus. Die geimpften Sauen bildeten Antikörper gegen das α- und β2-Toxin, die über das Kolostrum auf die Nachzucht übertragen wurden und neugeborene Ferkel signifikant (p < 0,05) vor einer Intoxikation mit einem α- und β2-toxinhaltigen Überstand eines heterologen C.-perfringens-Typ-A-Stammes schützten. Schlussfolgerung und klinische Relevanz: Die Ergebnisse unterstreichen die Bedeutung von α- und β2-toxinbildenden C.-perfringens-Typ-A-Stämmen im Durchfallgeschehen der Saugferkel. Im Intoxikationsmodell ließ sich eine deutliche Schutzwirkung der eingesetzten Vakzine vor dem α- und β2-Toxin nachweisen.

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18

Sharathchandra, R. G., N. P. Geetha, K. N. Amruthesh, K. Ramachandra Kini, B. R. Sarosh, N. P. Shetty, and H. S. Shetty. "Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum." Functional Plant Biology 33, no. 3 (2006): 267. http://dx.doi.org/10.1071/fp05197.

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Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.

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19

Sen, Prosenjit, Ramakrishnan Gopalakrishnan, Hema Kothari, Curtis Clark, Usha Pendurthi, and L. Vijaya Mohan Rao. "Factor VIIa Bound to Endothelial Cell Protein C Receptor Activates Protease Activated Receptor 1-Mediated Cell Signaling and Barrier-Protective Response In Endothelial Cells." Blood 116, no. 21 (November 19, 2010): 346. http://dx.doi.org/10.1182/blood.v116.21.346.346.

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Abstract Abstract 346 Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C and activated protein C (APC). In addition to controlling coagulation by modulating the protein C-mediated anticoagulant pathway, EPCR has been shown to play a critical role in supporting APC-induced cell signaling, which could be responsible for some of the non-hemostatic functions of EPCR and APC. Recent studies from our laboratory and others have shown that factor VIIa (FVIIa), a coagulation factor whose primary function is to initiate tissue factor (TF)-dependent coagulation, also binds to EPCR on endothelium. At present, the physiological significance of this interaction is unclear. APC binding to EPCR has been shown to provide cytoprotective effects via protease activated receptor (PAR) 1-mediated cell signaling. In earlier studies using exogenously expressed PAR1 and PAR2 reporter constructs in a heterologus cell model system, we were unable to find measurable n-terminal cleavage (activation) of PARs by FVIIa bound to EPCR. It is possible that transfected PAR constructs may segregate differently on the cell surface membrane than that of endogenous PARs, and thus may have decreased susceptibility for cleavage by FVIIa-EPCR. In the present study, we have investigated whether FVIIa, upon binding to EPCR on endothelial cells, activates endogenous PAR1 and induces PAR1-mediated cell signaling. To determine whether FVIIa cleaves endogenously expressed PAR1 on endothelial cells, unperturbed cultures of human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of FVIIa (0-40 nM) and the cleavage of PAR1 at the cell surface was measured quantitatively in a cell-surface ELISA using a cleavage-specific PAR1 monoclonal antibody. The data show that FVIIa, in a dose- and time-dependent manner, cleaves PAR1 on endothelial cells. FVIIa cleavage of PAR1 on endothelial cells is dependent on FVIIa binding to EPCR, as prevention of FVIIa binding to EPCR by pretreating HUVEC with EPCR polyclonal antibody completely abolished FVIIa cleavage of PAR1. Similarly, silencing EPCR with EPCR-specific siRNA fully attenuated FVIIa cleavage of PAR1. FVIIa cleavage of PAR1 on endothelial cells is independent of TF as pretreatment of HUVEC with anti-TF antibodies or transduction of HUVEC with adenovirus encoding TF had no significant effect on FVIIa cleavage of PAR1. The efficiency of PAR1 cleavage by FVIIa appears to be comparable to that of APC, as both at 10 nM cleave PAR1 to a similar extent. FVIIa (10 nM) cleaves only a fraction of PAR1 (∼25 to 30%) on endothelial cell surface; increasing either FVIIa concentration or duration of treatment has not resulted in additional cleavage of remaining PAR1. Low expression of PAR2 in endothelial cells and lack of cleavage specific antibodies to PAR2 prevented us from determining whether FVII bound to EPCR also cleaves PAR2. FVIIa (10 nM) induced p44/42 MAPK activation in HUVEC and this activation was dependent on EPCR and PAR1 but not PAR2, as silencing EPCR or PAR1 but not PAR2 attenuated FVIIa-induced p44/42 MAPK phosphorylation. In additional studies, FVIIa (10 nM) was found to elicit protection against thrombin-induced barrier disruption in endothelial cells as analyzed in a dual-chamber system using Evans blue-labeled BSA or measurements of transendothelial electrical resistance. FVIIa-induced barrier-protective effect is EPCR-dependent. F-actin staining of HUVEC exposed to thrombin showed formation of transcellular actin stress fibers, cellular contractions and paracellular gap formation. Pretreatment of HUVEC with FVIIa maintained actin at the cell periphery, and reduced formation of central stress fibers and paracellular gaps. FVIIa-induced p44/42 MAPK activation and barrier protective effect are mediated via Rac1, as specific inhibitors against Rac1 or transduction of Rac1 dominant negative mutant abolished these FVIIa-induced effects. Consistent with in vitro findings, in vivo studies in mice showed that administration of FVIIa prior to LPS attenuated the LPS-induced vascular leakage in lung and kidney. Overall, our present data provide strong and convincing evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier protective effect. These findings are novel and assume a great clinical significance as FVIIa is used prophylactically for prevention of bleeding in hemophiliacs. Disclosures: No relevant conflicts of interest to declare.

