Using Community-Based Participatory Research to Disseminate a Mass Media Campaign Into Rural Communities.

The authors present the results of a media documentary, Weight of the Nation, disseminated in rural communities in the Brazos Valley region of east central Texas. Researchers relied on a community-based participatory research strategy to assure community participation in the implementation and evaluation of the media documentary in rural communities. To measure the short-term effects of the documentary, the research team used a mixed-methods approach of quantitative panel data from a pre/post survey, qualitative meeting notes, and observations from facilitated discussion groups. Results showed short-term increases in behavioral intention, as well as an increase in self and collective efficacy of participants to make healthy changes at individual and community levels to reduce obesity. The findings suggest that Weight of the Nation is a catalyst for increasing awareness about obesity and initiating changes in intention and efficacy perceptions.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme: differential expression of alternatively spliced variants in Drosophila tissues.

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A. We performed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1 mRNA were found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K(0.5)) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.

Assessing theoretical risk and benefit suggested by genetic association studies of CCR5: experience in a drug development programme for maraviroc.

The proliferation of published gene association studies of the CCR5delta32 mutation is of relevance to drug development of a CCR5 antagonist for HIV, in highlighting potential safety concerns. We conducted an initial review of all non-HIV gene association studies of CCR5-delta32, followed by detailed meta-analyses in the three disease areas most commonly reported. Our review indicated no consistent evidence of increased risk of susceptibility to hepatitis C virus infection or multiple sclerosis among individuals with CCR5-delta32 mutation, and suggested treatment with a CCR5 inhibitor is unlikely to have related adverse effects. There was, however, evidence to suggest rheumatoid arthritis as a potential therapeutic target for a CCR5 antagonist. Clinical evidence would be required to confirm these findings.

Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N-C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N-C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N-C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N-C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24-->Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40-47) as being involved in the chemical N-C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.

Report on EU-USA workshop: how systems biology can advance cancer research (27 October 2008).

The main conclusion is that systems biology approaches can indeed advance cancer research, having already proved successful in a very wide variety of cancer-related areas, and are likely to prove superior to many current research strategies. Major points include: Systems biology and computational approaches can make important contributions to research and development in key clinical aspects of cancer and of cancer treatment, and should be developed for understanding and application to diagnosis, biomarkers, cancer progression, drug development and treatment strategies. Development of new measurement technologies is central to successful systems approaches, and should be strongly encouraged. The systems view of disease combined with these new technologies and novel computational tools will over the next 5-20 years lead to medicine that is predictive, personalized, preventive and participatory (P4 medicine).Major initiatives are in progress to gather extremely wide ranges of data for both somatic and germ-line genetic variations, as well as gene, transcript, protein and metabolite expression profiles that are cancer-relevant. Electronic databases and repositories play a central role to store and analyze these data. These resources need to be developed and sustained. Understanding cellular pathways is crucial in cancer research, and these pathways need to be considered in the context of the progression of cancer at various stages. At all stages of cancer progression, major areas require modelling via systems and developmental biology methods including immune system reactions, angiogenesis and tumour progression.A number of mathematical models of an analytical or computational nature have been developed that can give detailed insights into the dynamics of cancer-relevant systems. These models should be further integrated across multiple levels of biological organization in conjunction with analysis of laboratory and clinical data.Biomarkers represent major tools in determining the presence of cancer, its progression and the responses to treatments. There is a need for sets of high-quality annotated clinical samples, enabling comparisons across different diseases and the quantitative simulation of major pathways leading to biomarker development and analysis of drug effects.Education is recognized as a key component in the success of any systems biology programme, especially for applications to cancer research. It is recognized that a balance needs to be found between the need to be interdisciplinary and the necessity of having extensive specialist knowledge in particular areas.A proposal from this workshop is to explore one or more types of cancer over the full scale of their progression, for example glioblastoma or colon cancer. Such an exemplar project would require all the experimental and computational tools available for the generation and analysis of quantitative data over the entire hierarchy of biological information. These tools and approaches could be mobilized to understand, detect and treat cancerous processes and establish methods applicable across a wide range of cancers.

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity.

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

A novel brain-expressed protein related to carnitine palmitoyltransferase I.

Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the N-terminus of the muscle isoform influence the kinetic properties of the enzyme.

The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases; the sequences of ovine CPT1A and CPT1B cDNAs have the accession numbers Y18387 and AJ272435 respectively and the partial adipose tissue and liver CPT1A clones have the accession numbers Y18830 and Y18829 respectively. Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid beta-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes ( CPT1A and CPT1B ). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5'-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the K (m) for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

Structural and functional genomics of the CPT1B gene for muscle-type carnitine palmitoyltransferase I in mammals.

Muscle-type carnitine palmitoyltransferase I (M-CPT I) is a key enzyme in the control of beta-oxidation of long-chain fatty acids in the heart and skeletal muscle. Because knowledge of the mammalian genes encoding M-CPT I may aid in studies of disturbed energy metabolism, we obtained new genomic and cDNA data for M-CPT I for the human, mouse, rat, and sheep. The introns of these compact genes are 80% (mouse versus rat) and 60% (mouse versus human) identical. Sheep and goat, but not cow, pig, rodent, or human promoter sequences contain a short interspersed repeated sequence (SINE) upstream of highly conserved regulatory elements. These elements constitute two promoters in humans, sheep, and mice, and, contrary to previous reports, there is a second promoter in rats as well. Thus, the transcriptional organization of these genes is more uniform than previously supposed, with interspecies differences in the 5'-ends of the mRNAs reflecting differences in splicing; only in humans extensive splicing and splice variation is found in the 5'- and 3'-untranslated regions. In the mouse, intron retention was detected in heart, muscle, and testes and may indicate an additional mechanism of regulation of M-CPT I expression. Splice variation in the coding region was previously proposed to lead to expression of CPT I enzymes with altered malonyl-CoA sensitivity (Yu, G. S., Lu, Y. C., and Gulick, T. (1998) Biochem. J. 334, 225-231). However, when expressed in the yeast Pichia pastoris, none of three earlier described splice variants had CPT I activity. Therefore, the involvement of splice variation of M-CPT I in the modulation of malonyl-CoA inhibition of fatty acid oxidation may be less relevant than hitherto assumed.