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PubMed1992

Genetic variants in the putidaredoxin-cytochrome P-450cam electron-transfer complex: identification of the residue responsible for redox-state-dependent conformers.

Camphor is hydroxylated in Pseudomonas putida by a three-component system comprised of an oxidase, cytochrome P-450cam, and a two-protein electron-transfer chain, putidaredoxin and putidaredoxin reductase [Tyson et al. (1972) J. Biol. Chem. 274, 5777-5784]. The enzymatic removal of putidaredoxin's C-terminal tryptophan is known to cause a much reduced rate of enzymatic activity in the reconstituted camphor hydroxylase system [Sligar et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3906-3910]. To further study the role of tryptophan in the association and/or electron-transfer reactions of putidaredoxin, the gene coding for the iron-sulfur protein was altered so that the tryptophan codon was either deleted or replaced by Phe, Tyr, Asp, Leu, Val, or Lys. Although the initial evaluation of these variant proteins [Davies et al. (1990) J. Am. Chem. Soc. 112, 7396-7398] showed much reduced velocities of electron transfer between P-450cam and the nonaromatic C-terminal proteins, the relative contributions of the binding specificity and intracomplex electron-transfer rates were not addressed. We report here a complete kinetic characterization of these proteins where the dependence of the rate constant on the putidaredoxin concentration was used to determine the intracomplex electron-transfer rate constants and the association energies for all the putidaredoxins in both oxidation states. The sum of forward and reverse intracomplex electron-transfer rate constants varies from 4.90 s-1 for the Lys C-terminal variant to 172 s-1 for the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed2026

Biallelic ABCA13 Loss-of-Function Variants in a Child With Neurodevelopmental Delay: A Case Report.

ABCA13 encodes ATP-binding cassette subfamily A member 13, one of the largest members of the ABC transporter family. Rare ABCA13 variants have been reported in neuropsychiatric and neurodevelopmental phenotypes, including schizophrenia, bipolar disorder, autism spectrum disorder, intellectual disability, and developmental delay; however, the mode of inheritance remains uncertain, and most published cases have focused on heterozygous variants in the context of a possible dominant or susceptibility model. We report a 5-year-old boy with neurodevelopmental delay, skeletal foot deformities, nonspecific dysmorphic features, convergent strabismus, and behavioral abnormalities. Initial genome sequencing analysis was nondiagnostic. Reanalysis after 6 months identified two rare predicted loss-of-function variants in ABCA13 in trans: c.2510del, p.(Leu837TyrfsTer21), a frameshift variant, and c.12064C>T, p.(Arg4022Ter), a nonsense variant previously reported in a patient with unexplained intellectual disability. The identification of compound heterozygous predicted loss-of-function variants supports the possibility that biallelic disruption of ABCA13 may contribute to neurodevelopmental disease, whereas previously reported heterozygous variants may represent incompletely penetrant risk alleles, susceptibility factors, or candidate findings rather than fully penetrant dominant causes. This case expands the emerging clinical and genetic spectrum associated with ABCA13 and supports further evaluation of a recessive model in patients with intellectual disability and neurodevelopmental delay.

ABCA13↗

PubMed2026

Idiopathic neonatal arterial ischaemic stroke: a trio-based whole-exome sequencing study.

OBJECTIVE: To assess the contribution of rare coding genetic variants to idiopathic neonatal arterial ischaemic stroke (NAIS). DESIGN: Observational genetic study using trio-based whole-exome sequencing (WES). SETTING: Multicentre study. PATIENTS: 23 newborns diagnosed with idiopathic NAIS and their biological parents. INTERVENTIONS: WES-trio with a customised workflow for filtering and interpreting variants in de novo autosomal dominant and recessive inheritance models. MAIN OUTCOME MEASURES: Identification of pathogenic (P) or likely pathogenic (LP) variants potentially associated with NAIS. RESULTS: We identified 28 unique rare de novo variants in 28 genes across 23 newborns with NAIS. Under the autosomal recessive model, no candidate genes were identified. No common P/LP variant across the 23 newborns was detected. In-silico predictors and comprehensive knowledge-driven analysis highlighted PIK3CD (p.Gln431Arg) as a candidate gene in one patient with perforant stroke. However, no more cases were identified with PIK3CD variants, and functional studies are warranted to assess its pathogenicity impact. CONCLUSIONS: Trio-based WES did not identify a monogenic cause for idiopathic NAIS. Coding variants therefore appear unlikely to explain the underlying genetic base of the disease. Furthermore, PIK3CD (p.Gln431Arg) may contribute to perforant stroke, although it requires further association evidence. As the potential role of non-coding or structural variants in NAIS remains possible, genome-wide long-read sequencing approaches may provide further insights into the genetic architecture of this condition.