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Sowa, Miriam A., Nadja Kreuter, Nadine Sella, Julia Manhard, Alexander Siegl, Edgar Weichhard, Martin Rühl, Holger Zorn, and Martin Gand. "Ersatz von prägastrischen Esterasen bei der Käseherstellung ‐Identifizierung, heterologe Expression und Anwendung einer Lipase aus Pleurotus citrinopileatus." Lebensmittelchemie 75, S1 (March 2021). http://dx.doi.org/10.1002/lemi.202155012.

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Umami, Rifqiyah Nur, Apon Zaenal Mustopa, Linda Sukmarini, Hasim Danuri, Andini Setyanti Putri, and Krisna Dwi Aria Wibowo. "CLONING, EXPRESSION, AND PARTIAL PURIFICATION OF PLANTARICIN W LOCUS PRODUCED BY Lactobacillus plantarum S34." BERITA BIOLOGI 16, no. 1 (July 7, 2017). http://dx.doi.org/10.14203/beritabiologi.v16i1.2174.

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Lactobacillus plantarum S34 dilaporkan mempunyai aktivitas antibakteri yang terkait dengan produksi bakteriosin. Bagian dari gen yang menyandikan salah satu lokus bakteriosin yang diproduksi oleh L. plantarum S34, disebut dengan plantarisin W (plnW), diamplifikasi dari plasmid dan dikloning menggunakan sistem vektor pGEM®-T Easy ke dalam Escherichia coli DH5?. Sekuens nukleotida plnW (± 405 pb) diidentifikasi sebagai protein integral membran. Lebih lanjut, plnW diekspresikan secara heterologus sebagai fusi protein dengan His(6)-tag tioredoksin menggunakan vektor ekspresi pET-32a(+) ke dalam E. coli BL21 (DE3) pLysS. Protein fusi rekombinan plnW terdapat dalam sitoplasma sel, tetapi selain fraksi terlarut terdapat juga fraksi tidak terlarut berupa badan inklusi. Purifikasi parsial dilakukan menggunakan kromatografi afinitas ligan Co2+ untuk fraksi terlarut dan metode elektroelusi gel poliakrilamid untuk fraksi tidak terlarut. Massa molekul berukuran kurang lebih 33 kDa terdeteksi berdasarkan pemisahan SDS-PAGE dan dikonfirmasi dengan Western blot sebagai protein fusi rekombinan plnW. Protein yang sudah terpurifikasi bermanfaat untuk mengetahui kaitan antara struktur dan fungsi bakteriosin.

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Deeba, Farah, Tasawar Sultana, Nadia Majeed, and Syed Muhammad Saqlan Naqvi. "Heterologous expression of a plant WRKY protein confers multiple stress tolerance in E. coli Bir bitkinin heterolog ifadesi WRKY proteini çoklu stres yaratır E. coli’de tolerans." Turkish Journal of Biochemistry 45, no. 2 (April 28, 2020). http://dx.doi.org/10.1515/tjb-2018-0483.

