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At least 613 records · Page 34Linked to original sources
PubMed1987

Apolipoprotein E polymorphism, serum lipids and occurrence of "double pre-betalipoproteinemia' (DPBL) in subjects from two different populations.

The association between apolipoprotein E phenotype and the presence of 2 electrophoretic populations of very low density (VLDL) lipoproteins from human sera, double pre-beta(VLDL)lipoproteinemia (DPBL), was studied in 2 groups of subjects, one from Italy and one from Finland. In both populations the prevalence of DPBL was significantly higher in subjects with E4/4 and E4/3 phenotypes than in the other phenotypes not containing the E4 isoprotein. This finding suggests that the presence of a structural variant of the apo E protein, the isoprotein E4, may be causally related to the appearance of DPBL phenomenon.

Adolescent↗
PubMed1993

Capillary zone electrophoresis and packed capillary column liquid chromatographic analysis of recombinant human interleukin-4.

Capillary zone electrophoresis (CZE) and packed capillary column liquid chromatography (micro-LC) have been applied to the analysis of the recombinant human protein interleukin-4 (rhIL-4). Separations for both the parent protein and its enzymatic digest were developed for the purpose of characterizing protein purity and identity. CZE separations of the intact protein were investigated over the pH range of 4.5 to 8.0 using uncoated fused silica capillaries. Gradient reversed-phase micro-LC was performed using 0.32 mm packed capillary columns at flow-rates of 5-6 microliters/min. Emphasis was placed on the ability of these methods to separate close structural variants and degradation products of the protein. Peptide mapping of the tryptic digest of rhIL-4 using a combination of CZE and micro-LC provided complimentary high resolution methods for establishing protein identity. Reproducible separations were achieved using sub-picomol amounts of sample. The advantages and problems encountered with these two techniques for characterizing rhIL-4 were assessed.

Capillary Action↗
PubMed1981

A simplified two-dimensional electrophoretic technique.

The two-dimensional electrophoretic O'Farrell technique can be simplified in terms of effort and equipment. This simplification derives mainly from the fact that the first dimension (isoelectric focusing) is run in a vertical slab gel. Since all tracks are cast in a single template, a reproducible and uniform first dimension is obtained, allowing easy and accurate comparison among tracks after standard staining and/or radioautographic procedures. After the first dimension, the focused tracks are cut and, following equilibration, simply laid on top of the second dimensional slab gel, whose spacers are 0.1 mm thicker than those used in the first separation. Thus, basically the same equipment can be used for both dimensions. This modified technique has been experimentally validated since it has allowed the detection of structural variants of two proteins related to the mouse complement system, namely, the fourth component and the sex-limited protein.

Animals↗
PubMed1986

Isolation and characterization of apolipoproteins A-I, A-II, and A-IV.

A number of different analytical techniques are now available for the isolation of apoA-I, apoA-II, and apoA-IV. The choice of a particular technique is dependent on the instrumentation available, and the quantity of isolated apolipoprotein required. The isolation and characterization of the separate isoforms and the precursor isoproteins of the individual apolipoproteins are detailed, and methods for the evaluation of the purity of the separate apolipoproteins presented. A method for the evaluation of apolipoproteins in plasma is now available which permits the identification of structural variants of plasma apolipoproteins in patients with dyslipoproteinemias.

Amino Acid Sequence↗
PubMed1981

Genetic organization of transposon Tn10.

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.

Base Sequence↗
PubMed1992

Fibrinogen genotype and risk of peripheral atherosclerosis.

There is conflicting evidence about the influence of fibrinogen genotype on plasma fibrinogen concentrations, and the relation between genotype and atherosclerotic disease has not been studied. In a population-based case-control study we aimed to find out whether certain fibrinogen genotypes are associated with an increased risk of peripheral atherosclerosis. 121 subjects with peripheral arterial disease and 126 healthy controls matched for age and sex were selected from a random population sample aged 55-74 years in the Edinburgh Artery Study. Mean fibrinogen concentrations were higher in cases than in controls (3.12 [95% confidence interval 2.99-3.26] vs 2.75 [2.64-2.85], p less than 0.001). A greater proportion of cases than controls were homozygous or heterozygous for an allele at the beta fibrinogen locus (4.2 kb allele, Bcl I digestion); the allele frequency was 0.197 in cases and 0.097 in controls (p less than 0.005). Extended haplotypes for 4.2 kb heterozygotes were also associated with an increased risk of peripheral arterial disease. However, haplotype had only a small effect on the association of plasma fibrinogen concentration with disease, and the relation of haplotype with disease was independent of age, sex, social class, smoking status, plasma fibrinogen, alcohol consumption, body mass index, and diabetes mellitus. We conclude that variation at the beta fibrinogen locus is associated with an increased risk of peripheral atherosclerosis. The influence is not mediated simply by way of increased fibrinogen concentrations but could be due to a structurally variant fibrinogen or linkage disequilibrium with a neighbouring gene.

Aged↗
PubMed1993

Production of N-terminal and C-terminal human serum transferrin in Escherichia coli.

