Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig–human xenotransplantation

2010 ◽  
Vol 427 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Kwon-Ho Song ◽  
Yun-Jeong Kang ◽  
Un-Ho Jin ◽  
Yong-Il Park ◽  
Sung-Min Kim ◽  
...  
Keyword(s):  
Small Intestine ◽  
Functional Characterization ◽  
Ectopic Expression ◽  
Vascular Endothelial ◽  
Reading Frame ◽  
Transfected Cells ◽  
Acetylneuraminic Acid ◽  
Rejection Response ◽  
Key Enzyme ◽  
Human Xenotransplantation

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig–human xenotransplantation.

scholarly journals Identification and functional characterization of a spliced rhesus rhadinovirus gene with homology to the K15 gene of Kaposi's sarcoma-associated herpesvirus

2009 ◽  
Vol 90 (5) ◽  
pp. 1190-1201 ◽  
Author(s):  
Linding Wang ◽  
Marcel Pietrek ◽  
Melanie M. Brinkmann ◽  
Anika Hävemeier ◽  
Irina Fischer ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Functional Characterization ◽  
Ectopic Expression ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Reading Frame ◽  
Protein Isoform ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
The Right ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus related to the human Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8). This study identified an alternatively spliced gene at the right side of the RRV genome (strain 17577) between open reading frame 75 and the terminal repeat region. Of its eight exons, the first seven encoded up to 12 transmembrane domains, whilst the eighth exon encoded a predicted C-terminal cytoplasmic domain. Structurally and positionally, this RRV gene therefore resembles the K15 gene of KSHV; it was provisionally named RK15 to avoid confusion with other RRV17577 genes. In ectopic expression studies, the 55 kDa RK15 protein isoform activated the JNK and NF-κB pathways, like the 45 kDa KSHV K15-encoded protein isoform. In contrast to K15, which activates angiogenic and inflammatory cytokines such as interleukin (IL)-8, IL-6 and CCL20, the range of cellular transcripts activated by the RRV K15 homologue was much more restricted, but included IL-6, IL-8 and FGF21. These data suggest functional differences between terminal membrane proteins at the right end of the genomes of Old World primate gamma-2 herpesviruses.

scholarly journals Overexpression of Trypanosoma rangeli trypanothione reductase increases parasite survival under oxidative stress

2016 ◽  
Vol 2 ◽  
Author(s):  
I.T. BELTRAME-BOTELHO ◽  
P.H. STOCO ◽  
M. STEINDEL ◽  
B. ANDERSSON ◽  
E.F. PELOSO ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Functional Characterization ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Single Copy ◽  
Trypanothione Reductase ◽  
Trypanosoma Rangeli ◽  
Reading Frame ◽  
Key Enzyme

SUMMARYThe infectivity and virulence of pathogenic trypanosomatids are directly associated with the efficacy of their antioxidant system. Among the molecules involved in the trypanosomatid response to reactive oxygen or nitrogen species, trypanothione reductase (TRed) is a key enzyme. In this study, we performed a molecular and functional characterization of the TRed enzyme fromTrypanosoma rangeli(TrTRed), an avirulent trypanosome of mammals. TheTrTRed gene has an open reading frame (ORF) of 1473 bp (~490 aa, 53 kDa) and occurs as a single-copy gene in the haploid genome. The predicted protein contains two oxidoreductase domains, which are equally expressed in the cytosol of epimastigotes and trypomastigotes. Nicotinamide adenine dinucleotide phosphate (NADPH) generation is reduced and endogenous H2O2production is elevated inT. rangeliChoachí strain compared withT. cruziY strain epimastigotes. Oxidative stress induced by H2O2does not induce significant alterations inTrTRed expression. Overexpression ofTrTRed did not influencein vitrogrowth or differentiation into trypomastigotes, but mutant parasites showed increased resistance to H2O2-induced stress. Our results indicate thatT. rangeliconstitutively expresses TRed during the entire life cycle, with reduced levels during infective and non-replicative trypomastigote stages.

scholarly journals A Cucumber AGAMOUS-LIKE 15 (AGL15) MADS-Box Gene Mediates Abnormal Leaf Morphology in Arabidopsis