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AbstractObjectiveOsWRKY71, a WRKY protein from rice, is reported to function during biotic stresses. It is requisite to further enquire the efficiency and mechanism of OsWRKY71 under various environmental stresses. Stress indicators such as salt, cold, heat, and drought were studied by overexpressing the OsWRKY71 in E. coli.Materials and methodsDNA binding domain containing region of OsWRKY71 was cloned and expressed in E. coli followed by exposure to stress conditions. OsWRKY71 was also assessed for its role in abiotic stresses in rice by qPCR.ResultsRecombinant E. coli expressing OsWRKY71 was more tolerant to stresses such as heat, salt and drought in spot assay. The tolerance was further confirmed by monitoring the bacterial growth in liquid culture assay demonstrating that it encourages the E. coli growth under salt, drought, and heat stresses. This tolerance may be the consequence of OsWRKY71 interaction with the promoter of stress related genes or with other proteins in bacteria. The RT-qPCR analysis revealed the up-regulation of OsWRKY71 gene in rice upon interaction to cold, salt, drought and wounding with maximum up-regulation against salinity.ConclusionThus, the defensive role of OsWRKY71 may accord to the development and survival of plants during different environmental stresses.

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BUDIANI, Asmini, Djoko SANTOSO, Hajrial ASWIDINNOOR, and Antonius SUWANTO. "Aktivitas ACCase mesokarp kelapa sawit dan kloning fragmen gen penyandi ACCase subunit biotin karboksilase ACCase activity of oil palm mesocarp and cloning of gene fragment encoding biotin carboxylase subunit of ACCase." E-Journal Menara Perkebunan 74, no. 1 (March 8, 2016). http://dx.doi.org/10.22302/iribb.jur.mp.v74i1.119.

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Summary Genetic engineering to produce high yielding oil palm might be done by over expressing gene encoding key enzyme for oil biosynthesis in the oil palm mesocarp, one of which is ACCase. The objective of this research was to analyze ACCase activity of mesocarp from several developmental stages of fruit and to clone conserved region cDNA of gene encoding biotin carboxylase subunit of ACCase (BC-htACCase) from oil palm mesocarp. Activity of ACCase was analyzed by HPLC. Amplification of cDNA was done by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer on several annealing temperature and MgCl2 concentration. The cDNA fragment of RT-PCR product was cloned, sequenced and analyzed to confirm that the cloned cDNA was conserved region of BC-htACCase. The result showed that ACCase activity increased from the 14 week to the 20 week-old fruit, and then decreased. Using heterologous degenerate primers, cDNA fragments of BC-htACCase conserved region (469 bp) can be specifically amplified at 60 oC annealing temperature with 2 mM MgCl2 concentration.The result of BlastX analysis showed that the sequence of cloned cDNA fragment was highly homologous with the conserved region of BC-htACCase from Glycine max, Arabidopsis thaliana, Nicotiana tabacum, and Brassica napus with 243, 237, 236, 231 bit score, and E. value 2e-63, 1e-61, 2e-61 and 5e-60, respectively. Ringkasan Rekayasa genetika untuk menghasilkan bibit kelapa sawit berdaya hasil tinggi dapat ditempuh dengan meningkatkan ekspresi gen penyandi enzim kunci biosintesis minyak pada kelapa sawit, salah satunya adalah ACCase. Tujuan penelitian ini adalah menguji aktivitas ACCase mesokarp beberapa tahap perkem-bangan buah sawit dan mengklon fragmen cDNA daerah konservatif gen penyandi ACCase heteromerik subunit biotin karbok-silase (BC-htACCase) dari mesokarp buah sawit. Aktivitas ACCase dianalisis dengan HPLC. Amplifikasi cDNA dilakukan dengan teknik RT-PCR menggunakan primer degene-rate heterologus pada berbagai suhu penempelan dan konsentrasi MgCl2. Fragmen cDNA hasil RT-PCR diklon, disekuen dan dianalisis untuk mengkonfirmasi bahwa cDNA terklon adalah daerah konservatif BC-htACCase. Hasil penelitian menunjukkan bahwa aktivitas ACCase meningkat dari buah berumur 14 minggu hingga buah berumur 20 minggu, kemudian menurun kembali Dengan primer degenerate heterologus, fragmen cDNA daerah konservatif BC-htACCase (469 pb) dapat diamplifikasi secara spesifik pada suhu penempelan 60 oC dan konsentrasi MgCl2 2 mM. Hasil analisis BlastX dari sekuen DNA fragmen terklon menunjuk-kan bahwa sekuen tersebut mempunyai homologi tinggi antara lain dengan gen penyandi BC-htACCase dari Glycine max, Arabidopsis thaliana, Nicotiana tabacum dan Brassica napus, masing-masing dengan skor 243, 237, 236, 231 bit, dan E. value 2e-63, 1e-61, 2e-61 dan 5e-60.