Humans↗

PubMed1999

On-line high-performance liquid chromatography/mass spectrometric investigation of amyloid-beta peptide variants found in Alzheimer's disease.

Abeta peptides are the major components of amyloid deposits in Alzheimer's disease. The presence of N-terminally truncated Abeta variants in amyloid may be a critical factor in Alzheimer's disease pathogenesis. These Abeta variants are less soluble and more amyloidogenic than full-length Abeta, making their separation, purification and identification difficult. High-performance liquid chromatography (HPLC) at elevated temperatures, coupled to electrospray ionization (ES) mass spectrometry (MS), enables rapid separation and identification of N-terminally truncated Abeta variants. This methodology provides a potential tool for exploring the importance of these Abeta variants in both the pathogenesis and diagnosis of Alzheimer's disease.

Alzheimer Disease↗

PubMed2004

DNA delivery to cells in culture: generation of adenoviral libraries for high-throughput functional screening.

In functional genomics, the use of expression libraries of DNA variants in combination with potent screening techniques is a powerful tool for gene discovery. They allow study of gene and protein function, generation of peptide variants with novel properties, as well as identification of functional short DNA and RNA motifs. In proteomics, generation of large expression libraries of protein variants with random substitutions ("directed evolution") and further screening for novel or improved functions has been commonly used for isolation of proteins with novel characteristics, for improving enzymes, for rapid isolation of antibodies, and for functional protein studies. Most commonly, peptide libraries are expressed and screened in prokaryotic systems. Such systems have the advantage of rapid and simple generation of clones expressing single variants, allow high diversity (up to 10(11)), and can be combined with phage- or cell-surface display technique (2). The main disadvantage of bacterial systems is the absence of posttranslational modifications and native folding of many mammalian proteins, leading to limited applications, particularly when enzyme-substrate-, protein-protein, or protein-RNA interactions are to be studied.

Adenoviridae↗

PubMed1986

Analysis of antigenic determinants on internal and external proteins of influenza virus and identification of antigenic subpopulations of virions in recent field isolates using monoclonal antibodies and immunogold labelling.

An electron microscopic immunogold labelling technique employing monoclonal antibodies has been applied to the antigenic analysis of influenza A and B viruses. Reassortant influenza A H3N2 viruses containing haemagglutinin molecules from viruses isolated between 1968 and 1982 were analysed with a panel of monoclonal antibodies raised against viruses which appeared over the same period. The immunogold labelling technique clearly demonstrated the antigenic drift in the haemagglutinin molecule that occurred between 1968 and 1982. When the technique was applied to the examination of viruses from a more geographically restricted influenza epidemic in a semi-closed community, antigenic variants were found. Furthermore the technique enabled the identification of distinct antigenic variant subpopulations within a single clinical isolate. Analysis of the HA of MDCK cell or egg grown virus by this procedure provided data to support the hypothesis that the host cell exerts selective pressure on subpopulations of virus resulting in the emergence of antigenic variants.

Antibodies, Monoclonal↗

PubMed1999

The cytomorphology of papillary serous carcinoma of the endometrium in cervical smears.

BACKGROUND: An investigation into the determination of cytomorphologic criteria that may distinguish papillary serous carcinoma of the endometrium (PSC) from typical endometrioid carcinoma (TEC) in cervical smears was undertaken. Preoperative identification of this poor prognostic variant of endometrial carcinoma may influence the surgical management of these cases and the choice of adjuvant therapy. METHODS: The cervical smears of 12 cases of histologically confirmed PSC; 12 cases of TEC, including 2 villoglandular/papillary variants, and 6 cases of mixed PSC and papillary endometrioid carcinoma were reviewed. In all cases an initial diagnosis of malignancy had been made on the cervical smears. Twenty-seven criteria were evaluated and the relation between the cytologic characteristics and the type of adenocarcinoma, the degree of association, and the intergroup homogeneity were tested. RESULTS: Features strongly associated with PSC were hypercellular smears with a background tumor diathesis, papillae, bare nuclei, and cells with large pleomorphic nuclei and bulky dense cytoplasm. In contrast, TEC showed a relatively monomorphic population of cells with moderately enlarged oval nuclei and delicate cytoplasm. In the mixed tumors, the features were similar to those of PSC, suggesting preferential exfoliation of the PSC component of the tumor. CONCLUSIONS: Assessment of the cytomorphology of PSC and TEC of the endometrium in Papanicolaou stained cervical smears is possible using statistically significant diagnostic criteria.

Carcinoma, Endometrioid↗

PubMed1985

The use of capillary gas chromatography/mass spectrometry for the determination of acidic dopamine metabolites in human brain.

