Influence of dietary status on liver palmitoyl-CoA hydrolase, peroxisomal enzymes, CoASH and long-chain acyl-CoA in rats

In the livers of fasted rats, the activity of mitochondrial palmitoyl-CoA hydrolase was increased whereas the microsomal palmitoyl-CoA hydrolase activity decreased. Refeeding with a high-carbohydrate diet (glucose), the corresponding enzyme activities were decreased while refeeding with a high-fat diet (sheep tallow) increased the enzyme activities over the control values. The increased content of long-chain acyl-CoA and free CoASH under fasting and fat-refeeding was mainly attributed to the mitochondrial fraction with the remainder in the light mitochondrial fraction which contains peroxisomes. The results suggest a correlation of the compartmentation of the palmitoyl-CoA hydrolase and the content and compartmentation of the CoA derivatives in the liver under different nutritional states. The peroxisomal palmitoyl-CoA oxidase activity was increased by fasting. Fat-refeeding increased the activity even more; 1.8-fold as compared to the fasting animals. On the other hand, the activities of other peroxisomal enzymes which are not directly involved in the fatty acid metabolism such as urate oxidase were decreased to approximately the same extent by fasting. Re-feeding with glucose and fat further decreased the corresponding enzyme activity, particularly seen in the glucose-refed group.     

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators

The present study has confirmed previous findings of long-chain acyl-CoA hydrolase activities in the mitochondrial and microsomal fractions of the normal rat liver. In addition, experimental evidence is presented in support of a peroxisomal localization of long-chain acyl-CoA hydrolase activity. (a) Analytical differential centrifugation of homogenates from normal rat liver revealed that this activity (using palmitoyl-CoA as the substrate) was also present in a population of particles with an average sedimentation coefficient of 6740 S, characteristic of peroxisomal marker enzymes. (b) The subcellular distribution of the hydrolase activity was greatly affected by administration of the peroxisomal proliferators clofibrate and tiadenol. The specific activity was enhanced in the mitochondrial fraction and in a population of particles with an average sedimentation coefficient of 4400 S, characteristic of peroxisomal marker enzymes. Three populations of particles containing lysosomal marker enzymes were found by analytical differential centrifugation, both in normal and clofibrate-treated rats. Our data do not support the proposal that palmitoyl-CoA hydrolase and acid phosphatase belong to the same subcellular particles. In livers from rats treated with peroxisomal proliferators, the specific activity of palmitoyl-CoA hydrolase was also enhanced in the particle-free supernatant. Evidence is presented that this activity at least in part, is related to the peroxisomal proliferation.     

Effects of a specific nutrient combination on ESBL resistance

Background and aimExtended-spectrum beta-lactamases are the main cause of resistance in Enterobacteriaceae to beta lactam antibiotics. The aim of this study was to evaluate the antimicrobial effect of EpiQuercican supplement, combined with different antimicrobial agents, on ESBL-producing isolates and determine the underlying molecular mechanism of resistance in these isolates.Materials and methodsEleven ESBL producing Enterobacteriaceae isolates were collected from Saudi Arabia hospitals between 2016 and 2017 and disk diffusion test was performed in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines to determine the susceptibility of the isolates to 5 different antibiotics in the presence of EpiQuercican supplement. Polymerase chain reaction was performed for detection of ESBL genes, and efflux pump inhibitor was used to study the mechanism of resistance in these isolates.ResultsThe best synergistic effect was obtained when the supplement was combined with carbapenems followed by 4th generation cephalosporins. Either no effect or antagonistic effect was seen with most of the isolates when the supplement was added to the 3rd generation of cephalosporins. Among the tested genes responsible for ESBL production in this study, our results indicated the predominance of TEM genes (73%) followed by CTX-M genes (9%). As for the mechanism of resistance in ESBL isolates, 4 isolates showed to use efflux pumps as their main mechanism of resistance.ConclusionThe EpiQuercican supplement showed some promising results, yet its antibacterial mechanism of action needs to be elucidated further.     

Wnt signaling mediates regional specification in the vertebrate face

At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.     

