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At least 487 records · Page 27Linked to original sources
PubMed1993

Mammalian calponin. Identification and expression of genetic variants.

Calponin is a smooth muscle specific, actin-, tropomyosin- and calmodulin-binding protein thought to be involved in some way in the regulation or modulation of contraction. Here we describe the cloning and bacterial expression of two calponin species from murine and porcine smooth muscle tissues. Primary and secondary structural analyses of the deduced amino acid sequences revealed a high degree of homology to avian calponin with the exception of a short and variable C-terminal segment. The sequence data demonstrate that the two mammalian calponin variants do not arise via alternative splicing but are encoded by different genes.

Amino Acid Sequence↗
PubMed1996

Alzheimer's presenilin 1 gene expression in platelets and megakaryocytes. Identification of a novel splice variant.

The presenilin 1 (PS1) gene located on chromosome 14 has been linked with the majority of early-onset FAD. The normal biological role of PS1 as well as the mechanism by which mutations in PS1 cause FAD remains unknown. PS1 expression in platelets and the Dami megakaryocytic cell line was examined by Western blot analysis and RT-PCR. Using an anti-N-terminus PS1 antibody we detected PS1 immunoreactive bands of 44, 32 and 27 kDa in both cell types. After RT-PCR we observed that platelets and megakaryocytes carry at least four different PS1 transcripts. One of them is a novel PS1 splice variant that lacks the coding sequence for exon 10 resulting in a shorter 409 amino acid protein.

Alternative Splicing↗
PubMed1992

Identification of growth hormone molecular variants in chicken serum.

It has been described that pituitary growth hormone shows molecular and functional heterogeneity. In birds, size and charge variants of chicken growth hormone (cGH) have been shown in the chicken pituitary gland and in purified preparations of the hormone. Here we demonstrate the existence of cGH molecular isoforms in chicken serum, thus suggesting that they are secreted from the gland. The isolation of total cGH present in sera was performed by immunoaffinity chromatography employing a specific monoclonal antibody against cGH. Different analytical electrophoretic methods (SDS-polyacrylamide gel electrophoresis, isoelectric focusing, bidimensional polyacrylamide gel electrophoresis) followed by Western blot and immunostaining were employed to characterize the serum cGH isoforms, and compared to those present in a fresh pituitary extract. Several identical immunoreactive bands comigrated in both serum and the gland extract in the different systems (SDS-PAGE, MW 16, 22, 26, 29, 52, 62, 66 kDa; IEF, pIs 8.1, 7.5, 7.1, 6.8, 6.2), thus revealing a high correspondence of molecular isoforms of the hormone in the two tissues. Additionally, a glycosylated variant of chicken growth hormone (G-cGH) was also revealed in the serum after concanavalin A-Sepharose chromatography.

Animals↗
PubMed1995

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.

Amino Acid Sequence↗
PubMed1984

The identification of monoclonal class switch variants by sib selection and an ELISA assay.

Monoclonal antibodies can now be generated against a wide variety of antigens. However, a potentially useful monoclonal antibody may be of the wrong class or subclass for a particular task. Antibodies of the desired class can be obtained by identifying rare subclones in which the variable region has been rearranged to a new constant region. We describe here the use of sib selection and an enzyme-linked immunoassay (ELISA) for isolating such class and subclass switch variants from 2 IgM and 1 IgG3 producing hybridoma. The relative simplicity of ELISA assays makes it feasible to apply this approach to many different hybridomas. Since commercial affinity purified class and subclass specific antibodies are now available, the method can be used by any laboratory. Furthermore, the technique does not require that the switch variants express surface immunoglobulin and enriches for hybridomas secreting higher amounts of antibody.

Animals↗
PubMed1992

Identification of rare immunoglobulin switch variants using the ELISA spot assay.

We describe here the use of the ELISA spot assay to identify, quantify, and isolate rare hybridoma subclones that have switched to expressing a new class or subclass of Ig. This technique is less labor intensive and time consuming than sib selection and standard ELISA and eliminates the many false positives that complicated those techniques. The use of the ELISA spot assay also allows screening large populations of cells and accurate quantitation of the rate of isotype switching.

Animals↗
PubMed1993

Use of immunoplot analysis for the identification of immunodominant non-variant antigens of Trypanosoma brucei rhodesiense.

The application of the 'immunoplot' technique to the analysis of a complex series of immunoblots is described. A number of isolates of Trypanosoma brucei rhodesiense blood forms from Uganda, Kenya and Zambia were separated by SDS-PAGE under reducing and non-reducing conditions and transferred by immunoblotting onto nitrocellulose paper. The separated antigens from each isolate were then probed with sera from European and endemic controls, and a large panel of sera from confirmed trypanosomiasis patients. The resulting series of immunoblots was examined by the 'immunoplot' technique, which involves comparing frequencies of recognition of each band by the sera of controls and patients. A number of antigens of possible diagnostic interest were identified; a bad of 37/38 kDa was found to be recognised with high frequency (> 55%) by the sera of patients and virtually not at all by control sera under both reducing and non-reducing conditions. When this band was combined with one of the other frequently recognised bands, recognition by patient sera was raised to 92% (reduced) and 76% (non-reduced). This did not appear to be isolate-specific. The 'immunoplot' technique has potential for further development and wider use in immunoepidemiology and immunodiagnostic research.

