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Biomedical subjects

M Knight

Publications and source records attributed to M Knight.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of the myoglobin and its coding gene of the mollusc Biomphalaria glabrata.

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature. The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508). The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

Amino Acid Sequence↗
PubMed1998

The correlation of clinical phenotype in Friedreich ataxia with the site of point mutations in the FRDA gene.

Most cases of Friedreich ataxia (FRDA) are due to expansions of a GAA trinucleotide repeat sequence in the FRDA gene coding for frataxin, a protein of poorly understood function which may regulate mitochondrial iron transport. However, between 1% and 5% of mutations are single base changes in the sequence of the FRDA gene, causing missense, nonsense, or splicing mutations. We describe three new mutations, IVS4nt2 (T to G), R165C, and L182F, which occur in patients in association with GAA expansions. These cases, and a further five reported cases of point mutations causing FRDA, demonstrate that splicing, nonsense, or initiation codon mutations (which cause a complete absence of functional frataxin) are associated with a severe phenotype. Missense mutations, even in highly evolutionally conserved amino acids, may cause a mild or severe phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm.

Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I.

Amino Acid Sequence↗
PubMed1998

Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication.

Individuals with the recessive condition trimethylaminuria exhibit variation in metabolic detoxication of xenobiotics by hepatic flavin-containing monooxygenases. We show here that mutations in the human flavin-containing monooxygenase isoform 3 gene ( FMO3 ) impair N -oxygenation of xenobiotics and are responsible for the trimethylaminuria phenotype. Three disease-causing mutations in nine Australian-born probands have been identified which share a particular polymorphic haplotype. Nonsense and missense mutations are associated with a severe phenotype and are also implicated in impaired metabolism of other nitrogen- and sulfur-containing substrates including biogenic amines, both clinically and when mutated proteins expressed from cDNA are studied in vitro . These findings illustrate the critical role played by human FMO3 in the metabolism of xenobiotic substrates and endogenous amines.

Adolescent↗
PubMed1998

Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia.

OBJECTIVE: To determine whether circulating markers of oxidative stress are elevated in pre-eclampsia when appropriate precautions are taken to prevent in vitro oxidation DESIGN: A prospective study. SETTING: Nuffield Department of Obstetrics and Gynaecology, Oxford and The William Harvey Institute, London. SAMPLE: Three groups of women: those with pre-eclampsia (n = 19), control pregnant women (n = 19) matched for gestation, age and parity and a group of non pregnant individuals of reproductive age (n = 7). METHODS: Citrated plasma was stored at -80 degrees C with 20 micromol beta hydroxytoluene to prevent auto-oxidation. Plasma samples were assayed for levels of 8 epi-prostaglandin F2alpha, lipid hydroperoxides, malondialdehyde and also the lipid soluble antioxidant vitamin E. RESULTS: There were no differences in 8 epi-prostaglandin F2alpha, lipid peroxide or malondialdehyde levels between the groups of women with pre-eclampsia and those acting as pregnant controls. However, lipid hydroperoxides and malondialdehyde were significantly raised in both pre-eclampsia and normal pregnancy, compared with nonpregnant women. Vitamin E levels were similar in women with pre-eclampsia and those with a normal pregnancy, but in both groups levels were significantly higher than in nonpregnant women. CONCLUSION: Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia.

Adult↗
PubMed1998

Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies.

OBJECTIVE: To investigate whether syncytiotrophoblast microvilli (STBM) are shed into the maternal circulation in increased amounts in pre-eclamptic pregnancies as a possible cause of maternal vascular endothelial dysfunction. DESIGN: A time-resolved fluoroimmunoassay was developed to measure STBM levels in peripheral and uterine venous plasma from normal pregnant and pre-eclamptic women. Three colour flow cytometry was used to assess the microparticulate nature of the STBM in pregnancy plasma. The effects of these plasmas on endothelial cell proliferation was compared and a correlation with the levels of STBM detected was sought. SETTING: A laboratory investigation using clinical samples obtained from an obstetric practice in a teaching hospital. SAMPLES: Peripheral venous plasma from 20 women with established pre-eclampsia, 20 normal pregnant women matched for age, gestation and parity, and 10 nonpregnant women of reproductive age. Paired uterine and peripheral venous plasma taken at caesarean section from 10 women with pre-eclampsia and 10 unmatched normal pregnant women. RESULTS: STBM were detected in the plasma of pregnant women by both flow cytometry and time-resolved fluoroimmunoassay. Significantly higher levels of STBM were found in women with established pre-eclampsia (P=0.01). STBM concentrations were higher in uterine venous plasma than in concurrently sampled peripheral venous plasma, confirming their placental origin. A significant correlation was found between the amount of STBM in the plasma and endothelial cell inhibitory activity. CONCLUSIONS: STBM are shed into the maternal circulation (microvillous deportation) and are present in significantly increased amounts in pre-eclamptic women. They may contribute to the endothelial dysfunction underlying the maternal syndrome of pre-eclampsia.

