Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.     

Two chromone-secoiridoid glycosides and three indole alkaloid glycosides from Neonauclea sessilifolia

From the dried roots of Neonauclea sessilifolia, two new chromone-secoiridoid glycosides, sessilifoside and 7"-O-beta-D-glucopyranosylsessilifoside, and three novel indole alkaloid glycosides, neonaucleosides A, B, and C, were isolated along with the main known glycosides, 5-hydroxy-2-methylchromone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, sweroside, loganin, grandifloroside, and quinovic acid 3 beta-O-beta-D-quinovopyranoside-28-O-beta-D-glucopyranoside. The structures of these new glycosides were determined by spectroscopic and chemical means. Neonaucleoside A and its C-3 epimer were prepared from secologanin and tryptamine.     

Randomization of cortical microtubules in root epidermal cells induces root hair initiation in lettuce (Lactuca sativa L.) seedlings

Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.     

A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternata

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce host-specific toxins and cause diseases on different plants. Previously, the gene cluster involved in host-specific AK-toxin biosynthesis of the Japanese pear pathotype was isolated, and four genes, named AKT genes, were identified. The AKT homologs were also found in the strawberry and tangerine pathotypes, which produce AF-toxin and ACT-toxin, respectively. This result is consistent with the fact that the toxins of these pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety. In this study, three of the AKT homologs (AFT1-1, AFTR-1, and AFT3-1) were isolated on a single cosmid clone from strain NAF8 of the strawberry pathotype. In NAF8, all of the AKT homologs were present in multiple copies on a 1.05-Mb chromosome. Transformation-mediated targeting of AFT1-1 and AFT3-1 in NAF8 produced AF-toxin-minus, nonpathogenic mutants. All of the mutants lacked the 1.05-Mb chromosome encoding the AFT genes. This chromosome was not essential for saprophytic growth of this pathogen. Thus, we propose that a conditionally dispensable chromosome controls host-specific pathogenicity of this pathogen.     

Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain

Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.     

RNase 3 (ECP) is an extraordinarily stable protein among human pancreatic-type RNases

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.     

Binding of ribosome recycling factor to ribosomes,comparison with tRNA

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.