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Results for “Functional characterization”

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PubMed1999

Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro.

beta-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation beta-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of beta-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by beta-catenin and the molecular basis for functional differences between beta-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of beta-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with beta-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of beta-catenin interact specifically and directly with the TATA-binding protein in vitro providing further evidence that a major function of beta-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes.

Animals↗

PubMed2026

Whole genome sequencing analysis and functional characterization of Lacticaseibacillus rhamnosus HP-B1083.

Lacticaseibacillus rhamnosus is an important strain for the biotransformation of natural products, and its crude extract exhibits biotransformation effect on glycosidic compounds such as baicalin. To further explore the potential of this strain, particularly given its previously demonstrated high-efficiency β-glucuronidase activity for baicalin conversion, whole-genome sequencing and functional annotation of Lacticaseibacillus rhamnosus HP-B1083 were performed in this study, and its acid tolerance, bile salt tolerance, short-term heat resistance and antibacterial activity were evaluated. The results showed that the strain possessed a circular chromosome with a full length of 3,090,505 bp and a GC content of 46.69%. Gene annotation revealed that the genome contained 2941 coding sequences (CDS) and 112 non-coding RNA genes, including 60 tRNA genes, 1 tmRNA gene, 36 misc_RNA genes and 15 rRNA genes. The functional annotations further reveal that this genome is rich in genes related to carbohydrate metabolism, hydrolases, and transferases, which is highly consistent with its phenotypic characteristics in glycoside transformation and the synthesis of antibacterial substances. In addition, acid tolerance, bile salt tolerance and short-term heat resistance experiments verified that HP-B1083 had acid resistance, bile salt resistance and short-term heat resistance. Antibacterial activity tests confirmed that HP-B1083 produced inhibition zone diameters over 10 mm against common foodborne pathogenic bacteria such as Escherichia coli and Bacillus cereus. Therefore, Lacticaseibacillus rhamnosus HP-B1083 has important application prospects in the development of functional foods, preparation of enzyme preparations and pharmaceutical industry.

Whole Genome Sequencing↗

PubMed1996

The extracellular matrix molecule tenascin-C: expression in vivo and functional characterization in vitro.

Tenascin-C is a member of a small family of closely related extracellular matrix molecules. The protein is highly conserved during evolution and is expressed in a variety of tissues. In the nervous systems of rodents and chickens, tenascin-C is predominantly expressed at early developmental ages and has been hypothesized to be functionally involved in different steps of neural development. For example, tenascin-C has been implicated in synaptogenesis, migration of different neural cell types, axonal growth in the developing and lesioned nervous system, and in the formation and maintenance of discrete anatomical boundaries. Experiments with monoclonal anti-tenascin-C antibodies or recombinantly expressed tenascin-C fragments have localized different functions of tenascin-C to different domains of the protein. Functional diversity of tenascin-C in vitro might additionally relate to a cell type-specific expression of different receptor(s) and/or to differences in intracellular signal transduction pathways. Surprisingly, mutant mice reported to completely lack expression of tenascin-C develop normally and reveal no detectable phenotype, suggesting that tenascin-C does not play a critical role in the intact organism. However, residual expression of an abnormal fragment of the tenascin-C protein has recently been detected in this mutant.

Animals↗

PubMed2026

Phenotypic, physiological and transcriptomic analysis of graded salt stress responses in Pyrus betulifolia Bunge and functional characterization of the hub gene PbSTY46.

Pyrus betulifolia Bunge is a salt‑tolerant rootstock for pear, but its salt‑tolerance mechanisms remain largely unknown. In this study, P. betulifolia seedlings were subjected to graded NaCl stress at concentrations of 0 (CK), 50 (T1), 100 (T2), and 200 (T3) mM. We integrated phenotypic observation, physiological assessment, transcriptomic profiling, and functional gene validation to systematically elucidate its salt tolerance mechanisms. Salt stress inhibited seedling growth and root traits in a concentration-dependent manner, and T3 caused the most severe damage. Osmotic solutes responded differentially: soluble sugars peaked under T2, while proline peaked under T3. Antioxidant enzymes showed tissue-specific biphasic responses and declined after prolonged T3 stress. Meanwhile, chlorophyll and photosynthesis decreased, whereas anthocyanin increased, indicating a metabolic shift from photosynthesis to photoprotection. Transcriptome analysis revealed distinct responses depending on stress intensity: mild stress induced membrane lipid remodeling, moderate stress activated circadian rhythm and hormone signaling, and severe stress enhanced phenylpropanoid biosynthesis and thiamine metabolism. Gene Set Enrichment Analysis (GSEA) further highlighted progressive enrichment of phenylpropanoid biosynthesis, heme binding, and oxidoreductase activity. Weighted Gene Co‑expression Network Analysis (WGCNA) identified a blue module significantly positively correlated with root traits, from which the hub gene PbSTY46 was identified. Functional validation via overexpression, loss‑of‑function mutants, and pharmacological interventions (MeJA/DIECA) confirmed that PbSTY46 acts through JA signaling to enhance antioxidant enzyme activities and thereby confer salt tolerance. Collectively, P. betulifolia adopts a "survival‑first" strategy that coordinates growth arrest, osmotic homeostasis, and ROS scavenging. These findings establish PbSTY46 as a key regulator that links JA signaling to antioxidant defense. Thus, PbSTY46 represents a promising candidate for marker‑assisted breeding of salt‑tolerant pear cultivars.

