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PubMed2002

Modeling of human hepatic CYP3A4 enzyme kinetics, protein, and mRNA indicates deviation from log-normal distribution in CYP3A4 gene expression.

AIMS: To determine whether the variability in cytochrome P(450) (CYP)3A4 metabolic function is exhibited at both transcription and translation levels and to examine the population distribution of CYP3A4 enzyme kinetics, protein, and mRNA. METHODS: Enzyme kinetics of testosterone 6beta-hydroxylation, immunoblot CYP3A4 protein, and CYP3A4 mRNA were determined in a microsomal bank of human livers. The distribution of these determinations was analyzed using cumulative distribution (probit) plots and normality test variable (NTV) to detect deviation from normality. RESULTS: Mean hepatic CYP3A4 protein and relative CYP3A4 mRNA were 35+/-23 pmol/mg and 79+/-59 (CYP3A4/beta-actin), respectively. Kinetic parameter estimates of testosterone 6beta-hydroxylation were 611+/-684 pmol/mg/min for maximum rate of the reaction (V(max)) and 206+/-48 micro M for the Michaelis constant (K(m)). The CYP3A4 gene expression and its activity exhibited a relatively high degree of interindividual variability. Furthermore, significant correlation between CYP3A4 protein and V(max) of testosterone 6beta-hydroxylation (r=0.82, P<0.001) as well as CYP3A4 protein and its mRNA (r=0.52, P<0.01) was observed. Cumulative distribution plots and histograms for the CYP3A4 protein, its mRNA, and maximum testosterone 6beta-hydroxylation exhibited evidences of deviation from log-normal distribution. The minimum NTV value for the distribution of CYP3A4 protein corresponding to the inflection point in the probit occurred at approximately 10% cumulative frequency. The percentage of low CYP3A4 protein phenotype was consistent for CYP3A4 activity and its mRNA. In contrast, the distribution of K(m) of testosterone 6beta-hydroxylation does not show evidence of bimodality. CONCLUSIONS: The distribution of CYP3A4 metabolic function, protein, and mRNA is non-normal and may represent a regulatory polymorphism in hepatic CYP3A4 gene expression. In contrast, assessment of the possibility of a structural variant of CYP3A4 through evaluation of the standard deviation relative to the mean kinetic constant value suggests that structural mutation of CYP3A4 may not be a major factor affecting interindividual variation in CYP3A4 metabolic function.

Adolescent↗
PubMed1986

Nucleotide sequence analysis of murine 21-hydroxylase genes: mutations affecting gene expression.

Steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] is a cytochrome P-450 enzyme required for the adrenal synthesis of mineralocorticoids and glucocorticoids. The gene encoding this protein is present in two copies (21-OHase A and B) in the S region of the murine major histocompatibility complex. Previous studies utilizing gene-specific oligonucleotide probes and gene transfer showed that only the 21-OHase A gene is expressed in the BALB/c mouse. Here, we present the complete primary structures of both BALB/c 21-OHase encoding genes. Comparison of the nucleotide sequences defines a deletion of 215 nucleotides spanning the second exon of the 21-OHase B gene; other nucleotide changes in the 21-OHase B gene introduce frame shifts and premature termination codons. Southern blot analysis of C57BL/6 and DBA/2J mice indicates that a similar deletion is present in these strains; however the C3H/HeJ strain is a structural variant. A hybrid gene composed of the 21-OHase B promoter placed 5' of the 21-OHase A structural sequences was efficiently transcribed following transfection into Y1 adrenocortical tumor cells. These findings demonstrate that the 21-OHase B gene promoter is functional and suggest that mutations within the 21-OHase B structural gene are responsible for its lack of expression.

Amino Acid Sequence↗
PubMed2000

Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-receptor type 1 expression in rat and human placenta.