Animals↗
PubMed1993

Identification and characterization of hydrophobic microcystins in Canadian freshwater cyanobacteria.

Hepatotoxic microcystins produced by cyanobacteria in freshwater lakes represent a significant health hazard to humans and agricultural livestock. Liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay analysis of blooms of Microcystis aeruginosa produced in a Canadian drinking water lake identified several PPase inhibitors with significantly greater hydrophobicity than microcystin-LR, based on their retention time on C18 reverse phase LC columns. Seven PPase inhibitors were purified to homogeneity by bioassay-guided fractionation involving Sephadex LH-20 chromatography and two-step reverse phase at pH 6.5 and 2.0. One of the PPase inhibitors, isolated in a final yield of 1.5 micrograms/g lyophilized cyanobacteria, was identified as microcystin-LL by amino acid analysis and mass spectrometry. A further PPase inhibitor (20 ng/g cyanobacteria) was identified as microcystin-LL but with D-Ala replaced by an unknown amino acid. Four PPase inhibitors (< 20 ng/g cyanobacteria) were characterized by amino acid analysis and identified as microcystin-LV, -LM, -LF and -LZ (where Z represents an unknown hydrophobic amino acid). A further microcystin was also identified (< 10 ng/g cyanobacteria) in which arginine was apparently absent. The biological activity of the seven microcystins as inhibitors of the catalytic subunit of protein phosphatase-1 (PP-1c) was compared with microcystin-LR and motuporin (a hydrophobic analogue of nodularin). All of the compounds inhibited PP-1c with IC50 values of 0.06-0.4 nM, consistent with their identification as microcystins. These findings further demonstrate the applicability of a sensitive PPase bioassay for the identification of variant microcystins in the natural environment.

Amino Acids↗
PubMed1986

Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates.

DNA prepared from four isolates of Trichinella was compared by genomic DNA cross-hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar.

Animals↗
PubMed1986

Identification of proteins encoded by variant surface glycoprotein expression site-associated genes in Trypanosoma brucei.

The variant surface glycoprotein (VSG) genes of Trypanosoma brucei may be transcribed from several distinct telomeric expression sites (ESs). The mechanism responsible for regulating potential expression sites is unknown. Two members of a pleomorphic family of expression site associated genes (ESAGs) have been cloned and sequenced. By examination of the DNA sequences we inferred that ESAGs encode amphiphilic glycoproteins. Fragments of two ESAGs were inserted into the Escherichia coli expression vectors pATH and pEX. Antisera to the resulting anthranilate synthetase ESAG protein (ESAGP) fusion protein immune precipitated a 46 kDa glycoprotein from detergent extracts of T. brucei. In the presence of tunicamycin, the size of the immune-precipitated protein was reduced to 36 kDa, corresponding to the molecular weight predicted by the ESAG sequence. The 36 kDa and 46 kDa proteins were absent from procyclic culture forms of T. brucei.

Animals↗
PubMed2006

Identification and characterization of the variants of metastasis-associated protein 1 generated following alternative splicing.

The metastasis-associated gene 1 (mta1) was identified initially in rat highly metastatic cancer cell lines and found to be a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. The gene for mouse mta1 was screened and its genomic structure was determined. It consists of 21 exons spanning 40 kb of genomic DNA. The full-length mouse Mta1 cDNA contained a 2145 nucleotide open reading frame encoding 715 amino acids. In addition to the full-length cDNA, several alternative splicing variants were found. Some differences in the splicing variants found were observed among various mouse organs and cells examined by the semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cDNAs of the splicing variants were inserted into green fluorescent protein (GFP) expression vector and the subcellular localization of the GFP-Mta1 fusion proteins were analyzed. Knowledge of the Mta1 gene expression pattern will be useful in better understanding its functional diversity.

Alternative Splicing↗
PubMed2005

Identification of novel Nox4 splice variants with impact on ROS levels in A549 cells.

NAD(P)H oxidases (Nox) generate reactive oxygen species (ROS) that function in host defense and cellular signaling. While analyzing the expression of Nox4 at the protein and the mRNA levels, we identified four novel Nox4 splice-variants Nox4B, Nox4C, Nox4D, and Nox4E, which are expressed in human lung A549 cell line and lung tissues. One Nox4 isoform lacks the first NAD(P)H binding site (Nox4B) while another lacks all FADH and NAD(P)H binding sites (Nox4C). Cells over-expressing NoxB or Nox4C exhibited a decrease in ROS levels. Thus, these isoforms have dominant negative characteristics for ROS generation. Two other splice-variants (Nox4D, Nox4E) lack the transmembrane domains, suggesting these as non-membrane associated isoforms. Nox4D contains all FADH and NAD(P)H binding domains and shows the same rate of ROS generation as Nox4 prototype. Taken together, we suggest that Nox4 exists as several isoforms that may have different functions in ROS-related cell signaling.