Salt Stress↗

PubMed2006

From yeast ammonium transporters to Rhesus proteins, isolation and functional characterization.

Saccharomyces cerevisiae possesses three ammonium transporters from the Mep/Amt family involved in ammonium acquisition and retention. We have shown that Rh proteins are structurally related to Mep/Amt proteins and that human RhAG and RhCG perform bi-directional ammonium transport upon heterologous expression in yeast. Using yeast as an expression tool, we have started a structure-function analysis of distinct members from the Mep/Amt/Rh super-family.

Animals↗

PubMed2005

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.

ATP-Binding Cassette Transporters↗

PubMed1999

Molecular cloning and functional characterization of a snake venom metalloprotease.

A cDNA clone, MT-d, encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library. MT-d-I protein containing both metalloprotease and disintegrin domains, and MT-d-II protein containing the metalloprotease domain only were expressed in Escherichia coli and refolded successfully into their functional forms. Each of the refolded enzyme species exhibited distinct substrate specificity. Proteolytic activity of the MT-d-1 was able to hydrolyse type I gelatin, type-III and V collagens in contrast with the catalytic function of MT-d-II. MT-d-I protein having metalloprotease activity was also able to inhibit platelet aggregation. Functionally active MT-d-I protein underwent autoproteolytic processing in vitro to produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity. Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins.

Amino Acid Sequence↗

PubMed1992

Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.

Somatostatin is a tetradecapeptide that is widely distributed in the body. It acts on multiple organs including brain, pituitary, gut, exocrine and endocrine pancreas, adrenals, thyroid, and kidneys to inhibit release of many hormones and other secretory proteins. In addition, it functions as a neuropeptide affecting the electrical activity of neurons. Somatostatin exerts its biological effects by binding to specific high-affinity receptors, which appear in many cases to be coupled to GTP-binding proteins. Here we report the cloning, functional expression, and tissue distribution of two different somatostatin receptors (SSTRs). SSTR1 and SSTR2 contain 391 and 369 amino acids, respectively, and are members of the superfamily of receptors having seven transmembrane segments. There is 46% identity and 70% similarity between the amino acid sequences of SSTR1 and SSTR2. Stably transfected Chinese hamster ovary cells expressing SSTR1 or SSTR2 exhibit specific somatostatin binding, with an apparently higher affinity for somatostatin-14 than somatostatin-28, and NH2-terminally extended form of somatostatin-14. RNA blotting studies show that SSTR1 and SSTR2 are expressed at highest levels in jejunum and stomach and in cerebrum and kidney, respectively. A SSTR1 probe hybridized to multiple DNA fragments in EcoRI digests of human and mouse DNA, indicating that SSTR1 and SSTR2 are members of a larger family of somatostatin receptors. Thus, the biological effects of somatostatin are mediated by a family of receptors that are expressed in a tissue-specific manner.

Amino Acid Sequence↗

PubMed2004

Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa.

The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp. NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.

Bacterial Proteins↗

PubMed1985

Isolation and functional characterization of crustacean larval salt gland.

A batch method for isolating viable salt glands from the naupliar brine shrimp (Artemia salina) has been developed. This protocol produces a final preparation consisting of approximately 185 isolated salt glands, representing 1 X 10(4) secretory cells/g wet wt nauplii, with a final purity of 88%. Assays of cell integrity and function indicate good retention of in situ characteristics. Vital dye was excluded by 95% of the cells for at least 24 h. The O2 consumption rate was 22.7 nM O2 X min-1 X mg protein-1 and could be altered predictably by compounds known to affect oxidative phosphorylation and ion transport. The specific activity of the Na+-K+-ATPase in the salt gland, measured here for the first time, was 9.1 mM Pi X h-1 X mg protein-1. This is a substantial proportion of the body total, 17%, as expected for an active ion-transporting epithelium.

Animals↗

PubMed1986

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum.

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Molecular and functional characterization of recombinant human gamma-glutamyltransferase. Coupling of its activity to glutathione levels in V79 cells.

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.

Amino Acid Sequence↗

PubMed2006

Functional characterization of Aspergillus nidulans homologues of Saccharomyces cerevisiae Spa2 and Bud6.

