Reporting of treatment fidelity in behavioural paediatric obesity intervention trials: a systematic review.

Behavioural interventions for paediatric obesity are promising, but detailed information on treatment fidelity (i.e. design, training, delivery, receipt and enactment) is needed to optimize the implementation of more effective interventions. Little is known about current practices for reporting treatment fidelity in paediatric obesity studies. This systematic review, in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, describes the methods used to report treatment fidelity in randomized controlled trials. Treatment fidelity was double-coded using the National Institutes of Health Fidelity Framework checklist. Three hundred articles (N = 193 studies) were included. Mean inter-coder reliability across items was 0.83 (SD = 0.09). Reporting of treatment design elements within the field was high (e.g. 77% of studies reported designed length of treatment session), but reporting of other domains was low (e.g. only 7% of studies reported length of treatment sessions delivered). Few reported gold standard methods to evaluate treatment fidelity (e.g. coding treatment content delivered). General study quality was associated with reporting of treatment fidelity (p < 0.01) as was the number of articles published for a given study (p < 0.01). The frequency of reporting treatment fidelity components has not improved over time (p = 0.26). Specific recommendations are made to support paediatric obesity researchers in leading health behaviour disciplines towards more rigorous measurement and reporting of treatment fidelity.

Cells of human aminopeptidase N (CD13) transgenic mice are infected by human coronavirus-229E in vitro, but not in vivo.

Aminopeptidase N, or CD13, is a receptor for serologically related coronaviruses of humans, pigs, and cats. A mouse line transgenic for the receptor of human coronavirus-229E (HCoV-229E) was created using human APN (hAPN) cDNA driven by a hAPN promoter. hAPN-transgenic mice expressed hAPN mRNA in the kidney, small intestine, liver, and lung. hAPN protein was specifically expressed on epithelial cells of the proximal convoluted renal tubules, bronchi, alveolar sacs, and intestinal villi. The hAPN expression pattern within transgenic mouse tissues matched that of mouse APN and was similar in mice heterozygous or homozygous for the transgene. Primary embryonic cells and bone marrow dendritic cells derived from hAPN-transgenic mice also expressed hAPN protein. Although hAPN-transgenic mice were resistant to HCoV-229E in vivo, primary embryonic cells and bone marrow dendritic cells were infected in vitro. hAPN-transgenic mice are valuable as a source of primary mouse cells expressing hAPN. This hAPN-transgenic line will also be used for crossbreeding experiments with other knockout, immune deficient, or transgenic mice to identify factors, in addition to hAPN, that are required for HCoV-229E infection.

Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.

Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase.

Succinate dehydrogenase (EC 1.3.99.1) is an intrinsic bacterial or inner mitochondrial membrane protein that catalyses the oxidation of succinate and donates electrons to the respiratory chain via quinone acceptors. It is a heterotetramer composed of a flavoprotein, an iron-sulfur, and two hydrophobic subunits. We purified succinate dehydrogenase by blue native gel electrophoresis, determined the amino-terminal sequence of the Sdh4p subunit and used this information to clone the SDH4 gene. It encodes a precursor protein of 181 amino acids that is converted to the 150-amino acid mature Sdh4p protein with a mass of 16,638 Da. Hydrophobicity analysis predicts that Sdh4p forms three transmembrane alpha-helices. We have constructed an SDH4 mutant by targeted gene disruption; it retains the ability to grow on rich glycerol medium. Western blot analysis of SDH4 disruption mutant membrane fractions indicates that membrane attachment of the flavoprotein and iron-sulfur subunits is impaired but not abolished. This membrane-bound enzyme is able to reduce ubiquinone, although less efficiently than the wild-type enzyme. These findings indicate that Sdh4p contributes both to the membrane attachment of the catalytic flavoprotein and iron-sulfur subunits and to electron transfer to ubiquinone.

Sequence homology between prokaryotic and eukaryotic forms of serine hydroxymethyltransferase.

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.

Structure and reactivity of cysteine residues in mitochondrial serine hydroxymethyltransferase.

Six cysteine-containing tryptic peptides were isolated and sequenced from rabbit liver mitochondrial serine hydroxymethyltransferase. 2 of the 6 cysteine residues were located on the surface of the enzyme. These 2 cysteine residues are sufficiently close to each other that they form a disulfide bond when oxidized by periodate. Another of the cysteine residues is exposed upon removal of the active site pyridoxal phosphate. The remaining three cysteines are buried and react with sulfhydryl reagents only when the enzyme is denatured. Blocking of one of the surface sulfhydryls with any of several sulfhydryl reagents results in increased catalytic activity when allothreonine is the substrate but decreased activity when serine and tetrahydrofolate are the substrates. The activation and inhibition effects are on Vmax and not on the affinity of the enzyme for its substrates. Of the six cysteine peptides from the mitochondrial enzyme, three show substantial homology with cysteine-containing peptides from the cytosolic form of the enzyme. For both enzyme forms, one of these homologous pairs is a cysteine residue on the surface of the enzyme. These results suggest that the mitochondrial and cytosolic forms of rabbit liver serine hydroxymethyltransferases are the products of separate genes.