A capillary gas chromatographic/mass spectrometric method was developed for the determination of homovanillic acid and 3,4-dihydroxyphenylacetic acid in discrete areas of human brain known to contain only small amounts of dopamine metabolites. The electron impact mass spectra of the trimethylsilyl derivatives of homovanillic acid and 3,4-dihydroxyphenylacetic acid and their deuterated isotopic variants were used for their identification and quantification.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2004

Ordered subset analysis in genetic linkage mapping of complex traits.

Etiologic heterogeneity is a fundamental feature of complex disease etiology; genetic linkage analysis methods to map genes for complex traits that acknowledge the presence of genetic heterogeneity are likely to have greater power to identify subtle changes in complex biologic systems. We investigate the use of trait-related covariates to examine evidence for linkage in the presence of heterogeneity. Ordered-subset analysis (OSA) identifies subsets of families defined by the level of a trait-related covariate that provide maximal evidence for linkage, without requiring a priori specification of the subset. We propose that examining evidence for linkage in the subset directly may result in a more etiologically homogeneous sample. In turn, the reduced impact of heterogeneity will result in increased overall evidence for linkage to a specific region and a more distinct lod score peak. In addition, identification of a subset defined by a specific trait-related covariate showing increased evidence for linkage may help refine the list of candidate genes in a given region and suggest a useful sample in which to begin searching for trait-associated polymorphisms. This method provides a means to begin to bridge the gap between initial identification of linkage and identification of the disease predisposing variant(s) within a region when mapping genes for complex diseases. We illustrate this method by analyzing data on breast cancer age of onset and chromosome 17q [Hall et al., 1990, Science 250:1684-1689]. We evaluate OSA using simulation studies under a variety of genetic models.

Breast Neoplasms↗

PubMed2003

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay.

The highly polymorphic human alpha-1-antitrypsin (AAT) gene, more recently named SERPINA1, codes for the most abundant circulating plasma serine protease inhibitor, protease inhibitor 1 (PI). Most studies determining AAT haplotype frequencies have been restricted first by the limited accuracy of the phenotypic method used and secondly by the analysis of predominantly Caucasian populations. Limited studies have been performed on African-based populations. Here a new comprehensive assay for genotyping the entire coding region, including splice junctions, of the AAT gene was designed. This assay, based on denaturing gradient gel electrophoresis (DGGE), allows for the complete analysis of a single individual in two lanes on a gel. Application of the assay resulted in the identification of nine known AAT variants as well as 13 novel sequence variants, five of which are single nucleotide polymorphisms (SNPs), occurring exclusively in the African-based populations. This is the first comprehensive analysis of the genetic diversity of the AAT gene in a cohort from sub-Saharan Africa.

Africa South of the Sahara↗

PubMed2002

BRCA1 and BRCA2 in Indian breast cancer patients.

Incidence of breast cancer in Indian women is not as high as in Western countries, nonetheless age-adjusted incidence rates (AAR) have risen from 17.9 to 24.9 per 100,000 from 1965 to 1985. Although these rates are still approximately one quarter to one third of incidence rates in North America and Europe, respectively, due to the large population of women at risk, nearly 80,000 new cases were diagnosed in India in 2000. Although identification of BRCA1 and BRCA2 has greatly increased our understanding of breast cancer genetics in populations of Western European descent, the role of these genes in Indian populations remains unexplored. Analysis of a series of 20 breast cancer patients from North India with either family history of breast and/or ovarian cancer (2 or more affected first degree relatives) or early age of onset (<35 years) led to identification of two novel splice variants (331+1G>T; 4476+2T>C) in BRCA1 (10%). In addition, two BRCA2 missense variants were each identified in more than one patient (two unrelated individuals each) and likely represent population-specific polymorphisms.

Adult↗

PubMed1993

Estrogen receptor mutations in breast cancer.

It is fairly well accepted that the presence of estrogen receptor (ER) identifies those breast cancer patients with a lower risk of relapse and better overall survival [Clark and McGuire, 1988], and the measurement of ER has become a standard assay in the clinical management of breast cancer. Receptor status also provides a guideline for those tumors which may be responsive to hormonal intervention [McGuire 1978; Osborne et al., 1980; Rose et al., 1985]. But only about half of ER-positive patients will respond to the various hormonal therapies available, and of those who do initially respond, most will eventually develop hormonally unresponsive disease following a period of treatment even though ER is often still present. Loss of ER from initially ER-positive tumors biopsied again at a later date has been estimated at only 19% [Gross et al., 1984]. Obviously the simple measurement of ER presence by ligand-binding assays does not provide us with an adequate estimate of the functional state of the receptor. In 1985 Sluyser and Mester hypothesized that the loss of hormone dependence of certain breast tumors may be due to the presence of mutated or truncated steroid receptors that activate transcription even in the absence of hormone [Sluyser and Mester, 1985]. Based on the recent identification of several ER sequence variants in human breast cancer cell lines and tumor specimens, we would now like to propose that some of these identified mutations play a role in receptor dysfunction in vivo, and will review those ER mutations which may prove to be important in breast cancer progression.

