scholarly journals Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase. Regulation of its transcript by interferons in vivo

1992 ◽  
Vol 283 (3) ◽  
pp. 863-870 ◽  
Author(s):  
M Terao ◽  
G Cazzaniga ◽  
P Ghezzi ◽  
M Bianchi ◽  
F Falciani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mouse Liver ◽  
De Novo ◽  
Xanthine Dehydrogenase ◽  
Amino Acid Residues ◽  
Cross Hybridization ◽  
Reading Frame ◽  
Ifn Alpha ◽  
High Conservation

The cDNA coding for xanthine dehydrogenase (XD) is isolated from mouse liver mRNA by cross-hybridization with a DNA fragment of the Drosophila melanogaster homologue. Two lambda bacteriophage overlapping clones represent the copy of a 4538-nucleotide-residue-long transcript with an open reading frame of 4005 nucleotide residues, coding for a putative polypeptide of 1335 amino acid residues. Comparison of the deduced amino acid sequence of the mouse XD with those of the Drosophila and the rat homologues shows a high conservation of this protein (55% identity between mouse and Drosophila, and 94% identity between mouse and rat). RNA blotting analysis demonstrates that interferon-alpha (IFN-alpha) and its inducers, i.e. poly(I).poly(C), bacterial lipopolysaccharide (LPS) and tilorone (2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one), increase the expression of XD mRNA in liver. Poly(I).poly(C) also induces XD mRNA in several other tissues in vivo. Protein synthesis de novo is not required for the elevation of XD mRNA after IFN-alpha treatment, since cycloheximide does not block the induction. The elevation of XD mRNA concentration is relatively fast and precedes the induction of both XD and xanthine oxidase (XO) enzymic activities.

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

scholarly journals The Acinetobacter baylyi hfq Gene Encodes a Large Protein with an Unusual C Terminus

2009 ◽  
Vol 191 (17) ◽  
pp. 5553-5562 ◽  
Author(s):  
Dominik Schilling ◽  
Ulrike Gerischer
Keyword(s):  
Amino Acid ◽  
Gene Localization ◽  
Amino Acid Residues ◽  
Acinetobacter Baylyi ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus ◽  
The Difference ◽  
Cell Phenotypes

ABSTRACT In gammaproteobacteria the Hfq protein shows a great variation in size, especially in its C-terminal part. Extremely large Hfq proteins consisting of almost 200 amino acid residues and more are found within the gammaproteobacterial family Moraxellaceae. The difference in size compared to other Hfq proteins is due to a glycine-rich domain near the C-terminal end of the protein. Acinetobacter baylyi, a nonpathogenic soil bacterium and member of the Moraxellaceae encodes a large 174-amino-acid Hfq homologue containing the unique and repetitive amino acid pattern GGGFGGQ within the glycine-rich domain. Despite the presence of the C-terminal extension, A. baylyi Hfq complemented an Escherichia coli hfq mutant in vivo. By using polyclonal anti-Hfq antibodies, we detected the large A. baylyi Hfq that corresponds to its annotated size indicating the expression and stability of the full protein. Deletion of the complete A. baylyi hfq open reading frame resulted in severe reduction of growth. In addition, a deletion or overexpression of Hfq was accompanied by the loss of cell chain assembly. The glycine-rich domain was not responsible for growth and cell phenotypes. hfq gene localization in A. baylyi is strictly conserved within the mutL-miaA-hfq operon, and we show that hfq expression starts within the preceding miaA gene or further upstream.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Design and Synthesis of Pyrazole-3-one Derivatives as Hypoglycaemic Agents

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Prasanna A. Datar ◽  
Sonali R. Jadhav
Keyword(s):  
Amino Acid ◽  
Docking Studies ◽  
Diabetic Rats ◽  
Hypoglycemic Activity ◽  
Amino Acid Residues ◽  
Docking Score ◽  
Design And Synthesis ◽  
Hypoglycaemic Agents ◽  
Standard Compound

Pyrazole-3-one compounds were designed on the basis of docking studies of previously reported antidiabetic pyrazole compounds. The amino acid residues found during docking studies were used as guidelines for the modification of aromatic substitutions on pyrazole-3-one structure. Depending on the docking score, the designed compounds were selectively prioritized for synthesis. The synthesized compounds were subjected to in vivo hypoglycemic activity using alloxan induced diabetic rats and metformin as a standard. Compound 4 having sulphonamide derivative was found to be the most potent compound among the series.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation