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC1) and type II receptors (VPAC1), which bind both PACAP and VIP. The placenta is a site of production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC1 gene by Northern hybridization analysis performed with a 32P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC1 complementary DNA. The results of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC1 gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC1 receptor (PAC1short), the rPAC1-hip or hop (which are indistinguishable with the primers used), and the rPAC1-hip-hop, whereas the human placenta expresses only the PAC1SV1 (or SV2) variant, structurally homologous to the rat PAC1 hip (or hop). Sequence analysis of the human PCR-amplified PAC1 variant was therefore carried out and revealed that human placenta only expresses the PAC1SV2 isoform. The presence and characterization of PACAP binding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [125I]PACAP27 as tracer. Scatchard analysis of the binding results revealed the presence of two binding sites, one with high affinity and low capacity (Kd 0.33+/-0.04 nM; Bmax 36.9+/-12.1 fmol/mg protein) and one with low affinity and high capacity (Kd 24+/-6.9 nM, Bmax 9.3+/-0.19 pmol/mg protein). The relative potencies of PACAP-related peptides for inhibition ofradioligand binding were: PACAP27 > or = PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and beta-endorphin, did not inhibit [125I]PACAP27 binding. In conclusion, in this study, we provide evidence for the expression of PACAP within rat and human placenta. We also demonstrate that both human and rat placenta express the PAC1 gene and that the human tissue has binding sites for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.

Animals↗
PubMed2005

Lipid carriers for gene therapy.

A wide variety of lipid molecules used as gene carriers has been reported and compared over the last twenty years. This review highlights a few examples of mechanistic analysis applied to the study of lipid carriers. The modular nature of the lipid structure offers itself up to a controlled, systematic analysis. Key to exploring structural variants is the understanding of the role each component and module plays in the formation of the lipoplex structure itself and their roles in the transfection pathway. Firstly, the lipid carrier must be able to package, and release into the cell its nucleic acid cargo. Uptake of lipoplexes into cells involves endocytic processes that lead inevitably to endosomal/liposomal degradation of the nucleic acid contents, unless lipid structures and designs are optimised to facilitate their release into the cytoplasm. Testing of possible endosomal escape mechanisms has led to improved lipid designs. For example, it was predicted that mixing of cationic lipids with shorter alkyl tails (<C18) from liposomes with the endosomal bilayer during the transfection process within the endosome should promote membrane fluidity, enhancing plasmid release and transfection efficiency. This hypothesis has been borne out numerous times. "Smart" molecules that respond to cellular cues such as endosomal pH have now been developed that appear to offer exciting opportunities for the future role of lipoplexes in clinical applications of gene therapy. The variability of cultured cell and model biological systems remains a challenge in designing improved lipids of general utility.

Alkylation↗
PubMed1978

Hemoglobin Legnano (alpha2 141 (HC3) Arg replaced by Leu beta2): a new abnormal human hemoglobin with high oxygen affinity.

An abnormal, fast moving hemoglobin was noted in a 34-yr-old male patient living in Legnano (northern Italy) affected with renal failure and iron deficiency anemia, not related to the presence of the Hb variant. Structural studies have demonstrated a previously undescribed amino acid substitution, alpha 141 Arg replaced by Leu. This new variant has been named Hb Legnano, and is characterized by an increased oxygen affinity and a low cooperativity, at least as far as preliminary functional studies carried out on whole blood have indicated. Family studies are reported: three other heterozygous carriers were observed among the relatives of the propositus, all showing a mild polycythemia which, however, does not seem to produce appreciable clinical consequences.

Adult↗
PubMed1990

Presence of a differentially expressed U3A RNA variant in mouse. Structure and evolutive implications.

A U3 RNA variant has been identified in mouse, the abundance of which relative to the previously characterized major form (U3B) appears to vary to a large extent depending upon the cell origin. Its partial sequence analysis shows that it is clearly related to the U3A form previously described in rat. Sequence comparisons suggest that the separation of the two forms of U3 genes now found in rat and mouse represent a relatively ancient event in rodent evolution. While mouse U3B RNA is encoded by four clustered genes, the U3A variant is encoded by a unique gene. Both mouse U3 RNAs differ substantially in primary structure (more than 10% divergence). Although rodent U3 RNAs exhibit a largely similar secondary structure, a specific difference between the A and B form can nevertheless be observed.