Agronomy ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 265
Author(s):  
Yong Zhou ◽  
Lingli Ge ◽  
Lifang Hu ◽  
Yingui Yang ◽  
Shiqiang Liu
Keyword(s):  
Functional Characterization ◽  
Ectopic Expression ◽  
Leaf Size ◽  
Reproductive Organs ◽  
Mads Box ◽  
Cucumis Sativus L ◽  
Reading Frame ◽  
Relationship Analysis ◽  
Mads Box Gene

The AGL15 subfamily MADS-box proteins play vital roles in various developmental processes, such as floral transition, somatic embryogenesis, and leaf and fruit development. In this work, an AtAGL15 ortholog, CsMADS26, was cloned from cucumber (Cucumis sativus L.). The open reading frame (ORF) of CsMADS26 is 669 bp in length, encoding a predicted protein of 222 amino acids. The CsMADS26 protein contains a highly conserved MADS-box domain and a variable C domain, as well as less conserved I and K domains. Phylogenetic relationship analysis revealed that CsMADS26 was clustered into the AGL15 clade of AGL15 subfamily. Expression analysis based on qRT-PCR showed that CsMADS26 is mainly expressed in reproductive organs including flowers and fruits. Transgenic Arabidopsis plants with ectopic expression of CsMADS26 exhibited curled rosette and cauline leaves, and the leaf size was much smaller than that of wild-type (WT) plants. These results provide clues for the functional characterization of CsMADS26 in the future.

scholarly journals Expression of Arabidopsis Ornithine Aminotransferase (AtOAT) Encoded Gene Enhances Multiple Abiotic Stress Tolerances in Wheat

2021 ◽  
Author(s):  
Alia Anwar ◽  
Ke Wang ◽  
Jing Wang ◽  
Lei Shi ◽  
Lipu Du ◽  
...  
Keyword(s):  
Heat Stress ◽  
Functional Characterization ◽  
Ectopic Expression ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Ornithine Aminotransferase ◽  
Proline Biosynthesis ◽  
Biotic Stresses ◽  
Proline Catabolism ◽  
Key Enzyme

Abstract Key Message The drought and salt tolerances of wheat were enhanced by ectopic expression of the Arabidopsis ornithine aminotransferase (AtOAT) encoded gene. The OAT was confirmed to play a role in proline biosynthesis in wheat.Abstract Proline (Pro) accumulation is a common response to both abiotic and biotic stresses in plants. Ornithine aminotransferase (OAT) is pyridoxal-5-phosphate dependent enzyme involved in plant proline biosynthesis. During stress condition, proline is synthesized via glutamate and ornithine pathways. The OAT is the key enzyme in ornithine pathway. In this study, an OAT gene AtOAT from Arabidopsis was expressed in wheat for its functional characterization under drought, salinity and heat stress conditions. We found that the expression of AtOAT enhanced the drought and salt stress tolerances of wheat by increasing the proline content and peroxidase activity. In addition, it was confirmed that the expression of AtOAT also played a partial tolerance to heat stress in the transgenic wheat plants. Moreover, quantitative real-time PCR (qRT-PCR) analysis showed that the transformation of AtOAT up-regulated the expression of the proline biosynthesis associated genes TaOAT, TaP5CS, and TaP5CR, and down-regulated that of the proline catabolism related gene TaP5CDH in the transgenic plants under stress conditions. Moreover, the genes involved in ornithine pathway (Orn-OAT-P5C/GSA-P5CR-Pro) were up-regulated along with the up-regulation of those genes involved in glutamate pathway (Glu-P5CS-P5C/GSA-P5CR-Pro). Therefore, we concluded that the expression of AtOAT enhanced wheat abiotic tolerance via modifying the proline biosynthesis by up-regulating the expression of the proline biosynthesis associated genes and down-regulating that of the proline catabolic gene under stresses condition.