1985 ◽  
Vol 5 (10) ◽  
pp. 2684-2696
Author(s):  
D H Smith ◽  
D M Kegler ◽  
E B Ziff
Keyword(s):  
Amino Acid ◽  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Protein ◽  
Dna Replication Origin ◽  
E1a Protein

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

scholarly journals Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Jonathan H. M. van der Meer ◽  
Ruben J. de Boer ◽  
Bartolomeus J. Meijer ◽  
Wouter L. Smit ◽  
Jacqueline L. M. Vermeulen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Intestinal Epithelium ◽  
De Novo ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Argininosuccinate Synthetase ◽  
Important Amino Acid ◽  
Tissue Specimens ◽  
Colorectal Cancer Patients

AbstractThe epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

scholarly journals Identification of antigenic epitopes for Guertu virus nucleocapsid protein

2021 ◽  
Author(s):  
Siyuan Wang ◽  
Xihong Yue ◽  
Alai Shalitanati ◽  
Abulimiti Moming ◽  
Shu Shen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epitope Mapping ◽  
Virus Detection ◽  
Polyclonal Antiserum ◽  
Detection Methods ◽  
Amino Acid Residues ◽  
High Conservation ◽  
Potential Pathogenicity ◽  
Severe Fever ◽  
Antigenic Epitopes

Abstract Guertu virus (GTV), a novel tick-borne virus with potential pathogenicity, was first isolated from Dermacentor nuttalli in Xinjiang, China, in 2014. GTV has been shown to infect animal and human cell lines and to be pathogenic in mice. The viral nucleoprotein (NP) is the most conserved immunogenic protein. Elucidating the B-cell epitopes (BCEs) in the immunodominant region of the NP is important for the development of virus detection methods and vaccines. In order to identify the minimal motifs of linear BCEs in the NP of the GTV DXM strain, we used an improved biosynthetic peptide (BSP) method to truncate GTV NP into 30 16mer-peptides with 8 overlapping amino acid residues spanning the full length of the protein. The peptides were analyzed by western blot using rabbit anti-GTV NP polyclonal antiserum, and four positive 16mer-peptides were obtained. The 16mer-peptides were then truncated into 31 8mer-peptides with 7 overlapping amino acid residues and 10mer-peptides with 9 overlapping amino acid residues to screen for BCEs that can react with the rabbit anti-GTV NP polyclonal antiserum. The results showed that there were 6 minimal BCE motifs, namely, Enp1, 88EKYGLVER95; Enp2, 88EKYGLVER95; Enp3, 162TTKILMEA169; Enp4, 187GASKAEVY194; Enp5, 191AEVYNSFR198; and Enp6, 236ETAAAAYRNL245. Positive sheep sera could recognize all six BCEs with anti-GTV antibodies. The BCEs were aligned with the sequences of eight representative severe fever with thrombocytopenia syndrome phlebovirus strains from different countries and regions that were evolutionarily closely related to GTV. The sequence identity of the BCEs ranged from 80–100%, thus showing high conservation. The fine epitope mapping of GTV NP can be used to explore the biological and immunological properties of GTV NP antigens and serve as basic data for the development of multi-epitope detection reagents and vaccine design for GTV.

scholarly journals In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*

Endocrinology ◽  
1997 ◽  
Vol 138 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Patricia Grasso ◽  
Matthew C. Leinung ◽  
Stacy P. Ingher ◽  
Daniel W. Lee
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Amino Acid ◽  
Food Intake ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Residues ◽  
Inactive Form ◽  
In Vivo Effects

Abstract In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

scholarly journals Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

1995 ◽  
Vol 15 (10) ◽  
pp. 5329-5338 ◽  
Author(s):  
K Onel ◽  
M P Thelen ◽  
D O Ferguson ◽  
R L Bennett ◽  
W K Holloman
Keyword(s):  
Amino Acid ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Ustilago Maydis ◽  
Radiation Sensitivity ◽  
Open Reading Frame ◽  
Exonuclease Activity ◽  
Amino Acid Residues ◽  
Spontaneous Mutation Rate ◽  
Reading Frame

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

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