Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β β β β (GSK3β β β β ) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3β β β β did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3β β β β was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3β β β β secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5 − − − − /− − − − mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.

SummaryTuberculosis and other mycobacterial infections are the most serious infectious diseases in terms of human fatalities. The high content of unique cellwall lipids helps these organisms to resist antimicrobial drugs and host defences. The biosynthesis of these lipids is discussed briefly. The recent advances in recombinant DNA technology have begun to help to elucidate the nature of some of the enzymes involved in this process and the genes that encode them. Gene disruption and other molecular genetic technologies are beginning to provide new approaches to test for the biological functions of these gene products and may lead to identification of new antimycobacterial drug targets.

The final step in hydrocarbon biosynthesis involves loss of CO from a fatty aldehyde. This decarbonylation is catalyzed by microsomes from Botyrococcus braunu. Among the several detergents tested for solubiliing the decarbonylase, octyl fi-glucoside (0.1%) was found to be the most effective and released 65% ofthe enzyme activity in soluble form. FPLC ofthe solubilized enzyme preparation with Superose 6 followed by ion-exchange FPLC with Mono Q resulted in 200-fold increase in specific activity with 7% recovery. The purified enzyme released nearly 1 mol of CO for each mol of hydrocarbon.SDS/PAGE of the enzyme preparation showed two protein bands of equal intensity at 66 and 55 kDa. The absorption spectrum of the enzyme with bands at 410 un, 425 um, 580 mn, and 620 un suggests the presence of a porphyrin. Electron microprobe analysis revealed that the enzyme contained Co. Purification of the decarbonylase from B. braunii grown in "CoC12 showed that 57Co coeluted with the decarbonylase.These results suggest that the enzyme contains Co that might be part of a Co-porphyrin, although a corrin structure cannot be ruled out. Co-protoporphyrin IX itself caused decarbonylation ofoctadecanal at 60'C, whereas the metal ion or protoporphyrin alone, or several other metal porphyrins, did not cause decarbonylation. These results strongly suggest that biosynthesis of hydrocarbons is effected by a microsomal Co-porphyrincontaining enzyme that catalyzes decarbonylation of aldehydes and, thus, reveal a biological function for Co in plants.Aliphatic nonisoprenoid hydrocarbons are ubiquitous in living organisms in both the plant and animal kingdoms (1, 2). Widespread occurrence of hydrocarbons in animals, accumulation of hydrocarbons under pathological conditions (3,4), demonstrated biosynthesis of hydrocarbons in mammalian nerve tissue (2), and decreased hydrocarbon synthesis associated with neurological disorders (5, 6) suggest important biological functions for this class of simple compounds.On the basis of the results obtained with specifically labeled precursors in higher plant tissues, it was proposed that n-hydrocarbons are produced by elongation of a fatty acid followed by the loss of the carboxyl carbon (7-9). Subsequent studies with insects (10) and mammals (2) supported this mechanism for alkane biosynthesis. Microsomal preparations from plant and animal tissues that generate alkanes have been shown to catalyze elongation offatty acids (11,12). The nature of the reaction that results in the loss of the carboxyl carbon remained obscure as the chemical nature of the immediate precursors of hydrocarbon was unknown until recently. Aldehydes with one carbon more than the alkanes were found to accumulate when hydrocarbon synthesis was inhibited by thiol compounds such as dithioerythritol (DTE) (13). In cell-free preparations that generate hydrocarbons, the observation that an aldehyde with one carbon more than the hydrocarbon was formed suggests that the aldehyde might be the immediate precursor of hydrocarbons (14). Ho...

SummaryCell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene ( mas ) encodes the enzyme that produces one class of such acids. Seven mas -like genes ( msl s) were identified in the genome. One of them, msl 3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl 3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl 3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface-exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead-like growth morphology without affecting the overall growth rate.

Neuronal polarity and spatial rearrangement of neuronal processes are central to the development of all mature nervous systems. Recent studies have highlighted the dynamic expression of Collapsin-Response-Mediator Proteins (CRMPs) in neuronal dendritic/axonal compartments, described their interaction with cytoskeleton proteins, identified their ability to activate L- and N-type voltage-gated calcium channels (VGCCs) and delineated their crucial role as signaling molecules essential for neuron differentiation and neural network development and maintenance. In addition, evidence obtained from genome-wide/genetic linkage/proteomic/translational approaches revealed that CRMP expression is altered in human pathologies including mental (schizophrenia and mood disorders) and neurological (Alzheimer's, prion encephalopathy, epilepsy and others) disorders. Changes in CRMPs levels have been observed after psychotropic treatments, and disrupting CRMP2 binding to calcium channels blocked neuropathic pain. These observations, altogether with those obtained from genetically modified mice targeting individual CRMPs and RNA interference approaches, pave the way for considering CRMPs as potential early disease markers and modulation of their activity as therapeutic strategy for disorders associated with neurite abnormalities.

Collapsin response mediator proteins (CRMPs) are intracellular proteins that mediate signals for several extracellular molecules, such as Semaphorin3A and neurotrophins. The phosphorylation of CRMP1 and CRMP2 by Cdk5 at Ser522 is involved in axonal guidance and spine development. Here, we found that the Ser522-phosphorylated CRMP1 and/or CRMP2 are enriched in the dendrites of cultured cortical neurons and P7 cortical section. To determine the physiological role of CRMPs in dendritic development, we generated CRMP2 knock-in mutant mice (crmp2 ki/ki ) in which the Ser residue at 522 was replaced with Ala. Strikingly, the cortical basal dendrites of double mutant crmp2ki/ki and crmp1 Ϫ/Ϫ mice exhibited severe abnormal dendritic patterning, which we defined as "curling phenotype." These findings demonstrate that the function of CRMP1 and CRMP2 synergistically control dendritic projection, and the phosphorylation of CRMP2 at Ser522 is essential for proper dendritic field organization in vivo.

A highly anionic peroxidase was strongly suggested to be involved in the deposition of the aromatic domain of suberin. cDNA containing the coding region of the suberization-associated anionic peroxidase from potato has been cloned and sequenced. The deduced amino acid sequence of the peroxidase shows that it is an anionic protein with considerable homology to other peroxidases. The amino acid sequence of two tryptic peptides obtained from the anionic peroxidase purified from suberizing potato tuber slices matched exactly with two segments of the amino acid sequence deduced from the nucleotide sequence of the cloned cDNA. The identity of the cloned cDNA is further supported by hybrid-selected translation and immunological recovery of the product with antibodies prepared against the purified anionic peroxidase. This anionic peroxidase was barely detectable at 2 days after wounding, and reached a maximal level at 8 days after wounding. Using the cDNA for the anionic peroxidase as a probe, we showed that the mRNA for the enzyme was induced in suberizing potato. The mRNA levels increased from an undetectable level in control tuber tissue to a maximal level in suberizing tuber tissue aged for four days. In suberizing tomato fruit the peroxidase mRNA showed induction and the level reached a maximum in three days. Ine data suggest that the induction of the peroxidase by wounding is preceded by transcriptional activation of the peroxidase gene or by increased stabilization of the mRNA. The time course of increase in mRNA for the anionic peroxidase was consistent with its postulated role in suberization.

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