The elucidation of the relationship of the structure of human serum transferrin to its iron-binding activity and the delineation of the interactions between transferrin and its receptor will require the construction and production of site-specific mutants of human serum transferrin to test the importance of specific structural motifs to the functions of transferrin. The N-terminal domain of transferrin has been previously produced in BHK cells, but the production of the C-terminal domain of transferrin has never been reported. The amino-terminal and carboxyl-terminal half-molecules of human serum transferrin have been cloned into the T7 expression vector pET11a. Contrary to previous reports, nTf and cTf can be easily produced in E. coli. The plasmids produce 38-kDa proteins that are approximately the sizes predicted for N-terminal and C-terminal half-molecules of transferrin, and both proteins react with anti-human serum transferrin antibodies. It is estimated that nTf represents 30-40% of total cellular protein after induction, while cTf represents less than 5% of total cellular protein. This demonstrates that recombinant forms of human serum transferrin can be produced in E. coli and suggests that it will be possible to use a bacterial system to produce other structural variants of transferrin.

Base Sequence↗
PubMed1984

Restriction in IgM expression--V. Fine structure analysis in the anti-lactose system.

A methodology for the analysis of the fine specificity of monoclonal anti-lactose IgM and IgG antibodies is described using structural variants of the homologous lactoside epitope. These variants are used as inhibitors of the binding of a reference ligand N-(5-dimethylaminonaphthalene-1-sulfonyl)-p-aminophenyl-beta-lactoside. Excitation of the antibody with bound ligand at 295 nm leads to resonance energy transfer to and fluorescence emission by the ligand. Titration of the antibody-ligand mixture with the inhibitor and measurement of the emission at 550 nm provide the data for the calculation of the binding constants of the inhibitors. A comparison of two IgM and two IgG antibodies showed that the higher affinity of the IgG antibodies arises from their specific interaction with both hexosides of the lactoside in contrast to IgM antibodies which do not engage the non-terminal hexoside as effectively. The quantitative significance of this difference is a differential free energy contribution of about -3 kcal/mole to the binding of lactoside by IgG. A finer discrimination between homologous and several cross-reactive molecules is evident with IgG antibody compared to IgM. The former exhibits about 100-fold greater difference in their binding constants than does IgM. These differences applied to biologically relevant multivalent interactions, where functional affinity governs complex formation, suggest a possible explanation for the IgM to IgG conversion characteristic of the humoral immune response.

Antibodies, Monoclonal↗
PubMed1984

Protein hormones: detection and extraction of functional forms from alkaline polyacrylamide gels.

A method is presented for rapidly staining zones (30 min) in alkaline polyacrylamide gels in the absence of fixatives. Native functional proteins are recovered in homogeneous form after excision of the visible zones from the polyacrylamide matrix. Removal of dye from excised zones is facilitated because only the surface of the gel is stained. Biological activity is then recovered from the gel slices by simple diffusion. The technique makes use of the sensitivity of Coomassie Brilliant Blue G-250 for the detection of proteins (less than 1 microgram). Structural variants of prolactin are isolated and recovered by this method. The method is applicable to studies requiring analytical and semi-preparative electrophoresis of proteins, especially pituitary hormones.

Animals↗
PubMed1986

Platelet MAO concentration and molecular activity: II. Comparison of normal and schizophrenic populations.

Platelet monoamine oxidase (MAO B) in 59 normal and 57 RDC-diagnosed medicated and unmedicated schizophrenic subjects was analyzed for whole platelet and extracted activities, specific concentration, and molecular activity. A novel radioimmunoassay using a monoclonal antibody elicited to human platelet MAO was used. Female schizophrenics showed no differences from female normals in MAO measures; however, these data could not be clearly evaluated because of confounding effects of age and drugs. Male schizophrenics treated with neuroleptics expressed significantly reduced whole platelet MAO activity, compared to untreated male patients. Compared with normal males, male schizophrenics showed significantly lowered molecular activities, along with elevated specific concentrations, which did not appear to be explained solely by drug usage. Additional mechanisms explaining the diminished molecular activity in male schizophrenics may be the presence of an endogenous irreversible inhibitor or a genetically determined, possibly structural, variant of MAO B.

Adult↗
PubMed1985

Role of heterochromatin variation in the instability of a marker chromosome during tumor progression.

Karyotypic evolution of the poorly metastasizing tumorigenic RSV-transformed B77-3T3 fibroblast line was investigated both in highly metastasizing clones (selected by growth in hard agar) and in spontaneous metastases. Analysis of structural chromosome aberrations associated with the transition from the nonmetastatic to the metastatic phenotype was focused on a readily identifiable marker chromosome (A), displaying an extracentromeric heterochromatic region as a main feature promoting genetic instability. Well-defined changes in the structure of this marker were observed, both in vitro and in vivo, and invariably involved C-heterochromatic variation. In the metastatic clones, a specific rearrangement of the A chromosome was selected. This structural variant (B) showed two extracentromeric C-positive regions and probably originated from duplication of the segment of A included between the centromere and the internal C-band. On the other hand, selection of a modified form of chromosome A, not displaying the interpolated C-heterochromatin, had occurred in the extremely rare B77-3T3 spontaneous metastases. The connection among heterochromatin variants, genetic instability, and chromosome aberrations is discussed.