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24

BUDIANI, Asmini, Djoko SANTOSO, Hajrial ASWIDINNOOR, and Antonius SUWANTO. "Aktivitas ACCase mesokarp kelapa sawit dan kloning fragmen gen penyandi ACCase subunit biotin karboksilase ACCase activity of oil palm mesocarp and cloning of gene fragment encoding biotin carboxylase subunit of ACCase." E-Journal Menara Perkebunan 74, no. 1 (March 8, 2016). http://dx.doi.org/10.22302/ppbbi.jur.mp.v74i1.119.

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Abstract:

Summary Genetic engineering to produce high yielding oil palm might be done by over expressing gene encoding key enzyme for oil biosynthesis in the oil palm mesocarp, one of which is ACCase. The objective of this research was to analyze ACCase activity of mesocarp from several developmental stages of fruit and to clone conserved region cDNA of gene encoding biotin carboxylase subunit of ACCase (BC-htACCase) from oil palm mesocarp. Activity of ACCase was analyzed by HPLC. Amplification of cDNA was done by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer on several annealing temperature and MgCl2 concentration. The cDNA fragment of RT-PCR product was cloned, sequenced and analyzed to confirm that the cloned cDNA was conserved region of BC-htACCase. The result showed that ACCase activity increased from the 14 week to the 20 week-old fruit, and then decreased. Using heterologous degenerate primers, cDNA fragments of BC-htACCase conserved region (469 bp) can be specifically amplified at 60 oC annealing temperature with 2 mM MgCl2 concentration.The result of BlastX analysis showed that the sequence of cloned cDNA fragment was highly homologous with the conserved region of BC-htACCase from Glycine max, Arabidopsis thaliana, Nicotiana tabacum, and Brassica napus with 243, 237, 236, 231 bit score, and E. value 2e-63, 1e-61, 2e-61 and 5e-60, respectively. Ringkasan Rekayasa genetika untuk menghasilkan bibit kelapa sawit berdaya hasil tinggi dapat ditempuh dengan meningkatkan ekspresi gen penyandi enzim kunci biosintesis minyak pada kelapa sawit, salah satunya adalah ACCase. Tujuan penelitian ini adalah menguji aktivitas ACCase mesokarp beberapa tahap perkem-bangan buah sawit dan mengklon fragmen cDNA daerah konservatif gen penyandi ACCase heteromerik subunit biotin karbok-silase (BC-htACCase) dari mesokarp buah sawit. Aktivitas ACCase dianalisis dengan HPLC. Amplifikasi cDNA dilakukan dengan teknik RT-PCR menggunakan primer degene-rate heterologus pada berbagai suhu penempelan dan konsentrasi MgCl2. Fragmen cDNA hasil RT-PCR diklon, disekuen dan dianalisis untuk mengkonfirmasi bahwa cDNA terklon adalah daerah konservatif BC-htACCase. Hasil penelitian menunjukkan bahwa aktivitas ACCase meningkat dari buah berumur 14 minggu hingga buah berumur 20 minggu, kemudian menurun kembali Dengan primer degenerate heterologus, fragmen cDNA daerah konservatif BC-htACCase (469 pb) dapat diamplifikasi secara spesifik pada suhu penempelan 60 oC dan konsentrasi MgCl2 2 mM. Hasil analisis BlastX dari sekuen DNA fragmen terklon menunjuk-kan bahwa sekuen tersebut mempunyai homologi tinggi antara lain dengan gen penyandi BC-htACCase dari Glycine max, Arabidopsis thaliana, Nicotiana tabacum dan Brassica napus, masing-masing dengan skor 243, 237, 236, 231 bit, dan E. value 2e-63, 1e-61, 2e-61 dan 5e-60.

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Du, Jianyang, David Silverman, Bruce Liang, and Lixia Yue. "Abstract 953: Ca 2+ -permeable TRP Channels In Human Cardiac Fibroblasts." Circulation 116, suppl_16 (October 16, 2007). http://dx.doi.org/10.1161/circ.116.suppl_16.ii_188-b.

