scholarly journals Molecular Characterization and Nutrition Regulation of the Neutral Amino Acid Transporter ASCT2 in Triploid Crucian Carp

Fishes ◽  
2021 ◽  
Vol 6 (4) ◽  
pp. 77
Author(s):  
Zhuangwen Mao ◽  
Shengwei Luo ◽  
Dafang Zhao ◽  
Xiang Zhou ◽  
Zilong Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Crucian Carp ◽  
Stop Codon ◽  
Expression Level ◽  
Amino Acid Residues ◽  
Expression Levels ◽  
Feed Formulation ◽  
Neutral Amino Acid Transporter ◽  
High Sequence Homology

AlaSerCys Transporter 2 (ASCT2), encoded by the SLC1A5 gene, plays an important role in the absorption of glutamine. In this study, the full-length cDNA sequence of ASCT2 was cloned from triploid crucian carp. It encodes 539 amino acid residues and a stop codon. Phylogenetic analysis revealed that the sequences of the ASCT2 ORF region in cyprinid fishes shared high sequence homology. Comparing the abundance of ASCT2 in different tissues, we found its expression level in muscle was significantly higher than that in intestine (p < 0.05). In addition, the expression levels of ASCT2 also appeared different in diurnal variation. Then we found the addition of 2.5% glutamate in a feeding diet significantly increased the expression levels of ASCT2 in intestine and muscle (p < 0.05). However, in glutamine experiments, the muscle showed the highest expression level of ASCT2 when fish were fed the diet containing 3.0% glutamine (p < 0.05). In vitro, ASCT2 was sensitive to glutamine and its expression level appeared down-regulated when the addition of glutamine was added to 0.1 mg/mL. Finally, we found that the diet with 29% protein level significantly increased the expression level of ASCT2 in intestine (p < 0.05). Nevertheless, different protein sources (fish meal and soybean meal) had no significant effect on the expression levels of ASCT2 in intestine and muscle (p > 0.05). These results provided data for the study of ASCT2 in triploid crucian carp regulated by feeding nutrition, which had a potential application in improving feed formulation in aquaculture.

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 A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans

2021 ◽  
Author(s):  
Barbara Vona ◽  
Neda Mazaheri ◽  
Sheng-Jia Lin ◽  
Lucy A. Dunbar ◽  
Reza Maroofian ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Amino Acid ◽  
Rna Splicing ◽  
Stop Codon ◽  
Missense Variant ◽  
Homozygosity Mapping ◽  
Deafness Gene ◽  
Expression Studies ◽  
Hearing Function

AbstractDeafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly in vitro and rich in methionine

Biologia ◽  
2007 ◽  
Vol 62 (4) ◽  
Author(s):  
Reda Sammour
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Seed Quality ◽  
Amino Acid Residues ◽  
Methionine Residues

AbstractThe main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.

scholarly journals In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4147
Author(s):  
Neha Gupta ◽  
Saurav Kumar Choudhary ◽  
Neeta Bhagat ◽  
Muthusamy Karthikeyan ◽  
Archana Chaturvedi
Keyword(s):  
Molecular Docking ◽  
Amino Acid ◽  
Guanylyl Cyclase ◽  
Extracellular Domain ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Guanylyl Cyclase C ◽  
Amino Acid Residues ◽  
Lead Compounds

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

scholarly journals Identification of NAD+ Synthetase from Streptococcus sobrinus as a B-Cell-Stimulatory Protein

2004 ◽  
Vol 186 (2) ◽  
pp. 419-426 ◽  
Author(s):  
Isabel Veiga-Malta ◽  
Margarida Duarte ◽  
Márcia Dinis ◽  
Pedro Madureira ◽  
Paula Ferreira ◽  
...  
Keyword(s):  
B Cell ◽  
Active Form ◽  
Lymphocyte Activation ◽  
Amino Acid Residues ◽  
Streptococcus Sobrinus ◽  
Reading Frame ◽  
High Sequence Homology ◽  
Culture Supernatants ◽  
Nad Synthetase

ABSTRACT Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD+ synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD+ synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD+ synthetase was also observed with other B-cell markers, such as CD19+, B220+, and CD21+. Cell proliferation follows the activation induced by the recombinant NAD+ synthetase.

scholarly journals Physical and Functional Interaction of the HECT Ubiquitin-protein Ligases E6AP and HERC2

2011 ◽  
Vol 286 (22) ◽  
pp. 19410-19416 ◽  
Author(s):  
Simone Kühnle ◽  
Ulrike Kogel ◽  
Sandra Glockzin ◽  
Andreas Marquardt ◽  
Aaron Ciechanover ◽  
...  
Keyword(s):  
Amino Acid ◽  
Angelman Syndrome ◽  
Amino Acid Residues ◽  
Cervical Carcinogenesis ◽  
Tight Control ◽  
Functional Interaction ◽  
Ubiquitin Protein Ligase ◽  
Protein Ligases ◽  
Translational Level

Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150–200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.

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