Tight junction proteins in gallbladder epithelium: different expression in acute acalculous and calculous cholecystitis.

There is a paucity of information of tight junction (TJ) proteins in gallbladder epithelium, and disturbances in the structure of these proteins may play a role in the pathogenesis of acute acalculous cholecystitis (AAC) and acute calculous cholecystitis (ACC). Using immunohistochemistry, we investigated the expression of TJ proteins claudin-1, -2, -3, and -4, occludin, zonula occludens (ZO-1), and E-cadherin in 9 normal gallbladders, 30 gallbladders with AAC, and 21 gallbladders with ACC. The number of positive epithelial and endothelial cells and the intensity of the immunoreaction were determined. Membrane-bound and cytoplasmic immunoreactivities were separately assessed. We found that TJ proteins were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, expression of cytoplasmic occludin and claudin-1 were decreased, as compared with normal gallbladder. In ACC, expression of claudin-2 was increased, and expression of claudin-1, -3, and -4, occludin, and ZO-1 were decreased, as compared with normal gallbladder or AAC. We conclude that there are significant differences in expression of TJ proteins in AAC and ACC, supporting the idea that AAC represents a manifestation of systemic inflammatory disease, whereas ACC is a local inflammatory and often infectious disease.     

Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.     

Polyamine-dependent alterations in the structure of microfilaments,golgi apparatus,endoplasmic reticulum,and proteoglycan synthesis in BHK cells

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [³H]glucosamine and [³⁵S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165-174, 1997. © 1997 Wiley-Liss, Inc.     

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.     

Relationships among Body Condition,Insulin Resistance and Subcutaneous Adipose Tissue Gene Expression during the Grazing Season in Mares

Obesity and insulin resistance have been shown to be risk factors for laminitis in horses. The objective of the study was to determine the effect of changes in body condition during the grazing season on insulin resistance and the expression of genes associated with obesity and insulin resistance in subcutaneous adipose tissue (SAT). Sixteen Finnhorse mares were grazing either on cultivated high-yielding pasture (CG) or semi-natural grassland (NG) from the end of May to the beginning of September. Body measurements, intravenous glucose tolerance test (IVGTT), and neck and tailhead SAT gene expressions were measured in May and September. At the end of grazing, CG had higher median body condition score (7 vs. 5.4, interquartile range 0.25 vs. 0.43; P=0.05) and body weight (618 kg vs. 572 kg ± 10.21 (mean ± SEM); P=0.02), and larger waist circumference (P=0.03) than NG. Neck fat thickness was not different between treatments. However, tailhead fat thickness was smaller in CG compared to NG in May (P=0.04), but this difference disappeared in September. Greater basal and peak insulin concentrations, and faster glucose clearance rate (P=0.03) during IVGTT were observed in CG compared to NG in September. A greater decrease in plasma non-esterified fatty acids during IVGTT (P<0.05) was noticed in CG compared to NG after grazing. There was down-regulation of insulin receptor, retinol binding protein 4, leptin, and monocyte chemoattractant protein-1, and up-regulation of adiponectin (ADIPOQ), adiponectin receptor 1 and stearoyl-CoA desaturase (SCD) gene expressions in SAT of both groups during the grazing season (P<0.05). Positive correlations were observed between ADIPOQ and its receptors and between SCD and ADIPOQ in SAT (P<0.01). In conclusion, grazing on CG had a moderate effect on responses during IVGTT, but did not trigger insulin resistance. Significant temporal differences in gene expression profiles were observed during the grazing season.     

The Human Obesity Gene Map: The 2002 Update

This is the ninth update of the human obesity gene map, incorporating published results through October 2002 and continuing the previous format. Evidence from single‐gene mutation obesity cases, Mendelian disorders exhibiting obesity as a clinical feature, quantitative trait loci (QTLs) from human genome‐wide scans and various animal crossbreeding experiments, and association and linkage studies with candidate genes and other markers is reviewed. For the first time, transgenic and knockout murine models exhibiting obesity as a phenotype are incorporated (N = 38). As of October 2002, 33 Mendelian syndromes relevant to human obesity have been mapped to a genomic region, and the causal genes or strong candidates have been identified for 23 of these syndromes. QTLs reported from animal models currently number 168; there are 68 human QTLs for obesity phenotypes from genome‐wide scans. Additionally, significant linkage peaks with candidate genes have been identified in targeted studies. Seven genomic regions harbor QTLs replicated among two to five studies. Attempts to relate DNA sequence variation in specific genes to obesity phenotypes continue to grow, with 222 studies reporting positive associations with 71 candidate genes. Fifteen such candidate genes are supported by at least five positive studies. The obesity gene map shows putative loci on all chromosomes except Y. More than 300 genes, markers, and chromosomal regions have been associated or linked with human obesity phenotypes. The electronic version of the map with links to useful sites can be found at http:obesitygene.pbrc.edu .     

