Distantly related cousins of MAP kinase: biochemical properties and possible physiological functions

MAP kinases have been established to be key regulators of cellular signal transduction systems and are conserved from baker's yeast to human beings. Until now, three major types of mammalian MAP kinases (ERK, p38, and JNK/SAPK) have been reported and extensively studied. Advancement of genomic research as well as homology cloning techniques has revealed that there are several other protein kinase families that are structurally modestly related to those conventional MAP kinases. Indeed, most of them possess the TXY motif characteristic to MAP kinases in their activation loop, and can be regarded as members of the MAP kinase superfamily, yet some of them show closest overall similarity to Cdks. These kinases, all of mammalian origin, include MAK, MRK, MOK, p42KKIALRE, p56KKIAMRE, NLK, DYRK/Mnb, and Prp4. Although most of their physiological roles remain unknown, recent progress starts shedding some light on their functions.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

A mutant with constitutive sexual organ development in<Emphasis Type="Italic"> Marchantia polymorpha</Emphasis> L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.     

Mitochondrial genomes and phylogeny of the ocean sunfishes (Tetraodontiformes: Molidae)

We determined the complete nucleotide sequences of the mitochondrial genomes for the three currently recognized species of ocean sunfish: Mola mola, Masturus lanceolatus, and Ranzania laevis (Tetraodontiformes: Molidae). Each genome contained the 37 genes as found in teleosts, with the typical gene order in teleosts. Bayesian, maximum-likelihood, and maximum-parsimony analyses were conducted with the data set comprising concatenated nucleotide sequences from 36 genes (excluding the ND6 gene) of three molids and four outgroups (three tetraodontiforms plus a caproid). The resultant trees supported monophyly of the Molidae and its intrarelationships ((Mola, Masturus), Ranzania), which were congruent with previous morphology-based hypotheses.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

PCR-Based Approach for Sequencing Mitochondrial Genomes of Decapod Crustaceans,with a Practical Example from Kuruma Prawn (<Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>)

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.     

Crystal structures of epothilone D-bound,epothilone B-bound,and substrate-free forms of cytochrome P450epoK

Epothilones are potential anticancer drugs that stabilize microtubules by binding to tubulin in a manner similar to paclitaxel. Cytochrome P450epoK (P450epoK), a heme containing monooxygenase involved in epothilone biosynthesis in the myxobacterium Sorangium cellulosum, catalyzes the epoxidation of epothilones C and D into epothilones A and B, respectively. The 2.10-, 1.93-, and 2.65-A crystal structures reported here for the epothilone D-bound, epothilone B-bound, and substrate-free forms, respectively, are the first crystal structures of an epothilone-binding protein. Although the substrate for P450epoK is the largest of a P450 whose x-ray structure is known, the structural changes along with substrate binding or product release are very minor and the overall fold is similar to other P450s. The epothilones are positioned with the macrolide ring roughly perpendicular to the heme plane and I helix, and the thiazole moiety provides key interactions that very likely are critical in determining substrate specificity. Interestingly, there are strong parallels between the epothilone/P450epoK and paclitaxel/tubulin interactions. Based on structural similarities, a plausible epothilone tubulin-binding mode is proposed.