Peroxisome proliferator-activated receptor gamma 2 and acyl-CoA synthetase 5 polymorphisms influence diet response

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness.     

Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. Group V is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.     

Sensing complex regulatory networks by conformationally controlled hairpin ribozymes

The hairpin ribozyme catalyses RNA cleavage by a mechanism utilizing its conformational flexibility during the docking of two independently folded internal loop domains A and B. Based on this mechanism, we designed hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides influencing the docking process. We incorporated a third domain C to assimilate alternate stable RNA motifs such as a pseudo-half-knot or an internal stem-loop structure. Small sequence changes in domain C allowed targeted switching of ribozyme activity: the same effector oligonucleotide can either serve as an inducer or repressor. The ribozymes were applied to trp leader mRNA, the RNA sequence tightly bound by l-tryptophan-activated trp-RNA-binding attenuation protein (TRAP). When domain C is complementary to this mRNA, ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. This approach allows to precisely sense the activity status of a protein controlled by its metabolite molecule.     

Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.     

The synovial proteome: analysis of fibroblast-like synoviocytes

The present studies were initiated to determine the protein expression patterns of fibroblast-like synovial (FLS) cells derived from the synovia of rheumatoid arthritis patients. The cellular proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the in-gel digested proteins were analyzed by matrix-assisted laser desorption ionization mass spectrometry. A total of 368 spots were examined and 254 identifications were made. The studies identified a number of proteins that have been implicated in the normal or pathological FLS function (e.g. uridine diphosphoglucose dehydrogenase, galectin 1 and galectin 3) or that have been characterized as potential autoantigens in rheumatoid arthritis (e.g. BiP, colligin, HC gp-39). A novel uncharacterized protein product of chromosome 19 open reading frame 10 was also detected as an apparently major component of FLS cells. These results demonstrate the utility of high-content proteomic approaches in the analysis of FLS composition.     

Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure

Studies on the status of multifunctional Ca(2+)-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 +/- 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr(17)) and serine-16 (PLB-Ser(16)) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr(17) and PLB-Ser(16) levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.     

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.     

Pregnancy outcome post renal transplantation

BACKGROUND: The success in performing organ transplantations and prevention of rejection has resulted not only in a substantial increase in life expectancy, but also improvement in the patients' quality of life. Thus, women who underwent organ transplantation are now reaching puberty and the age of reproduction. This has presented new challenges regarding the teratogenicity and the long-term effect of immunosuppressive medications used by these patients. Previous studies have shown that pregnancies after renal transplantation are associated with an increased risk for both the mother and the fetus. There is, however, very little information available on neonatal and long-term pediatric follow-up of babies born to mothers who have undergone renal transplantation and have been exposed to immunosuppressive medications, compared to controls. We report the experience of our center, the largest in Canada, regarding the prenatal and long-term postnatal outcome of pregnancies after renal transplantation. METHODS: This is a retrospective case series reporting the outcome of 44 consecutive pregnancies followed by the Toronto Renal Transplant Program. Follow-up data were gathered on the 32 live born children by either a return visit to the clinic or by telephone interview. Medical, as well as developmental information, was gathered on all children and the study group was compared to controls, matched for maternal age (+/-2 years) and smoking status, obtained through the Motherisk Program. RESULTS: Of the 44 pregnancies followed by us, there were 32 live-born children delivered by 26 mothers and 12 stillborn/abortuses. Twenty-six pregnancies were treated with cyclosporine, azathioprine and prednisone, 13 with azathioprine and prednisone and five with cyclosporine and prednisone. The mean gestational age at delivery in the study group was 36.5 +/- 2.7 weeks compared to 40.2 +/- 1.6 weeks in the control group (P < 0.001). The mean birthweight in the study group was 2.54 +/- 0.67 kg, compared to 3.59 +/- 0.53 kg in the control group (P < 0.0001). In the study group there was one child with multiple anomalies and four stillbirths compared to zero in the control group. There were also six spontaneous abortions and two therapeutic abortions in the study group. On follow-up (from 3 months to 11 years of age) there was one child with insulin-dependent diabetes mellitus, two children with asthma and one child with recurrent otitis media. Developmental follow-up revealed one child with moderate to severe sensorineural hearing loss, one child with a learning disability and one child with pervasive developmental disorder. In none of these cases were there signs of perinatal asphyxia. CONCLUSION: There are significantly more stillbirths, preterm deliveries and increased incidence of low birth weight in the transplant group. Most pregnancies in the study group went well, however, and their offspring had normal postnatal growth and development. Further studies with long-term pediatric follow-up are needed to delineate their outcome and rule out possible long term effects of the immunosuppressive medication on their growth, development, reproduction and general health.