Alternative Splicing↗
PubMed2006

ABCB1 genetic variability and methadone dosage requirements in opioid-dependent individuals.

BACKGROUND AND OBJECTIVES: The most common treatment for opioid dependence is substitution therapy with another opioid such as methadone. The methadone dosage is individualized but highly variable, and program retention rates are low due in part to nonoptimal dosing resulting in withdrawal symptoms and further heroin craving and use. Methadone is a substrate for the P-glycoprotein transporter, encoded by the ABCB1 gene, which regulates central nervous system exposure. This retrospective study aimed to investigate the influence of ABCB1 genetic variability on methadone dose requirements. METHODS: Genomic deoxyribonucleic acid was isolated from opioid-dependent subjects (n = 60) and non-opioid-dependent control subjects (n = 60), and polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction were used to determine the presence of single nucleotide polymorphisms at positions 61, 1199, 1236, 2677, and 3435. ABCB1 haplotypes were inferred with PHASE software (version 2.1). RESULTS: There were no significant differences in the allele or genotype frequencies of the individual single nucleotide polymorphisms or haplotypes between the 2 populations. ABCB1 genetic variability influenced daily methadone dose requirements, such that subjects carrying 2 copies of the wild-type haplotype required higher doses compared with those with 1 copy and those with no copies (98.3 +/- 10.4, 58.6 +/- 20.9, and 55.4 +/- 26.1 mg/d, respectively; P = .029). In addition, carriers of the AGCTT haplotype required significantly lower doses than noncarriers (38.0 +/- 16.8 and 61.3 +/- 24.6 mg/d, respectively; P = .04). CONCLUSION: Although ABCB1 genetic variability is not related to the development of opioid dependence, identification of variant haplotypes may, after larger prospective studies have been performed, provide clinicians with a tool for methadone dosage individualization.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Identification of a constitutively active variant of LuxO that affects production of HA/protease and biofilm development in a non-O1, non-O139 Vibrio cholerae O110.

Pathogenesis of Vibrio cholerae depends on the concerted action of numerous virulence factors that includes a secreted hemagglutinin (HA) protease. Recent studies have evidenced that the expression of these virulence factors as well as the genes responsible for biofilm development is subject to control by quorum sensing in this organism. At low cell density, LuxO, the pivotal regulator of quorum-sensing circuit, has been shown to be phosphorylated at aspartate-47. Working in concert with sigma-54, LuxO-P activates the downstream repressor, which turned out to be four sRNAs [Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., Wingreen, N.S., Bassler, B.L., 2004. The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69-82]. Subsequently, these sRNAs form complex with sRNA chaperone, Hfq. The Hfq-sRNA complex causes the destabilization of hapR mRNA transcript. HapR is a positive regulator of hapA that encodes HA/protease. At high cell density, dephosphorylation of LuxO impairs its function to activate the expression of sRNA, which in turn promotes HapR expression and causes protease production. It has been demonstrated that conversion of aspartate to glutamate (D47E) renders the LuxO molecule active without being phosphorylated. This variant of LuxO is referred as constitutively active LuxO or con-LuxO [Freeman, J.A., Bassler, B.L., 1999. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Mol Microbiol 31, 665-677]. Other than D47E, mutation at L104Q also develops con-LuxO [Vance, R.E., Zhu, J., Mekalanos, J.J., 2003. A constitutively active variant of the quorum-sensing regulator LuxO affects protease production and biofilm formation in Vibrio cholerae. Infect. Immun. 71, 2571-2576]. The purpose of this study was to investigate the cause of protease negative phenotype of a non-O1, non-O139 strain of V. cholerae O110. In the process of exploring the nature of the phenotype, a constitutively active variant of LuxO molecule was characterized which represses protease production and enhances biofilm formation by this strain. Unlike luxU, disruption of luxO restored the protease production, which showed the constitutively active nature of LuxO protein in this strain.

Amino Acid Sequence↗
PubMed2005

Identification of human herpesvirus 6 variants A and B by primer-specific real-time PCR may help to revisit their respective role in pathology.

BACKGROUND: Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood. OBJECTIVES: To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens. STUDY DESIGN: The VS-PCR was adapted from a real-time PCR assay, based on TaqMan technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27-35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts. RESULTS: The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders. CONCLUSIONS: We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants.

DNA Primers↗
PubMed2005

Exploring the genetics of longevity in the Old Order Amish.

Lifespan is a complex phenotype determined by the interaction of genetic and environmental factors. This makes the identification of variants in genes that influence longevity challenging. We believe that the Old Order Amish (OOA) of Lancaster, Pennsylvania is an excellent population for studying the genetics of longevity. They are a closed population derived from a limited number of founders. They have large families and maintain extensive genealogic records dating to the 1700 s. They eschew modern technology; their lifestyle is little changed over the last 250 years. Homogeneity of environment and lifestyle factors across time and across the OOA population minimizes the influence that environmental factors have in determining the differences in lifespan between individuals. We hypothesize that this reduction in environmental variability will make it easier to identify the genetic factors that influence lifespan. In this article, we describe our strategy for identifying variants in genes that influence longevity in the Amish and present the results of our studies to date.

Adult↗