Animals↗
PubMed1989

ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution.

This review summarizes present knowledge of the chemistry of histo-blood group ABH and related antigens. Recent advances in analytical carbohydrate chemistry (particularly mass spectrometry and NMR spectroscopy) and the introduction of monoclonal antibodies (MoAbs) have made it possible to distinguish structural variants of histo-blood group ABH antigens. Polymorphism of ABH antigens is induced by: (i) variations in peripheral core structure, of which four (type 1, 2, 3 and 4) are known in man; (ii) variation in inner core by branching process (blood group iI), leading to variation of unbranched vs. branched ABH determinants; (iii) biosynthetic interaction with other glycosyltransferases (Lewis, P. T/Tn blood systems) capable of acting on the same substrate as the ABH-defined transferases, and finally (iv) the nature of the glycoconjugate (glycolipid, glycoprotein of N- or O-linked type). ABH variants induced by item (i) above have been clearly distinguished qualitatively by MoAbs; e.g., at least six types of A determinants can be distinguished by qualitatively different classes of antibody. The variants induced by item (ii) create mono- vs. bivalent antigens which may be responsible for observed differences in antibody-binding affinity. Detailed studies of the chemistry of these antigens have increased our insight into blood groups, providing the basis for blood group iI and A subgrouping, as well as a relation between the ABH and Lewis, P, and T/Tn systems. A survey of the literature on distribution patterns of ABH variants is presented. It has been assumed that expression of histo-blood group antigens is developmentally regulated. Relationships between histo-blood group expression, development, differentiation and maturation, as well as malignant transformation, are discussed.

ABO Blood-Group System↗
PubMed1991

Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa.

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III-Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT-III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III-Hamilton is capable of only the first of these events.

Alleles↗
PubMed1986

Six variants of HLA-B27 identified by isoelectric focusing.

Six variant forms of HLA-B27 were identified among 68 unrelated B27-positive donors by isoelectric focusing (IEF) gel analysis. Each of the six IEF variants was distinguished by charge heterogeneity of desialated B27 heavy chains immunoprecipitated with specific monoclonal antibody (MAb). Charge differences varied from single to several charge units, indicating that these variants may have substantially different amino acid compositions. Informative family study showed that three B27 variant molecules were genetically determined. The uniqueness of these variant molecules was also demonstrable using a panel of alloantisera and MAbs recognizing B27-associated epitopes. Six distinct serological reactivity patterns were observed. Five of these serological patterns correlated with four of the IEF-defined variants, two of these patterns being associated with one IEF variant form. The sixth serological pattern was shared by the remaining two IEF variants. Combining the results of the electrophoretic and serological analyses, it is apparent that there are more than six structural variants within the B27 alloantigen family. Some B27 variant forms were found only in individuals of particular racial origin, indicating that unique genetic variations might occur in different racial groups. In a preliminary analysis of patients with ankylosing spondylitis, no apparent correlation was observed between any specific B27 variants and disease susceptibility.

Gene Frequency↗
PubMed2006

Structural variation of the human genome.

There is growing appreciation that the human genome contains significant numbers of structural rearrangements, such as insertions, deletions, inversions, and large tandem repeats. Recent studies have defined approximately 5% of the human genome as structurally variant in the normal population, involving more than 800 independent genes. We present a detailed review of the various structural rearrangements identified to date in humans, with particular reference to their influence on human phenotypic variation. Our current knowledge of the extent of human structural variation shows that the human genome is a highly dynamic structure that shows significant large-scale variation from the currently published genome reference sequence.

Genetic Variation↗
PubMed2001

The influence of mannose binding lectin polymorphisms on disease outcome in early polyarthritis. TIRA Group.