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In our laboratory, recent single cell electrophysiologic studies have demonstrated the absence of voltage-gated Ca 2+ channels in human cardiac fibroblasts. The more positive membrane potential found in these cells suggests that Ca 2+ entry occurs through a different mechanism. We hypothesized that non-voltage-gated Ca 2+ -permeable TRP channels are responsible for Ca 2+ entry in human cardiac fibroblasts. With informed consent, right atrial biopsies were obtained from patients undergoing cardiac surgery (n=4:.3M, 1F; mean age 65±8 yrs, EF 63±5%, LVEDP 24±4 mm Hg). Fibroblasts were dissociated and cultured for 7 to 10 days. We found that TRPC1, TRPC4, TRPC6, TRPV4, TRPV5, TRPV6, TRPM4 and TRPM7 were detectable at message levels by RT-PCR. Functional expression of these channels was evaluated by patch-clamp technique. An outward rectifying current with typical I–V relation of TRPM7 was readily recorded in the fibroblasts. The averaged current density was 14.5±0.8 pA/pF (mean±SEM, n=60 from four patients). These currents exhibit similar properties to those of heterologues expressed TRPM7, including permeation to Ca 2+ and Mg 2+ , single channel conductance, and inhibition by 2-APB. Moreover, the TRPM7-like current in human fibroblasts was activated by acidic pH conditions, suggesting that this channel function may be enhanced under ischemic conditions. To study whether this TRPM7-like current promotes fibroblast proliferation, we designed siRNA specifically targeting human TRPM7. Fibroblasts treated with TRPM7 siRNA for five days showed a 76.3% decrease in current density, indicating that transcription of trpm7 produces the message for the Ca 2+ -pemeable channel in human cardiac fibroblasts. Consistent with the decreased current amplitude, Ca 2+ influx in siRNA treated fibroblasts was significantly smaller than those of control fibroblasts, further suggesting that TRPM7-like current contributes to calcium signaling in these cells. Taken together, our results indicate that TRPM7 provides the molecular basis of the Ca 2+ -permeable cation channel in human cardiac fibroblast, and that TRPM7 may play a crucial role in the fibrogenesis cascade in human heart.

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TRIASTANTO, Oktaviany Ferry, Muhammad JUSUF, and Djoko SANTOSO. "Identifikasi homolog TcAGL-15 untuk penanda embriogenesis tanaman kakao melalui pendekatan bioinformatika Identification of TcAGL-15 homolog in the embryogenic culture from the zygotic embryo explant." E-Journal Menara Perkebunan 74, no. 2 (March 8, 2016). http://dx.doi.org/10.22302/iribb.jur.mp.v74i2.102.

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Summary One of the major problems encountered in micropropagation of cacao through tissue culture is very low frequency of embryo formation. Embryogenesis is believed to have key regulatory gene determining the process. Understanding such gene may help to solve problems in the regeneration process. One of the genes reported to involve in the embryogenesis is AGAMOUS-like 15 (AGL-15). This gene has an important role in the regulation of early embryogenesis in several plants. This experiment aimed to identify AGL-15 homolog in cacao through bioinformatics approach. The first step of this experiment is to identify the AGL-15 homolog using hetero-logous primers from DNA genomic isolated from leaves of cacao plants. The sequence of the AGL-15 fragment was used in designing specific primers for longer AGL-15 fragment. These primers were then used to identify AGL-15 gene using total RNA isolated from cultured zygotic embryos. Differential pattern of AGL-15 gene expression was observed in zygotic embryos cultured for five weeks. AGL-15 heterologous primers designed from several plants could be used to identify cacao AGL-15 homolog. The putative cacao AGL-15 gene could be identified from zygotic embryos. The differential pattern of the AGL-15 gene expression is a strong indication that AGL-15 can be used as an embryogenesis marker in cacao plants.Ringkasan Salah satu kendala perbanyakan kakao melalui kultur jaringan adalah sulitnya embriogenesis, yang diduga melibatkan satu atau lebih gen kunci yang menentukan proses tersebut. Keberhasilan mengidentifikasi gen-gen kunci akan membantu menyelesaikan masalah dalam regenerasi embrio kakao. Salah satu gen yang diduga terlibat dalam proses ini adalah AGAMOUS-like 15 (AGL-15). Gen ini berperan pada regulasi selama masa awal perkembangan embrio beberapa tanaman. Penelitian ini bertujuan untuk mengidentifikasi homolog AGL-15 pada kakao melalui pen-dekatan bioinformatika dan RT-PCR. Pene-litian diawali dengan identifikasi homolog AGL-15 dari DNA genomik daun kakao meng-gunakan primer heterologus. Sekuen fragmen homolog AGL-15 yang diperoleh, kemudian digunakan untuk merancang primer spesifik AGL-15 yang berukuran lebih panjang. Primer ini selanjutnya digunakan untuk meng-identifikasi gen AGL-15 dari RNA total embrio zigotik. Pengamatan pola pita gen AGL-15 dilakukan pada kultur in vitro embrio zigotik yang berumur lima minggu. Primer hetero-logus gen AGL-15 yang berasal dari berbagai tanaman, mampu mengidentifikasi keberadaan homolog gen tersebut pada tanaman kakao. Fragmen homolog AGL-15 putatif tanaman kakao teridentifikasi pada tingkat RNA embrio. Dengan adanya pola diferensial dari ekspresi gen AGL-15 hingga lima minggu pertama perkembangan embrio, ada indikasi kuat bahwa fragmen homolog AGL-15 dapat menjadi penanda embriogenesis pada tanaman kakao.

