Structure-activity relationships of RGD mimetics as fibrinogen-receptor antagonists

The activities of a series of RGD mimetics, which contained a variety of cationic structures, for the inhibition of platelet aggregation and fibrinogen-receptor binding were measured. The stability of the coulombic ion-pairing complex of the model compounds with the acetate anion as a model for the receptor was calculated in terms of the ionic interaction energy. The results suggest that stability is one of the significant factors which govern the inhibitory potency of fibrinogen-receptor binding. The distance between cationic and anionic groups might also affect the potency. A compound which contained an amidinophenyl structure as the cationic moiety showed exceptionally high inhibitory activity, suggesting that some other factors, in addition to coulombic interaction and the distance, affect the potency.     

Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice

We have analyzed the pathfinding of thalamocortical axons (TCAs) from dorsal thalamus to neocortex in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. In wild-type mice, we identified four cell domains that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, we studied Mash-1 mutant mice to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTP(delta) domain in ventral thalamus is correspondingly reduced and the normally graded expression of RPTP(delta) in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTP(delta) expression. A small proportion of TCAs extend around the RPTP(delta) domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon.     

Characteristics of the production of active oxygen species from adherent and non-adherent neutrophils

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.     

Periodically fluctuating protein kinases phosphorylate CLOCK, the putative target in the suprachiasmatic nucleus

We studied nuclear protein phosphorylation in the rat suprachiasmatic nucleus (SCN) and found that a nuclear fraction of the SCN contained histone H1 kinase activity that periodically fluctuated with a diurnal rhythm, reaching a maximum at the midpoint of the light phase and a minimum at the midpoint of the dark phase. A p13suc1-bound fraction from the SCN nuclear fraction also exhibited diurnally fluctuating histone H1 kinase activity. Using in situ kinase assay, three histone H1 kinases, p45PFK, p100PFK, and p200PFK (termed periodically fluctuating protein kinases, or PFKs) were found in the p13suc1-bound fractions. p45PFK exhibited the highest level of light/dark cycle phosphorylation activity fluctuation. p45PFK highly phosphorylated the Ser-Pro-rich region of CLOCK, the putative physiological target. These results suggest that PFKs, especially p45PFK, are involved in circadian clock-related signal transduction and gene expression, through the phosphorylation of target proteins such as CLOCK.     

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.     

Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform

Filamentous tau aggregates are hallmarks of tauopathies, e.g., frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). Since FTDP-17 tau gene mutations alter levels/functions of tau, we overexpressed the smallest human tau isoform in the CNS of transgenic (Tg) mice to model tauopathies. These mice acquired age-dependent CNS pathology similarto FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Inclusions were present in cortical and brainstem neurons but were most abundant in spinal cord neurons, where they were associated with axon degeneration, diminished microtubules (MTs), and reduced axonal transport in ventral roots, as well as spinal cord gliosis and motor weakness. These Tg mice recapitulate key features of tauopathies and provide models for elucidating mechanisms underlying diverse tauopathies, including Alzheimer's disease (AD).     

The role of branching glycan of human alpha1-acid glycoprotein in enantioselective binding to basic drugs as studied by capillary electrophoresis

The role of the branching glycan structure of human alpha1-acid glycoprotein (AGP) in the interaction with basic drugs was investigated in terms of enantioselectivity in binding ability. AGP was separated by concanavalin A lectin affinity chromatography into two subfractions, the unretained AGP (UR-AGP) which has no biantennary glycan chain and the retained AGP (R-AGP) which possesses biantennary oligosaccharide chain(s). The unbound concentrations of propranolol (PRO) enantiomers and verapamil (VER) enantiomers in UR-AGP solution and R-AGP solution were determined by high-performance frontal analysis combined with capillary electrophoresis. It was found that (S)-PRO is bound to UR-AGP and R-AGP more strongly than (R)-PRO, whereas the reverse applies to VER enantiomers, and that such enantioselectivity is common to these proteins. This suggests that the branching type of glycan chains of AGP does not play significant role in the chiral recognition in binding these basic drugs.     

Post-glacial migration of silver fir (Abies alba Mill.) in the south-western Alps

Aim  Previous studies have failed to reconstruct the regional post-glacial migration pattern of Abies alba in southern France. Based on the first exhaustive compilation of palaeoecological data in this region, we present the state-of-the-art and attempt to synthesize the available information concerning glacial refugia and post-glacial migration, and analyse the information with regard to climate and orography.
Location  South-western Alps and adjacent areas, southern France.
Methods  The work compiles the available palaeoecological data in the south-western Alps (52 sites, 290 radiocarbon dates). The post-glacial migration pattern of Abies alba is reconstructed based on 22 selected palynological analyses (11 well-dated reference sites and 11 supplementary ones).
Results  The geographical patterns of approaching area limit, immigration and expansion are reconstructed at the scale of the southern French Alps.
Main conclusions  Despite previous assertions, the evidence of refugia in southern France is non-existent. The late-glacial records of fir pollen, previously interpreted in French Mediterranean regions and on adjacent foot-hills as possibly reflecting regional refugia, most probably correspond to reworking phenomena or long-distance pollen transport. Fir migration, originating in the Apennine refugia and through the south-western extremity of the Alps, was extremely rapid in the southern French Alps, only spanning a few centuries between 10,100 and 9800 cal. yr bp . The subsequent spread of fir populations was controlled by local parameters, such as the aridity of the inner valleys, which resulted in a delayed expansion in comparison to other regions. Abies almost disappeared from the south-western Alps during the Roman era, around 2000 cal. yr bp .     

spoIVH (ykvV), a requisite cortex formation gene, is expressed in both sporulating compartments of Bacillus subtilis

It is well known that the ykvU-ykvV operon is under the regulation of the sigma(E)-associated RNA polymerase (Esigma(E)). In our study, we observed that ykvV is transcribed together with the upstream ykvU gene by Esigma(E) in the mother cell and monocistronically under Esigma(G) control in the forespore. Interestingly, alternatively expressed ykvV in either the forespore or the mother cell increased the sporulation efficiency in the ykvV background. Studies show that the YkvV protein is a member of the thioredoxin superfamily and also contains a putative Sec-type secretion signal at the N terminus. We observed efficient sporulation in a mutant strain obtained by replacing the putative signal peptide of YkvV with the secretion signal sequence of SleB, indicating that the putative signal sequence is essential for spore formation. These results suggest that YkvV is capable of being transported by the putative Sec-type signal sequence into the space between the double membranes surrounding the forespore. The ability of ykvV expression in either compartment to complement is indeed intriguing and further introduces a new dimension to the genetics of B. subtilis spore formation. Furthermore, electron microscopic observation revealed a defective cortex in the ykvV disruptant. In addition, the expression levels of sigma(K)-directed genes significantly decreased despite normal sigma(G) activity in the ykvV mutant. However, immunoblotting with the anti-sigma(K) antibody showed that pro-sigma(K) was normally processed in the ykvV mutant, indicating that YkvV plays an important role in cortex formation, consistent with recent reports. We therefore propose that ykvV should be renamed spoIVH.