The importance of polarized growth for fungi has elicited significant effort directed at better understanding underlying mechanisms of polarization, with a focus on yeast systems. At sites of tip growth, multiple protein complexes assemble and coordinate to ensure that incoming building material reaches the appropriate destination sites, and polarized growth is maintained. One of these complexes is the polarisome that consists of Spa2, Bud6, Pea2, and Bni1 in Saccharomyces cerevisiae. Filamentous hyphae differ in their development and life style from yeasts and likely regulate polarized growth in a different way. This is expected to reflect on the composition and presence of protein complexes that assemble at the hyphal tip. In this study we searched for polarisome homologues in the model filamentous fungus Aspergillus nidulans and characterized the S. cerevisiae Spa2 and Bud6 homologues, SpaA and BudA. Compared to the S. cerevisiae Spa2, SpaA lacks domain II but has three additional domains that are conserved within filamentous fungi. Gene replacement strains and localization studies show that SpaA functions exclusively at the hyphal tip, while BudA functions at sites of septum formation and possibly at hyphal tips. We show that SpaA is not required for the assembly or maintenance of the Spitzenkörper. We propose that the core function of the polarisome in polarized growth is maintained but with different contributions of polarisome components to the process.

Amino Acid Sequence↗

PubMed2001

Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status.

The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other P(II) homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

ADP Ribose Transferases↗

PubMed1988

Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA.

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.

Animals↗

PubMed1996

Expression of rat liver tryptophan 2,3-dioxygenase in Escherichia coli: structural and functional characterization of the purified enzyme.

The hepatic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the key regulatory enzyme that, through irreversible degradation, controls the flux of tryptophan through physiologically relevant pathways. This enzyme is composed of four identical subunits and in its fully assembled tetrameric form requires 2 mol of heme (Fe(+2)-protoporphyrin IX)/mol of protein for functional competence. Using a full-length cDNA for the rat liver TDO subunit (pUC119/TDO) as the template, TDO cDNA was amplified by polymerase chain reaction (PCR) and incorporated into the expression vector pTrc99A after introduction of convenient restriction sites as well as modification of the second codon AGT to GCT to optimize its bacterial expression. DH5 alpha F' strain Escherichia coli cells transfected with this pTrc99A/TDO construct expressed soluble, functionally active, tetrameric TDO protein in high yields. The enzyme was isolated from 30,000g supernatant of cell lysates, purified by ion-exchange chromatography, and its spectral and catalytic properties were assessed in terms of its substrate and prosthetic moiety specificities. In almost all aspects, the bacterially expressed enzyme was found to be identical to that of the rat liver. Heterologous expression of the fully functional enzyme, we trust, will enable future elucidation of its structure-function relationships.

Aminolevulinic Acid↗

PubMed2005

Molecular cloning and functional characterization of zebrafish ATM.

Ataxia-telangiectasia mutated (ATM) is the gene product mutated in ataxia-telangiectasia (A-T), which is an autosomal recessive disorder with symptoms including neurodegeneration, cancer predisposition and premature aging. ATM is thought to play a pivotal role in signal transduction in response to genotoxic DNA damage. To study the physiological and developmental functions of ATM using the zebrafish model system, we cloned the zebrafish homolog cDNA of human ATM (hATM), zebrafish ATM (zATM), analyzed the expression pattern of zATM during early development, and further developed the system to study loss of zATM function in zebrafish embryos. Employing information available from the zebrafish genomic database, we utilized a PCR-based approach to isolate zATM cDNA clones. Sequence analysis of zATM showed a high level homology in the functional domains of hATM. The putative FAT, phosphoinositide 3-kinase-like, and FATC domains of zATM, which regulate ATM kinase activity and functions, were the most highly conserved regions, exhibiting 64-94% amino acid identity to the corresponding domains in hATM, while exhibiting approximately 50% amino acid identity outside these domains. The zATM gene is expected to consist of 62 coding exons, and we have identified at least 55 exons encompassing more than 100kb of nucleotide sequence, which encodes about 9 kb of cDNA. By in situ hybridization, zATM mRNA was detected ubiquitously with a dramatic increase at the 18-somite stage, then more specifically in the eye, brain, trunk, and tail at later stages. To inhibit zATM expression and function, we designed and synthesized splice-blocking antisense-morpholino oligonucleotides targeting the phosphoinositide 3-kinase-like domain. We demonstrated that this knockdown of zATM caused abnormal development upon ionizing radiation-induced DNA damage. Our data suggest that the ATM gene is structurally and functionally conserved in vertebrates from zebrafish to human.

Aging↗

PubMed1999

Development of a cold resistant mutant of plant growth promoting Pseudomonas fluorescens and its functional characterization.

A cold resistant mutant of plant growth promoting Pseudomonas fluorescens GRS(1), was isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The mutant was able to grow and promote root and shoot elongation of wheat both at 25 and 10 degrees C, a temperature at which the wild type was unable to proliferate and function. In an effort to determine the mechanistic basis for this behavior, it was observed that following growth at 10 degrees C, the mutant synthesized some new proteins. SDS-polyacrylamide gel electrophoresis of the protein synthesized by the mutant revealed the presence of one major protein with a molecular mass of 13.5 kDa. However, at this point the function of this protein in not known.

Journal Article↗