Oligoclonal Band Status in Scandinavian Multiple Sclerosis Patients Is Associated with Specific Genetic Risk Alleles

The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in multiple sclerosis (MS). We applied data from Norwegian, Swedish and Danish (i.e. Scandinavian) MS patients from a genome-wide association study (GWAS) to search for genetic differences in MS relating to OCB status. GWAS data was compared in 1367 OCB positive and 161 OCB negative Scandinavian MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. HLA-DRB1 genotypes were analyzed in a subset of the OCB positive (n = 2781) and OCB negative (n = 292) MS patients and compared to 890 healthy controls. Results from the genome-wide analyses showed that single nucleotide polymorphisms (SNPs) from the HLA complex and six other loci were associated to OCB status. In SNPs selected for replication, combined analyses showed genome-wide significant association for two SNPs in the HLA complex; rs3129871 (p = 5.7×10⁻¹⁵) and rs3817963 (p = 5.7×10⁻¹⁰) correlating with the HLA-DRB1*15 and the HLA-DRB1*04 alleles, respectively. We also found suggestive association to one SNP in the Calsyntenin-2 gene (p = 8.83×10⁻⁷). In HLA-DRB1 analyses HLA-DRB1*15∶01 was a stronger risk factor for OCB positive than OCB negative MS, whereas HLA-DRB1*04∶04 was associated with increased risk of OCB negative MS and reduced risk of OCB positive MS. Protective effects of HLA-DRB1*01∶01 and HLA-DRB1*07∶01 were detected in both groups. The groups were different with regard to age at onset (AAO), MS outcome measures and gender. This study confirms both shared and distinct genetic risk for MS subtypes in the Scandinavian population defined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS with implications for patient management, which need to be further studied.     

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.     

Effects of thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation. Studies in vivo and in vitro.

1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of beta-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial beta-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.     

Regulation of FynT function by dual domain docking on PAG/Cbp

In resting T-cells, the transmembrane adaptor protein PAG (phosphoprotein associated with glycosphingolipid-enriched microdomains) is constitutively tyrosine-phosphorylated, a state maintained by the Src family kinase FynT. PAG has a role in negative regulation of Src family kinases in T-cells by recruitment of Csk (C-terminal Src kinase) to the membrane via binding to PAG phosphotyrosine 317. The interaction between FynT and PAG is essential for PAG function; however, so far the FynT binding mode has been unknown. Here, we demonstrate that the FynT-PAG complex formation is a dual domain docking process, involving SH2 domain binding to PAG phosphotyrosines as well as an SH3 domain interaction with the first proline-rich region of PAG. This binding mode affects FynT kinase activity, PAG phosphorylation, and recruitment of FynT and Csk, demonstrated in Jurkat TAg cells after antibody stimulation of the T cell receptor. Furthermore, we show that TCR-induced tyrosine phosphorylation is regulated by SH3 domain modulation of the FynT-PAG interaction in human primary T-cells. Although FynT SH3 domain association is shown to be crucial for efficiently initiating PAG phosphorylation, we suggest that engagement of the SH2 domain on PAG renders FynT insensitive to Csk negative regulation. Thus, in T-cells, PAG is involved in positive as well as negative regulation of FynT activity.     

Vertical flux of particulate matter in an Arctic fjord: the case of lack of the sea-ice cover in Adventfjorden 2006–2007

Seasonal dynamics of suspended minerals, organic matter, particulate, and dissolved organic carbon (DOC), chlorophyll, and their vertical fluxes were studied in a small Arctic fjord (Adventfjorden, Spitsbergen) from November 2006 to October 2007. The fjord was usually covered with fast ice in winter, but remained open throughout the year since 2005. The open-water winter period caused increased wave action and resuspension of organic and mineral particles. The lack of sea-ice in spring accelerated the onset of the productive season. The earlier light signal also caused an earlier appearance of mesozooplanktonic organisms, accompanied by a significant increase of the DOC pool in the water. In the cold period (winter and spring) 47% of the annual organic matter settled on the sampling site, a lot coming from the spring diatom bloom in April. Summer melt (July, August) resulted in turbid and brackish fjord surface water with stratification and increase of both suspended particles and sedimentation, causing 60% of the annual minerals and 53% of the annual organic matter to settle at the sampling site. Increased dissolved organic carbon (DOC) through sloppy feeding on the mixo- and heterotrophic protista by the abundant mesozooplankton indicated intensive secondary production, resulting in a maximum fecal pellets flux of >5 mg C m⁻² day⁻¹. A warmer climate with reduced sea-ice cover in fjords will advance the onset of the spring bloom and will also result in a larger input of turbid melt water in summer, restricting the light availability and enhancing flocculation and thereby sedimentation.