Vascular smooth muscle cell detachment from elastin and migration through elastic laminae is promoted by chondroitin sulfate-induced “shedding” of the 67-kDa cell surface elastin binding protein

Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in AN-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a threedimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.     

INDUCIBILITY OF THYMIDINE KINASE BY THYMIDINE AS A FUNCTION OF INTERPHASE STAGE

Thymidine may act as an inducer of thymidine kinase activity in cells of higher plants. The general response is demonstrable in a randomly developing cell population such as is found in germinating wheat embryos. If a synchronously developing cell population is studied, however, potentially inducible cells are found to be susceptible to the inductive effect of thymidine only during about 10 per cent of the G1 period, and close to the interval when thymidine kinase activity normally appears.     

Stratified Whole Genome Linkage Analysis of Chiari Type I Malformation Implicates Known Klippel-Feil Syndrome Genes as Putative Disease Candidates

Chiari Type I Malformation (CMI) is characterized by displacement of the cerebellar tonsils below the base of the skull, resulting in significant neurologic morbidity. Although multiple lines of evidence support a genetic contribution to disease, no genes have been identified. We therefore conducted the largest whole genome linkage screen to date using 367 individuals from 66 families with at least two individuals presenting with nonsyndromic CMI with or without syringomyelia. Initial findings across all 66 families showed minimal evidence for linkage due to suspected genetic heterogeneity. In order to improve power to localize susceptibility genes, stratified linkage analyses were performed using clinical criteria to differentiate families based on etiologic factors. Families were stratified on the presence or absence of clinical features associated with connective tissue disorders (CTDs) since CMI and CTDs frequently co-occur and it has been proposed that CMI patients with CTDs represent a distinct class of patients with a different underlying disease mechanism. Stratified linkage analyses resulted in a marked increase in evidence of linkage to multiple genomic regions consistent with reduced genetic heterogeneity. Of particular interest were two regions (Chr8, Max LOD = 3.04; Chr12, Max LOD = 2.09) identified within the subset of “CTD-negative” families, both of which harbor growth differentiation factors (GDF6, GDF3) implicated in the development of Klippel-Feil syndrome (KFS). Interestingly, roughly 3–5% of CMI patients are diagnosed with KFS. In order to investigate the possibility that CMI and KFS are allelic, GDF3 and GDF6 were sequenced leading to the identification of a previously known KFS missense mutation and potential regulatory variants in GDF6. This study has demonstrated the value of reducing genetic heterogeneity by clinical stratification implicating several convincing biological candidates and further supporting the hypothesis that multiple, distinct mechanisms are responsible for CMI.     

Effective Part-Task Training as Evidence of Distinct Adaptive Processes with Different Time Scales

For some types of visuo-motor transformations like large visuo-motor rotations or the complex transformation of a sliding first-order lever, distinct adaptive processes have been hypothesized that produce a rapid, discrete approximation of the transformation and a slow, graded fine tuning, respectively. Here we investigate whether part-task training of only the second of these processes, namely the fine tuning, transfers to the subsequent performance in a condition with the full transformation of the sliding first-order lever. Therefore, we compared performance of three groups with different practice conditions during transfer to the full transformation. While two groups only practiced the fine tuning without the right-left inversion of the lever prior to transfer, a third group practiced the full lever transformation. Our results show a positive, but less than perfect transfer of the isolated practice of the fine tuning on performance with the full transformation. For the fine tuning itself, transfer was not reliably different from being perfect. The observation that the fine tuning can be acquired separately and added to the later adaptation to the left-right inversion of the lever supports the notion that these slow and fast processes progress rather independently. The additional finding that the preceding acquisition of the fine tuning also facilitates the subsequent rapid process could be due to generalized learning-to-learn or to a more precise assignment of movement errors to the process from which they originate.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Elemental Fingerprinting of Mussel Shells to Predict Population Sources and Redistribution Potential in the Gulf of Maine

As the climate warms, species that cannot tolerate changing conditions will only persist if they undergo range shifts. Redistribution ability may be particularly variable for benthic marine species that disperse as pelagic larvae in ocean currents. The blue mussel, Mytilus edulis, has recently experienced a warming-related range contraction in the southeastern USA and may face limitations to northward range shifts within the Gulf of Maine where dominant coastal currents flow southward. Thus, blue mussels might be especially vulnerable to warming, and understanding dispersal patterns is crucial given the species'' relatively long planktonic larval period (>1 month). To determine whether trace elemental “fingerprints” incorporated in mussel shells could be used to identify population sources (i.e. collection locations), we assessed the geographic variation in shell chemistry of blue mussels collected from seven populations between Cape Cod, Massachusetts and northern Maine. Across this ∼500 km of coastline, we were able to successfully predict population sources for over two-thirds of juvenile individuals, with almost 80% of juveniles classified within one site of their collection location and 97% correctly classified to region. These results indicate that significant differences in elemental signatures of mussel shells exist between open-coast sites separated by ∼50 km throughout the Gulf of Maine. Our findings suggest that elemental “fingerprinting” is a promising approach for predicting redistribution potential of the blue mussel, an ecologically and economically important species in the region.     

Fatty acid patterns of dog erythrocyte membranes after feeding of a fish-oil based DHA-rich supplement with a base diet low in n-3 fatty acids versus a diet containing added n-3 fatty acids

Background

Numerous signaling pathways function in the brain ventricular system, including the most important - GABAergic, glutaminergic and dopaminergic signaling. Purinergic signalization system - comprising nucleotide receptors, nucleotidases, ATP and adenosine and their degradation products - are also present in the brain. However, the precise role of nucleotide signalling pathway in the ventricular system has been not elucidated so far. The aim of our research was the identification of all three elements of purinergic signaling pathway in the porcine brain ventricular system.

Results

Besides nucleotide receptors on the ependymocytes surface, we studied purines and pyrimidines in the CSF, including mechanisms of nucleotide signaling in the swine model (Sus scrofa domestica). The results indicate presence of G proteins coupled P2Y receptors on ependymocytes and also P2X receptors engaged in fast signal transmission. Additionally we found in CSF nucleotides and adenosine in the concentration sufficient to P receptors activation. These extracellular nucleotides are metabolised by adenylate kinase and nucleotidases from at least two families: NTPDases and NPPases. A low activity of these nucleotide metabolising enzymes maintains nucleotides concentration in ventricular system in micromolar range. ATP is degraded into adenosine and inosine.

Conclusions

Our results confirm the thesis about cross-talking between brain and ventricular system functioning in physiological as well as pathological conditions. The close interaction of brain and ventricular system may elicit changes in qualitative and quantitative composition of purines and pyrimidines in CSF. These changes can be dependent on the physiological state of brain, including pathological processes in CNS.