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27

TRIASTANTO, Oktaviany Ferry, Muhammad JUSUF, and Djoko SANTOSO. "Identifikasi homolog TcAGL-15 untuk penanda embriogenesis tanaman kakao melalui pendekatan bioinformatika Identification of TcAGL-15 homolog in the embryogenic culture from the zygotic embryo explant." E-Journal Menara Perkebunan 74, no. 2 (March 8, 2016). http://dx.doi.org/10.22302/ppbbi.jur.mp.v74i2.102.

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Abstract:

Summary One of the major problems encountered in micropropagation of cacao through tissue culture is very low frequency of embryo formation. Embryogenesis is believed to have key regulatory gene determining the process. Understanding such gene may help to solve problems in the regeneration process. One of the genes reported to involve in the embryogenesis is AGAMOUS-like 15 (AGL-15). This gene has an important role in the regulation of early embryogenesis in several plants. This experiment aimed to identify AGL-15 homolog in cacao through bioinformatics approach. The first step of this experiment is to identify the AGL-15 homolog using hetero-logous primers from DNA genomic isolated from leaves of cacao plants. The sequence of the AGL-15 fragment was used in designing specific primers for longer AGL-15 fragment. These primers were then used to identify AGL-15 gene using total RNA isolated from cultured zygotic embryos. Differential pattern of AGL-15 gene expression was observed in zygotic embryos cultured for five weeks. AGL-15 heterologous primers designed from several plants could be used to identify cacao AGL-15 homolog. The putative cacao AGL-15 gene could be identified from zygotic embryos. The differential pattern of the AGL-15 gene expression is a strong indication that AGL-15 can be used as an embryogenesis marker in cacao plants.Ringkasan Salah satu kendala perbanyakan kakao melalui kultur jaringan adalah sulitnya embriogenesis, yang diduga melibatkan satu atau lebih gen kunci yang menentukan proses tersebut. Keberhasilan mengidentifikasi gen-gen kunci akan membantu menyelesaikan masalah dalam regenerasi embrio kakao. Salah satu gen yang diduga terlibat dalam proses ini adalah AGAMOUS-like 15 (AGL-15). Gen ini berperan pada regulasi selama masa awal perkembangan embrio beberapa tanaman. Penelitian ini bertujuan untuk mengidentifikasi homolog AGL-15 pada kakao melalui pen-dekatan bioinformatika dan RT-PCR. Pene-litian diawali dengan identifikasi homolog AGL-15 dari DNA genomik daun kakao meng-gunakan primer heterologus. Sekuen fragmen homolog AGL-15 yang diperoleh, kemudian digunakan untuk merancang primer spesifik AGL-15 yang berukuran lebih panjang. Primer ini selanjutnya digunakan untuk meng-identifikasi gen AGL-15 dari RNA total embrio zigotik. Pengamatan pola pita gen AGL-15 dilakukan pada kultur in vitro embrio zigotik yang berumur lima minggu. Primer hetero-logus gen AGL-15 yang berasal dari berbagai tanaman, mampu mengidentifikasi keberadaan homolog gen tersebut pada tanaman kakao. Fragmen homolog AGL-15 putatif tanaman kakao teridentifikasi pada tingkat RNA embrio. Dengan adanya pola diferensial dari ekspresi gen AGL-15 hingga lima minggu pertama perkembangan embrio, ada indikasi kuat bahwa fragmen homolog AGL-15 dapat menjadi penanda embriogenesis pada tanaman kakao.

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Journal articles on the topic 'Heterologe Expression'

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