Adult↗
PubMed1997

(Stearyl, Norleucine17)VIP hybrid antagonizes VIP receptors on non-small cell lung cancer cells.

The effects of VIP receptor antagonists were investigated using non-small cell lung cancer (NSCLC) cells. By Northern blot and RT-PCR, VIP1 receptors were detected on NSCLC cell line NCI-H1299. VIPhybrid,(N-Stearyl-Norleucine17) VIPhybrid ((SN) VIPhybrid) and PTC4495 inhibited 125I-VIP binding to NCI-H1299 cells with IC50 values of 500, 30 and 5000 nM respectively. (SN) VIPhybrid (1 microM) had no effect on basal cAMP but strongly inhibited the increase in cAMP caused by 10 nM VIP. The order of peptide potency to inhibit cAMP was (SN) VIPhybrid > VIPhybrid > PTC4495. (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 colony formation. Also, (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 xenograft formation in nude mice. These data suggest that (SN) VIPhybrid antagonizes VIP1 receptors on NSCLC cells.

Amino Acid Sequence↗
PubMed1997

Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria.

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNA-expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.

DNA, Complementary↗
PubMed1997

Prevalence of dental caries in Omani 6-year-old children.

OBJECTIVE: To determine the prevalence of dental caries in Omani 6-year-old children. DESIGN: Clinical caries cross-sectional survey, conducted by 16 trained and calibrated dentists. SETTING: Omani primary schools in December 1994. SUBJECTS: 3,114 subjects, randomly selected to achieve an overall 6.6 per cent sample of Omani 6-year-old children. OUTCOME MEASURES: Caries diagnosis based solely on clinical examination in accordance with criteria of British Association for the Study of Community Dentistry. RESULTS: Only 484 subjects (15.5 per cent) were caries-free; regional variations ranging from 4.4 per cent to 31 per cent. Overall, the national dmft averaged 4.61; the majority of caries experienced being in the form of untreated decay, with occlusal surfaces of first primary molars being the most commonly involved site. CONCLUSIONS: Compared with results from a survey of Omani 12-year-old children in 1993, a much smaller proportion of this 6-year-old sample were caries free, emphasising the need for continuance of existing, and the development of further, preventive programmes.

Child↗
PubMed1996

Schistosoma mansoni: use of a subtractive cloning strategy to search for RFLPs in parasite-resistant Biomphalaria glabrata.

A subtractive cloning strategy has been applied for the identification of two cDNA clones whose corresponding transcripts were elevated in Schistosoma mansoni-resistant (BS-90) compared to susceptible (M-line) snails. Clone pBS11 encoded a 1.9-kb transcript that was more elevated compared to a 500-bp transcript encoded by clone pBS12. Consequently, more attention was focused on the molecular characterization of clone pBS11. Results showed that the transcript encoded by this clone was expressed in the albumen gland and was developmentally regulated. Sequence analysis of pBS11 demonstrated the presence of an open reading frame that corresponded to a novel Biomphalaria glabrata albumen gland gene product. Comparative Southern analysis of the resistant and susceptible snail lines using pBS11 as probe indicated the presence of a BamHI and EcoRI RFLP between the two strains.

Animals↗
PubMed1996

Children's understanding of the distinction between real and apparent emotions: a training study.

The effects of two types of training on 4-year-old children's understanding of the distinction between experienced positive affect and neutral or negative display were examined. One type of training provided first-hand experience of possible discrepancies between facial expression and actual affect. The other training focused on the sorts of motives that might prompt the concealment of positive affect. Compared with a control group, both experimental groups showed modest gains.

Child, Preschool↗
PubMed1996

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

The oral health status of Omani 12-year-olds--a national survey.

This study reports the first ever national oral health survey of Omani 12-year-olds. Conducted in October 1993, of the 3,435 children examined, 1,438 (41.9%) were caries-free, although regional variations ranged from 24.8% to 61.9%. Overall, the national DMFT averaged 1.53, the majority of caries experienced being in the form of untreated decay, with occlusal surfaces of first permanent molars being the most commonly involved site. Oral hygiene was poor, only 11% of those examined being scored as plaque-free.

Child↗