Plasma carnitine ester profiles in Crohn's disease and ulcerative colitis patients with different IGR2230a_1 genotypes

An association has been repeatedly demonstrated between inflammatory bowel disease (IBD) and the IBD5 locus in the 5q31 chromosomal region. The aim of the present study was to examine the prevalence of the IGR2230a_1 intronic nucleotide polymorphism of the slc22a5 gene (coding for the OCTN2 carnitine transporter protein) lying within this region, and its possible relationship with the carnitine metabolism in Hungarian IBD patients and controls. We genotyped by restriction fragment length polymorphism 200 Crohn's disease (CD) and 246 ulcerative colitis (UC) patients, as well as 187 healthy controls. From plasma samples we determined detailed carnitine ester profiles of 76 CD, 43 UC patients and 45 control persons using electrospray ionization triple quadruple tandem mass spectrometry. The distribution of the genotypes was not significantly different in the CD or the UC group compared with the controls. We found no significant alterations of the carnitine profile in the carrier/non‐carrier or the homozygote/non‐homozygote comparisons in both the CD and the UC groups, stratified by IGR2230a_1 genotype. Our data suggest that this polymorphism alone is not associated with CD and UC in the Hungarian population, and has no effect on the carnitine metabolism.     

PACAP-Mediated Neuroprotection of Neurochemically Identified Cell Types in MSG-Induced Retinal Degeneration

Pituitary adenylate cyclase-activating polypeptide (PACAP) is neuroprotective in animal models of different brain pathologies and injuries, including cerebral ischemia, Parkinson's disease, and different types of retinal degenerations. We have previously shown that PACAP is protective against monosodium glutamate (MSG)-induced retinal degeneration, where PACAP-treated retinas has more retained structure and PACAP induces anti-apoptotic while it inhibits pro-apoptotic signaling pathways. The aim of the present study was to investigate cell-type specific effects of PACAP in MSG-induced retinal degeneration by means of immunohistochemistry. Rat pups received MSG (2 mg/g b.w.) applied on postnatal days 1, 5, and 9. PACAP (100 pmol in 5 microl saline) was injected into the right vitreous body, while the left eye received only saline. Retinas were processed for immunocytochemistry after 3 weeks. Immunolabeling was determined for vesicular glutamate transporter 1, tyrosine hydroxylase, calretinin, calbindin, parvalbumin, and vesicular gamma-aminobutyric acid (GABA) transporter. In the MSG-treated retinas, the cell bodies and processes in the inner nuclear, inner plexiform, and ganglion cell layers displayed less immunoreactivity for all antisera. Apart from photoreceptors, only one major retinal cell type examined in this study; the calbindin-immunoreactive horizontal cell seemed not to be affected by MSG application. After simultaneous application of MSG and PACAP, staining of retinas was similar to that of normal eyes, with no significant alterations in immunoreactive patterns. These findings further support the neuroprotective function of PACAP in MSG-induced retinal degeneration.     

Oral L-carnitine supplementation in low-birth-weight newborns: a study on neonates requiring combined parenteral and enteral nutrition

Effect of L-carnitine supplementation on plasma ketone body (KB) and triglyceride (TG) concentrations was studied in ten premature infants requiring combined enteral and parenteral nutrition. At the second week of life (9 to 14 days of age) the infants were randomly divided into two groups. Five of them (plasma carnitine value, 33.77 +/- 2.48 mumol/l; mean +/- SEM) received oral L-carnitine supplementation (60 mumol/kg daily) added to pasteurized pooled human milk for seven consecutive days; additional five (plasma carnitine value, 36.70 +/- 5.19 mumol/l) served as controls. Composition of the daily diet was nearly constant in the study period. On the seventh day, prior to an Intralipid infusion, plasma carnitine and ketone body levels were significantly increased in the supplemented group as compared to controls or to previous values of the same group. In response to lipid infusion the fat load induced ketone body production was significantly higher in the supplemented group as compared to controls, whereas the triglycerides reached higher levels in the control group. It is suggested that L-carnitine supplementation in low-weight newborns promotes ketone body formation from endogenous stores as well as from exogenous fat supply, and thus may enhance triglyceride utilization.     

Plasma carnitine ester profiles in Crohn's disease patients characterized for SLC22A4 C1672T and SLC22A5 G-207C genotypes