Molecular cloning and functional characterization of CvLCYE, a key enzyme in lutein synthesis pathway in Chlorella vulgaris

2021 ◽  
Vol 55 ◽  
pp. 102246
Author(s):  
Sulin Lou ◽  
Xin Lin ◽  
Chenglong Liu ◽  
Muhammad Anwar ◽  
Hui Li ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Chlorella Vulgaris ◽  
Functional Characterization ◽  
Key Enzyme

scholarly journals Cloning, Characterization, and RNA Interference Effect of the UDP-N-Acetylglucosamine Pyrophosphorylase Gene in Cnaphalocrocis medinalis

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 464
Author(s):  
Yuan-Jin Zhou ◽  
Juan Du ◽  
Shang-Wei Li ◽  
Muhammad Shakeel ◽  
Jia-Jing Li ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Developmental Stages ◽  
Phylogenetic Analyses ◽  
Digital Pcr ◽  
Cnaphalocrocis Medinalis ◽  
Chitin Synthesis ◽  
Reading Frame ◽  
Rnai Technology ◽  
Glyphodes Pyloalis ◽  
Key Enzyme

The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect.

scholarly journals Perforation of the Small Intestine after Introduction of Lenvatinib in a Patient with Advanced Hepatocellular Carcinoma

2020 ◽  
Vol 14 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Naomi Suzuki ◽  
Kazuto Tajiri ◽  
Yuka Futsukaichi ◽  
Shinichi Tanaka ◽  
Aiko Murayama ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Small Intestine ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Vegf Receptor ◽  
Advanced Hepatocellular Carcinoma ◽  
Vascular Endothelial ◽  
First Line ◽  
Endothelial Growth Factor ◽  
Line Standard

Lenvatinib is a first-line standard treatment for advanced hepatocellular carcinoma (HCC) with better anti-tumor effects than sorafenib, as shown by greater inhibition of the kinases of fibroblast growth factor receptor and vascular endothelial growth factor (VEGF) receptor. This report describes a patient with advanced HCC who experienced perforation of the small intestine 1 month after starting the treatment with lenvatinib. This patient likely had partial necrosis of a metastasis to the small intestine before starting lenvatinib treatment, with subsequent ischemic changes leading to perforation of the small intestine. Although metastasis of HCC to the small intestine is rare, patients with these metastases should be regarded as being at risk for perforation during lenvatinib treatment.

scholarly journals Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis

Structure ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Michele A McTigue ◽  
John A Wickersham ◽  
Chris Pinko ◽  
Richard E Showalter ◽  
Camran V Parast ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Vascular Endothelial ◽  
Key Enzyme ◽  
Endothelial Growth Factor

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5

1993 ◽  
Vol 264 (6) ◽  
pp. G1169-G1176 ◽  
Author(s):  
E. B. Rand ◽  
A. M. Depaoli ◽  
N. O. Davidson ◽  
G. I. Bell ◽  
C. F. Burant
Keyword(s):  
Small Intestine ◽  
Functional Characterization ◽  
Cdna Probe ◽  
Amino Acid Identity ◽  
Human Testis ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Rat Small Intestine ◽  
Cross Hybridization ◽  
Distal Small Intestine

cDNA clones encoding rat GLUT5-small intestinal facilitative hexose transporter were isolated from a jejunum library by cross-hybridization with a human GLUT5 cDNA probe. The cDNA sequence indicates that rat GLUT5 is composed of 502 amino acids and has 81.5% amino acid identity and 87.3% similarity with the sequence of human GLUT5. Expression of synthetic rat GLUT5 mRNA in Xenopus oocytes showed that rat GLUT5 was able to mediate the uptake of fructose and, to a lesser extent, of glucose. RNA blotting studies showed that GLUT5 mRNA was present in rat small intestine, kidney, and brain. Although GLUT5 mRNA is expressed in human testis, adipose tissue, and skeletal muscle, it could not be detected by RNA blotting in these rat tissues. Developmental studies showed low levels of GLUT5 mRNA in rat small intestine and kidney during the prenatal period with a rapid induction of GLUT5 expression occurring postnatally. In situ hybridization studies of GLUT5 mRNA expression in the small intestine revealed differential expression along the crypt-villus axis with the highest levels of mRNA being in the midvillus region. In addition, there was quantitatively more GLUT5 mRNA detected in the proximal as opposed to the distal small intestine.

Sign in / Sign up

Export Citation Format

Share Document