Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells

Two cDNA clones, cATMPK1 and CATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells. These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems.     

Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines-T136-8, K10, and C9-which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.     

Alterations of load-induced p38 MAP kinase activation in failing rat hearts.

Hemodynamic load-induced cardiac p38 mitogen-activated protein kinase (MAPK) activation was studied in normotensive control Dahl rats (n = 10) and hypertensive Dahl rats with heart failure (n = 16). The isolated heart from each animal was stretched on a Langendorff apparatus at an equivalent diastolic wall stress, and the p38-MAPK activity of the left ventricular (LV) myocardium was analyzed by immunoprecipitation-kinase assay. Compared to the control hearts, the stretch-induced p38-MAPK activities were significantly decreased, and inversely correlated with the LV diameter (r = -0.73, P < 0.01). Chronic treatment with an angiotensin II AT1-receptor antagonist, valsartan (10 mg/kg/day), ameliorated cardiac function and remodeling process in the failing hearts, which was associated with an improvement of the p38-MAPK activities. Thus, the mechano-signal transduction of p38-MAPK pathway is downregulated in the failing hearts, along with progressive ventricular remodeling. The data also suggest that the beneficial effects of the AT1-receptor antagonists are potentially mediated by the restoration of cardiac growth-related signal transduction.     

Adsorption study of metal ions onto crosslinked seaweed Laminaria japonica

An efficient and cost effective non-conventional adsorbent has been prepared from seaweed Laminaria japonica by crosslinking with epichlorohydrin. Its adsorption behavior for trivalent and divalent metal ions was studied and it was found to exhibit excellent selectivity towards several metal ions. As a typical example, binary mixture of Pb(II) and Zn(II) was studied by using a packed column, indicating that the Pb(II) ion can be easily separated from its mixture with a concentration factor of 74 times. The maximum adsorption capacity for Pb(II), Cd(II), Fe(III) was found to be 1.35, 1.1, 1.53 mol kg(-1), respectively, while 0.8 7 mol kg(-1) for both La(III) and Ce(III) from the single metal ion solution according to the adsorption isotherm. The obtained values are comparable to the commercially available synthetic chelating resins.     

Interactions of sodium and potassium ions with oligonucleotides carrying human telomeric sequence and pyrene moieties at both termini

The organization of human telomeric DNA is of intense interest because of its role in aging, cancer research and bioanalytical applications. The Htelom sequence 5'-G(3)(T(2)AG(3))(3)-3' has been use to prepare two pyrene-modified fluorescence probes with three- and six-carbon linkers: Py-Htelom-Py(C3) and Py-Htelom-Py(C6), respectively. Results of the circular dichroism (CD), native PAGE, steady-state fluorescence, and anisotropy measurements of sodium and potassium quadruplex formation by these pyrene-modified conjugates are presented and discussed in order to clarify which conformation facilitates or renders the pyrene/pyrene or G-tetrad/pyrene stacking interaction. The CD spectra and native PAGE images suggested that conjugation of pyrene moieties has negligible effect on the folding properties of Htelom oligonucleotide. CD melting profiles and thermodynamic parameters revealed that both sodium and potassium quadruplexes are stabilized by the anchoring of pyrene tags with potassium ion being more effective than its sodium counterpart. Monomer emission of pyrene dominated in all investigated systems with fluorescence intensity being sensitive to the nature and concentration of cation and this phenomenon was attributed to the quenching processes and to the particular topologies of sodium and potassium quadruplexes. Strong quenching observed in the presence of KCl was attributed to the peculiarity of the potassium hybrid-type quadruplex, which enables effective stacking of pyrene moieties on the exposed guanine tetrads, thus facilitating static or electron transfer quenching. Plausibility of stacking interactions between pyrene and G-tetrad in a hybrid-type potassium quadruplex was further supported by the anisotropy measurements and molecular modeling results.     

Production of 4-hydroxyfuranones in simple media by fermentation

Both 2,5-dimethyl-3(2H)-furanone (DMHF) and 2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (EMHF) were produced at concentrations up to 4 mg l^–1 and 20 mg l^–1 respectively by yeast fermentation of a heated mixture of a single amino acid and a single sugar added to a yeast extract/peptone/glucose (YPG) medium. About 1 mg DMHF 1^–1 was also produced from precursors in the autoclaved YPG medium but EMHF formation depended entirely on the presence of a heated ribose/amino acid mixture. Glutamate was the best precursor amino acid for both furanones. Formation of EMHF showed a positive, non-linear response to ribose/glutamate concentration from 20 to 200 mM in the heated mixture.     

Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4. Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation

Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.     

Dendritic spinules in rat nigral neurons revealed by acetylcholinesterase immunocytochemistry and serial sections of the dendritic spine heads

Dendritic spinules of rat nigral neurons were visualized at electron microscopic level by acetylcholinesterase immunocytochemistry and serial sections of the nigral dendrites. The spinules (at least 150 nm in length and 10-20 nm in width) which protruded from the spine heads are found in extracellular space in the neuropil and particularly between nerve terminals of the presynaptic neurons and fine glial processes. The nigral spinules are, however, not observed as invaginated processes in the nerve terminals. The dendritic spinule may be endowed with synaptic plasticity and metabolic exchange between nerve terminals and glial processes.