Animals↗
PubMed1994

Structural characterization and biological activity of recombinant human epidermal growth factor proteins with different N-terminal sequences.

The primary structures and molecular homogeneity of recombinant human epidermal growth factors from different suppliers were characterized and their biological activities evaluated by a standard DNA synthesis assay. Molecular weight determinations using 252Cf-plasma-desorption and electrospray mass spectrometry in combination with N- and C-terminal sequence analysis and determination of intramolecular disulfide bridges revealed that one recombinant protein had the correct human-identical structure (54 aa residues; 6347 Da). In contrast, a second recombinant protein (7020 Da) was found to contain a pentapeptide (KKYPR) insert following its N-terminal methionine. This structural variant showed a significant reduction in its capacity to stimulate DNA synthesis.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of endorphins from the pituitary of the spiny dogfish Squalus acanthias.

Opioid-like immunoreactive material was extracted from the pituitary and brain of the Spiny Dogfish Shark Squalus acanthias. The immunoreactive material in the pituitary extracts was purified to apparent homogeneity by reverse phase high performance liquid chromatography and subsequently characterized by amino acid analysis, Edman degradation and fast atom bombardment mass spectrometry. The largest opioid-like peptide isolated contained 30 amino acids and showed 80 percent homology with salmon endorphin-II but less than 50 percent homology with human beta-endorphin. Three structural variants of this molecule were also characterized. These variants were shown to be shorter N-terminal fragments, two of which corresponded to cleavage products at the single basic residues arginine and lysine. Cleavage at a single lysine residue has not been reported for posttranslational processing of beta-endorphin in mammals and could represent a modification seen only in lower vertebrates. The remaining fragment corresponded to a loss of 3 residues from the C-terminus of the parent molecule. No alpha-N-acetylated peptides were detected. These results provide the first unequivocal confirmation of beta-endorphin in an elasmobranch and provide evidence of novel N-terminal variants of beta-endorphin.

Acetylation↗
PubMedDate not supplied

Chromatographic and immunological evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla) brain and pituitary.

Gonadotropin-releasing hormone (GnRH) peptides in the brain and pituitary of the European eel (Anguilla anguilla) were investigated by reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Two GnRH molecular forms were demonstrated in brain and pituitary extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2 and COOH termini of mammalian GnRH as well as by antibodies to the middle region. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Salmon GnRH and chicken GnRH I were not detected. The occurrence of mammalian GnRH in teleost fish suggests that this molecular form is more ancient than was previously suspected and arose earlier than in primitive tetrapods, or that it has arisen in the eel through random mutation of salmon GnRH. The lack of salmon GnRH in the eel brain indicates that this molecular form is not common to all teleost species. The finding in eel brain of chicken GnRH II, which has previously been described in species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

Anguilla↗
PubMedDate not supplied

Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica).

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

Amino Acid Sequence↗
PubMedDate not supplied

Gonadotropin-releasing hormone in elasmobranch (electric ray, Torpedo marmorata) brain and plasma: chromatographic and immunological evidence for chicken GnRH II and novel molecular forms.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.

Animals↗
PubMed1996

Allelic association analysis of the 5-HT2C receptor gene in bipolar affective disorder.

We have examined a structural variant of the 5-HT2C receptor (Cys23Ser) for allelic association with bipolar affective disorder in 88 cases and 113 controls. Overall, there was no significant difference in allele frequencies between the two groups, indicating that the 5-HT2C gene is not a major risk factor for bipolar affective disorder. However, when the subjects were analysed according to sex, there was a small excess of the serine ser23 allele in female cases (P = 0.04) and this effect was also seen if the ser23 allele was considered recessive (P = 0.03). A small increase in significance was found if only female cases with a known family history were included (P = 0.01). These results suggest that the ser23 allele may increase susceptibility to bipolar affective disorder in women.

Alleles↗
PubMed1988

The use of high field NMR to determine homogeneity in a preparation of human C5a produced by Escherichia coli.

The polypeptide backbone of human C5a was prepared by recombinant DNA techniques. Standard biochemical analysis guided the protein separation to give a sample of C5a which was deemed homogeneous. However, nuclear magnetic resonance (NMR) studies showed the material to be significantly heterogeneous. Reanalysis by high performance liquid chromatography (HPLC) corroborated the NMR results. Further separation by HPLC and analysis by NMR spectroscopy guided the isolation of rC5a to greater than 92% purity. NMR analysis, immunochemical and biological evaluation of the impurities showed them to be C5a structural variants. These results indicate that conventional methods of protein chemistry can fail to reveal heterogeneity in recombinant proteins, and in some circumstances NMR spectroscopy can aid in their purification.

Chromatography, High Pressure Liquid↗