Flycatchers Copy Conspecifics in Nest-Site Selection but Neither Personal Experience nor Frequency of Tutors Have an Effect

Using the behavior of others in guiding one''s own behavior is a common strategy in animals. The prevailing theory predicts that young age and the inexperience of an individual are expected to increase the probability of adopting the behaviors of others. Also, the most common behavior in the population should be copied. Here, we tested the above predictions by examining social information use in the selection of nest-site features with a field experiment using a wild cavity nesting bird, the collared flycatcher (Ficedula albicollis). We used an experimental design in which geometric symbols depict nest-site features. By manipulating the apparent symbol choices of early settled individuals and monitoring the choices of later arriving birds, we can study social information use without bias from learned or innate preferences. Flycatchers were found to use social information in the selection of nest-site features, with about 60% of the population preferring the manipulated conspecific choices. However, age and experience as explanatory factors suggested by the social information use theory did not explain the choices. The present result, in concert with earlier similar experiments, implies that flycatchers may in some situations rely more on interspecific information in the selection of nest-site characteristics.     

Genomic scan for maximal oxygen uptake and its response to training in the HERITAGE Family Study.

This study aimed to identify human genomic regions that are linked to maximal oxygen uptake (VO(2 max)) in sedentary individuals or to the responsiveness of VO(2 max) to a standardized endurance training program. The results of a genomic scan based on 289 polymorphic markers covering all 22 pairs of autosomes performed on the Caucasian families of the HERITAGE Family Study are presented. The mean spacing of the markers was 11 cM, and a total of 99 families and 415 pairs of siblings were available for the study. VO(2 max) in the sedentary state was adjusted for the effects of age, sex, body mass, fat mass, and fat-free mass, whereas the VO(2 max) response was adjusted for age and baseline level of the phenotype. Two analytic strategies were used: a single-point linkage procedure using all available pairs of siblings (SIBPAL) and a multipoint variance components approach using all the family data (SEGPATH). Results indicate that linkages at P values of 0.01 and better are observed with markers on 4q, 8q, 11p, and 14q for VO(2 max) before training and with markers on 1p, 2p, 4q, 6p, and 11p for the change in VO(2 max) in response to a 20-wk standardized endurance training program. These chromosomal regions harbor many genes that may qualify as candidate genes for these quantitative traits. They should be investigated in this and other cohorts.     

Intra-annual tracheid formation of Norway spruce provenances in southern Finland

We studied the intra-annual wood formation in a Norway spruce provenance experiment in southern Finland from 2004–2008. Two Finnish provenances, northern and southern, as well as German and Hungarian provenances were included. Timing of tracheid formation and differentiation, and tracheid dimensions were determined from periodically extracted microcores. The aim was to determine the differences between the years and provenances in the timing of the xylogenesis and in the xylem characteristics. Year-to-year variation was high both in timing of tracheid formation and xylem characteristics, while between-provenance differences were small. The onset of tracheid formation varied from early May to late June in different trees in different years. The onset of tracheid formation was not closely related to the annual variations of temperature sum. In all the years, daily temperatures exceeded the threshold +5°C for several weeks before the onset of tracheid formation. The highest tracheid formation rate occurred after the summer solstice in all years and generally coincided with the highest daily temperatures during the growing season. Tracheid production ceased early in 2006 due to a mid-summer drought. Cell differentiation continued late in autumn as non-mature tracheids were still observed around mid-September. No clear differences between the provenances in the timing of tracheid formation were observed, although the Finnish provenances tended to initiate tracheid formation slightly earlier than the other provenances. The tree-ring widths of the Finnish provenances were also wider, while tracheid diameter of the German provenance was slightly smaller. Our results indicate that between-tree variation in the timing of wood formation is high compared with the latitude effect of seed source.     

Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A

The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.