Crohn's disease (CD) is a chronic inflammatory bowel disorder caused by environmental and genetic factors. The purpose of this study was to analyse the possible influence of functional variants of genes of OCTN cation transporters on the carnitine ester profile of patients with CD. Genotyping for SLC22A4 1672C --> T, SLC22A5-207G --> C mutations and three common NOD2 variants (R702W, G908R and 1007finsC) were performed in 100 adult CD patients and in ninety-four healthy controls by direct sequencing. The carnitine ester profile was determined using ESI triple quadrupole tandem MS. Contrary to the NOD2/CARD15 mutations, none of the SLC variants showed increased prevalence in the CD group, the prevalence of TC haplotype did not differ between the patients and the controls. In the mixed group of CD patients the fasting propionyl- (0.243 (sem 0.008) v. 0.283 (sem 0.014) micromol/l), butyryl- (0.274 (sem 0.009) v. 0.301 (sem 0.013)) and isovalerylcarnitine (0.147 (sem 0.006) v. 0.185 (sem 0.009)) levels were decreased; while the level of octenoyl- (0.086 (sem 0.006) v. 0.069 (sem 0.005)), myristoleyl- (0.048 (sem 0.003) v. 0.037 (sem 0.003)), palmitoyl- (0.140 (sem 0.005) v. 0.122 (sem 0.004)) and oleylcarnitine (0.172 (sem 0.006) v. 0.156 (sem 0.008); P < 0.05 in all comparisons) were increased. After sorting the patients into SLC22A genotype-specific subgroups, no significant differences could be observed between them. The carnitine ester profile data suggest selective involvement of the carnitine esters in CD patients, probably due to their altered metabolism.     

Rapid time-stamped analysis of filament motility

The in vitro motility assay is a valuable tool to understand motor protein mechanics, but existing algorithms are not optimized for accurate time resolution. We propose an algorithm that combines trace detection with a time-stamped analysis. By tracking filament ends, we minimize data loss from overlapping and crossing filaments. A movement trace formed by each filament end is created by time-stamping when the filament either first (filament tip) or last (filament tail) occupies a pixel. A frame number vs. distance curve is generated from this trace, which is segmented into regions by slope to detect stop-and-go movement. We show, using generated mock motility videos, accurate detection of velocity and motile fraction changes for velocities?<?0.05 pixels per frame, without manual trace dropping and regardless of filament crossings. Compared with established algorithms we show greatly improved accuracy in velocity and motile fraction estimation, with greatly reduced user effort. We tested two actual motility experiments: (1) adenosine triphosphate (ATP) added to skeletal myosin in rigor; (2) myosin light chain phosphatase (MLCP) added to phasic smooth muscle myosin. Our algorithm revealed previously undetectable features: (1) rapid increase in motile fraction paralleled by a slow increase in velocity as ATP concentration increases; (2) simultaneous reductions in velocity and motile fraction as MLCP diffuses into the motility chamber at very low velocities. Our algorithm surpasses existing algorithms in the resolution of time dependent changes in motile fraction and velocity at a wide range of filament lengths and velocities, with minimal user input and CPU time.     

Freeze‐dried human serum albumin improves the adherence and proliferation of mesenchymal stem cells on mineralized human bone allografts

Mineralized scaffolds are widely used as bone grafts with the assumption that bone marrow derived cells colonize and remodel them. This process is slow and often unreliable so we aimed to improve the biocompatibility of bone grafts by pre‐seeding them with human mesenchymal stem cells from either bone marrow or dental pulp. Under standard cell culture conditions very low number of seeded cells remained on the surface of freeze‐dried human or bovine bone graft or hydroxyapatite. Coating the scaffolds with fibronectin or collagen improved seeding efficiency but the cells failed to grow on the surface until the 18th day. In contrast, human albumin was a very potent facilitator of both seeding and proliferation on allografts which was further improved by culturing in a rotating bioreactor. Electron microscopy revealed that cells do not form a monolayer but span the pores, emphasizing the importance of pore size and microstructure. Albumin coated bone chips were able to unite a rat femoral segmental defect, while uncoated ones did not. Micro‐hardness measurements confirmed that albumin coating does not influence the physical characteristics of the scaffold, so it is possible to introduce albumin coating into the manufacturing process of lyophilized bone allografts. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:489–496, 2012     

Gene-gene and gene-environment interplay represent specific susceptibility for different types of ischaemic stroke and leukoaraiosis

Stroke is a very frequent entity. It is the third leading cause of death and the leading cause of adult disability in the developed world. At a population level, the common sporadic form of ischaemic stroke is underpinned by both environmental and genetic risk factors. Typically, in clinical practice, environmental risk factors such as hypertension, diabetes mellitus, smoking, alcohol consumption, and other factors, are usually considered to be more important than genetic factors. However, it is the interplay of both environmental and common genetic factors [such as the Leiden V, methylenetetrahydrofolate reductase C677T, apolipopotein E 4, endothelial nitric oxide synthase G894T, angiotensin-converting enzyme I/D and angiotensin II type 1 receptor A1166C mutations and polymorphisms] that leads to the development of ischaemic stroke. Indeed, a complex network of interactions between genetic factors and clinical risk factors can be supposed. This review evaluates the possible roles of gene-gene and gene-environment interactions concerning the above genetic factors in the evolution of ischaemic stroke and leukoaraiosis. A knowledge of the specific genetic patterns which are associated with a significant risk of ischaemic stroke or leukoaraiosis may also draw attention to a large population at an increased risk of circulatory disorders. This may facilitate the choice of more effective and specific prevention on the basis of the genotype.