Breast Neoplasms↗

PubMed1995

Utilization of monoclonal-antibody-based assay (HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies.

Sickle cell disease covers a group of conditions in which pathology may be attributed to the presence of sickle hemoglobin (HbS). The identification of HbS and other variants including those in combination with HbS is commonly achieved by cellulose acetate electrophoresis at alkaline pH. Because many hemoglobin variants with similar charges have similar electrophoretic migration patterns, they are difficult to differentiate by electrophoresis. The HemoCard assays address this concern through the use of monoclonal antibodies capable of specifically recognizing the unique amino acid substitution in the variant hemoglobin. The panel of HemoCard monoclonal antibodies confirms the absence and presence of HbA, HbC, HbE, HbS, and other sickling hemoglobin variants. The combination of alkaline cellulose acetate electrophoresis and HemoCard assays allows the technologist to reach a final conformation of both common and much less common sickle cell disease genotypes, combinations of HbS with other hemoglobins that ordinarily do not produce sickle cell disease, and other clinically important hemoglobinopathies including HbE/beta-thalassemia and hemoglobin C disease.

Anemia, Sickle Cell↗

PubMed2005

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.

Allergens↗

PubMed1994

A new Italian case of lipoprotein lipase deficiency: a Leu365- > Val change resulting in loss of enzyme activity.

We describe a second Italian family with primary Lipoprotein Lipase deficiency. A new mutation in exon 8 causes a Leu365- > Val change resulting in severe mass reduction and loss of enzyme activity. We suggest that this change interferes with the correct folding and stability of the protein and impairs the assembly of the active homodimer. The procedures applied are useful to screen a large sample of population for genetic variants and allow the clear identification of asymptomatic heterozygous subjects at risk from atherosclerosis disease.

Adult↗

PubMed2002

Altering retroviral tropism using a random-display envelope library.

Tissue-specific gene delivery is an important aspect of many gene therapy applications. The experiments reported here constitute the first successful demonstration that cell-specific entry can be obtained by screening a random library of retroviral envelope proteins produced from a mammalian cell system. The library consisted of 10(6) different subgroup A feline leukemia virus envelope protein variants with 10 randomly substituted amino acids in the receptor-determining region. Selecting the library for fully functional envelope proteins able to mediate stable gene transfer resulted in the identification of a single envelope protein variant (EF). Subsequent examination of the host range of EF revealed that it was highly specific for D17 canine osteosarcoma cells. This was in contrast to the host ranges of the parental subgroup A and closely related subgroup C envelope proteins. Interference assays on D17 cells further indicated that receptor usage by EF was also altered compared with the A and C envelope proteins. The EF envelope protein thus isolated should be useful for studying gene therapy treatments of osteosarcoma in a large-animal model.

Amino Acid Sequence↗

PubMed1989

Characterization of embryo globulins encoded by the maize Glb genes.

Two of the most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins. One is a Mr 63,000 protein encoded by the Glb1 gene and the other is a Mr 45,000 component encoded by the Glb2 gene. Both proteins accumulate to high levels during embryo development and are rapidly degraded during the early stages of seed germination. Amino acid composition analysis indicates that these proteins may serve as storage reserves to provide sources of nitrogen and carbon to the germinating embryo. Amino-terminal sequence analysis of the final Glb1 gene product, GLB1, and its immediate precursor, GLB1', indicates that the latter is proteolytically cleaved near the amino terminus to form GLB1. In addition to these biochemical studies, we describe the identification of a novel maize variant which lacks the protein product of the Glb2 gene.

Amino Acid Sequence↗

PubMed2005

Isolation and characterization of the leucine-rich proteoglycan nyctalopin gene (cNyx) from chick.

We describe the isolation and molecular characterization of the chick ortholog of nyctalopin (NYX), the gene responsible for X-linked complete congenital stationary night blindness (CSNB1, also known as cCSNB). Chick Nyx (cNyx) comprises four exons spanning approximately 6.2 kb on Chromosome 1 and encodes a protein of 473 amino acids that shares 55% identity overall with its human counterpart. cNyx is expressed in both the developing and the fully differentiated retina. Transcripts are localized primarily to cells within the outer half of the inner nuclear layer (INL) and the ganglion cell layer (GCL), a pattern consistent with the principal electrophysiologic findings in CSNB1 that suggest a main defect in depolarizing ON-bipolar cells normally located in the outer half of the INL. Expression (albeit weaker) was also detected in the cerebrum and cerebellum and in non-neuronal tissues. Finally, we also report the identification of three novel splice variants, one of which predominates in the retina.

Amino Acid Sequence↗