OBJECTIVE: To determine whether variant alleles of the mannose binding lectin (MBL) gene causing low serum concentrations of MBL are associated with increased susceptibility to rheumatoid arthritis (RA) and erosive outcome in an inception cohort of patients with early polyarthritis. METHODS: MBL and HLA-DRB1 alleles were determined by polymerase chain reaction in 68 Danish patients with incident early polyarthritis observed for one year. The associations between MBL and specific HLA-DRB1 genotypes and disease outcomes were analyzed. RESULTS: Among the patients with early polyarthritis 7.4% (5/68) and 41.2% (28/68) were homozygous and heterozygous for MBL variant alleles, compared with 2.8% (7/250) and 34.4% (86/250) of healthy controls (p = 0.09), while the corresponding figures in the patients with RA were 10% (5/50) and 42% (21/50) (p = 0.03), and in the patients with erosive RA 18.8% (3/16) and 35.3% (6/16), respectively (p = 0.004). Patients with early polyarthritis homozygous for MBL variant alleles had an increased risk of having erosive RA at inclusion by a factor of 4.7 (p = 0.02) and after one year by a factor of 3.6 (p = 0.04). MBL deficiency was associated with increased levels of C-reactive protein (CRP) and IgM rheumatoid factor (RF) at inclusion (p < 0.05). HLA-DRB1 alleles were not found to be associated with disease outcome. CONCLUSION: MBL variant alleles appear to be weak susceptibility markers for RA, and patients with early polyarthritis and homozygous for MBL structural variant alleles have a higher risk of developing early erosive RA. These findings, together with the positive association between MBL variant alleles and the increased serum levels of IgM RF and CRP, point at the MBL gene as a relevant locus in the pathophysiology of RA.

Adult↗
PubMed1997

The use of on-line capillary electrophoresis/electrospray ionization with detection via an ion trap storage/reflectron time-of-flight mass spectrometer for rapid mutation-site analysis of hemoglobin variants.

Capillary electrophoresis/electrospray ionization using an ion trap storage/reflectron time-of-flight mass spectrometer detector (CE/ESI-IT/reTOF) is used to provide a rapid and sensitive method for analyzing structural variants in the hemoglobin (Hb) beta-chain. The Hb alpha- and beta-chains are separated and the beta-chain is digested by trypsin. The digest is analyzed by CE/ESI-IT/reTOF where a comparison of the total ion electrophorograms and mass spectra of the mutant and normal hemoglobins (Hbs) can detect the presence of a mutation site. In addition, collision-induced dissociation in the vacuum interface-skimmer region can be used to pinpoint the identity of such a site. The unique capability of the CE/ESI-IT/reTOF system for accurately detecting fast separations with narrow peaks that may be under 1 s full width at half maximum is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.

Amino Acid Sequence↗
PubMed1993

Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects in the central nervous system.

N-CAM is abundantly expressed in the nervous system in the form of numerous structural variants with characteristic distribution patterns and functional properties. N-CAM-180, the variant having the largest cytoplasmic domain, is expressed by all neurons. The N-CAM-180-specific exon 18 has been deleted to generate homozygous mice unable to express this N-CAM form. The most conspicuous mutant phenotype was in the olfactory bulb, where granule cells were both reduced in number and disorganized. In addition, precursors of these cells were found to be accumulated at their origin in the subependymal zone at the lateral ventricle. Analysis of the mutant in this region suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway. Subtle but distinct abnormalities also were observed in other regions of the brain.

Actins↗
PubMed1996

Intestinal fatty acid-binding protein: the structure and stability of a helix-less variant.

The structure of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) exhibits a beta-clam topology comprised of two five-stranded antiparallel beta-sheets surrounding a large solvent-filled cavity into which the ligand binds. It also contains two alpha-helices that span residues E15-A32 and join beta-strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed delta 17-SG, has a high beta-sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of delta 17-SG revealed patterns similar to those observed for wild-type I-FABP, except for the selective absence of resonances and through-space interactions assigned to the helical domain. The delta 17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of delta 17-SG were essentially identical to those of wild-type. We conclude that delta 17-SG is a helix-less, essentially all-beta-sheet variant of I-FABP and that the helical domain is not a required element of the beta-clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., & Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding.

Amino Acid Sequence↗
PubMed1993

Solution structure of a variant of human pancreatic secretory trypsin inhibitor determined by nuclear magnetic resonance spectroscopy.

The structure of a variant of human pancreatic secretory trypsin inhibitor (PSTI) has been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and a combination of distance geometry and molecular dynamics simulations. After complete assignment of the 1H signals, the nuclear Overhauser data imply the existence of a rather well-determined tertiary structure stabilized by a central alpha-helix and a short three-stranded beta-sheet. The tertiary structure of the amino terminus and of the loop 11-17 could not be defined by n.m.r. data, suggesting a high flexibility in these areas. As the crystal structures of two complexes of human PSTI variants and that of an uncomplexed variant are also known a comparison of the PSTI tertiary structure in solution and in the crystal is now possible.

Amino Acid Sequence↗
PubMed1984

Allotypes of murine factor B controlled by a locus within the S region of the H-2 complex.

Bf protein was directly precipitated from a mixture of EDTA-plasma from 13 different, inbred strains and rabbit IgG anti-mouse Bf, and was isolated by SDS-PAGE. The gel pieces containing Bf protein were digested by trypsin after labeling with 125I-Na, and then peptides of Bf protein with each mouse strain were compared by two-dimensional peptide mapping. The results for the peptide patterns with the standard B10 congenic strains (all of which Bf phenotypes had been already designated as Bf.1 because of the identical isoelectric point values) revealed two distinct peptide patterns. The peptide pattern of Bf protein appeared to be identical in each of the inbred strains, but only one of the 36 spots in the fingerprint observed either with B10 or B10.BR (designated tentatively as Bf.1 (b.k.) ) was not detected in that from B10.D2 and B10.S (designated tentatively as Bf.1 (d.s.) ). The results of peptide mapping with intra-H-2 recombinant inbred strains show that this structural variant was mapped to the S region, which is direct evidence that allotypes of murine Bf are encoded by a structural gene within the MHC.

Animals↗
PubMed1997

MR imaging of normal variants and interpretation pitfalls of the elbow.

Discrimination of normal anatomic landmarks from true disease is one of the fundamental tenets of adept MR imaging. The radiologist is thus compelled to accumulate a comprehensive knowledge of normal structures, variants, and potential MR imaging interpretation pitfalls. In this article the authors focus on a number of normal, bony, ligamentous, and tendinous structures that can simulate disease at the elbow. A discussion of the particular anatomy responsible for the appearance of each of these interpretation pitfalls is provided. In addition, ways to distinguish these pitfalls from true elbow disease are discussed.

Diagnosis, Differential↗
PubMed1997

[Hemoglobin Haná or alpha 2 beta 2 63 (E7) His-Asn: a new unstable hemoglobin variant with a paradoxically different clinical manifestations in smokers and non-smokers in the same family].

The authors describe the identification and the clinical manifestations of a new structural variant of haemoglobin found in three subjects from two generations of a Moravian family. It is manifested by mild haemolytic anaemia with Heinz bodies in the proband and a slightly elevated value of methaemoglobin. The sequential analysis of the beta-globin gene provided evidence that the cause is mutation CAT-AAT in codon 63 which leads to the exchange of distal histidine /E7/ for asparagine. The ratio of beta X:beta A is 38:62. The mother with the same mutation is asymptomatic. The relative amount of beta X:beta A mRNA transcripts in mother and daughter are equal. This indicates that the clinical differences are not due to a different gene expression. The mother is a heavy smoker with an elevated CO-Hb level that probably protect the mutant against oxidative denaturation and increases thus the stability of Hb-Haná. The authors discuss also the finding of two abnormal stripes assessed by three electrophoretic methods.

Anemia, Hemolytic, Congenital↗