CRMPs: critical molecules for neurite morphogenesis and neuropsychiatric diseases.

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.

Biopolyester membranes of plants: cutin and suberin.

Cutin, a biopolyester composed of hydroxy and epoxy fatty acids, is the barrier between the aerial parts of higher plants and their environment. Suberin a polymer containing aromatics and polyesters, functions as a barrier in underground parts, wound surfaces, and a variety of internal organs. The composition and probable structure of these polymers are discussed. The biosynthesis of the hydroxy, epoxy, and dicarboxylic acids of the polyesters from the common cellular fatty acids is elucidated. An extracellular enzyme transfers the hydroxy and epoxyacyl moieties from their coenzyme A derivatives to the growing polyester. The enzymes acting in the biodegradation of the polyesters have been isolated from fungi, pollen, and mammals and characterized. The function and possible practical implications of these polyester barriers are briefly discussed.

Prevention of fungal infection of plants by specific inhibition of cutinase.

Specific antibodies prepared against cutinase from Fusarium solani pisi and diisopropylfluorophosphate, a potent inhibitor of this enzyme, prevented infection of the host (pea epicotyl) by this organism, without affecting the viability of the spores. This finding shows that enzymatic penetration of cuticle is involved in pathogenesis.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Malonyl-CoA decarboxylase from the mammary gland of lactating rat. Purification, properties and subcellular localization.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was purified 500--600-fold from the mammary gland extracts by (NH4)2SO4 precipitation, gel filtration with Sepharose 4B, anion-exchange chromatography with QAE-Sephadex, and chromatography with NADP-Agarose. This enzyme (spec. act. 200--300 nmol/min per mg protein) had a molecular weight of approx. 170 000. It did not cross-react with rabbit antiserum prepared against either fatty acid synthetase from the mammary gland or malonyl-CoA decarboxylase from the uropygial gland of goose. The decarboxylase showed a pH optimum near 8.5--9.0 and a Km of 0.33 mM, decarboxylated neither malonic acid nor methylmalonyl-CoA and was inhibited by thiol directed reagents but not by avidin. Sucrose density gradient centrifugation of the gland homogenate showed that the major peak of decarboxylase activity coincided with that of cytochrome oxidase. Breakage of mitochondria released greater than 80% of the decarboxylase activity into the 105,000 X g supernatant, suggesting that malonyl-CoA decarboxylase may be located in the mitochondrial matrix.

Isolation of a Protein Containing Covalently Linked Large and Small Subunits of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Botryococcus braunii.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and a 66-kD protein were co-purified from solubilized microsomal preparations of the green alga Botryococcus braunii by Green A agarose, sucrose density gradient, MonoQ, and gel filtration. The 66-kD protein remained intact after 6 M urea treatment and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It could be detected in the soluble fraction of the cell-free extract but appeared to be more abundant in the microsomal preparations. It cross-reacted with antibodies raised against Rubisco holoenzyme, large and small subunits, indicating that the 66-kD protein contains both the large and the small subunits of Rubisco. The N-terminal amino acid sequence of this protein and that of a proteolytic fragment showed high homology with the mature Rubisco small subunits, and the sequence of another proteolytic fragment showed high homology with that of the Rubisco large subunit. It is concluded that the 66-kD protein is produced by cross-linking of large and small sub-units of Rubisco in the cell.

Abolition of an Inducible Highly Anionic Peroxidase Activity in Transgenic Tomato.

Locally induced expression of a highly anionic peroxidase has previously been correlated temporally and spatially with suberization of tissues responding to pathogen assault, wounding, or exogenously applied abscisic acid or fungal elicitors. DNA sequences corresponding to the 5[prime] regions of two tomato (Lycopersicon esculentum) genes encoding homologous anionic peroxidases were fused, inserted into a pTi-based plasmid designed to express a composite antisense transcript, and introduced into tomato via Agrobacterium-mediated transformation. RNA gel-blot analyses showed high expression of the antisense transcript in most transgenic plants and no detectable induction of native anionic peroxidase transcripts in wounded or abscisic acid or pathogen-treated tissues. Plants and fruits expressing the antisense transcript appeared normal in all respects. Electrophoretic analysis of anionic proteins from selected transgenic plants showed no detectable anionic peroxidase protein or activity. Depolymerization of polymeric material from the wound periderm of transgenic tomato fruits and analysis of the aliphatic products by gas-liquid chromatography/mass spectrometry showed that the content and composition of C16/C18 [omega]-hydroxy and dicarboxylic acids, characteristic of suberin, were not affected by the absence of the anionic peroxidase. Autofluorescence generated from cell wall phenolics at the wound lesion was also not affected by the absence of the highly anionic peroxidase.

Chemical Signals from Avocado Surface Wax Trigger Germination and Appressorium Formation in Colletotrichum gloeosporioides.

The surface wax of the host, avocado (Persea americana) fruit, induced germination and appressorium formation in the spores of Colletotrichum gloeosporioides. Waxes from nonhost plants did not induce appressorium formation in this fungus, and avocado wax did not induce appressorium formation in most Colletotrichum species that infect other hosts. Bioassays of the thin-layer chromatographic fractions of the avocado wax showed that the fatty alcohol fraction was the main appressorium-inducing component. Testing of authentic n-C8 to n-C32 fatty alcohols revealed that C24 and longer-chain alcohols induced appressorium formation. Gas-liquid chromatography/mass spectrometry analysis of free fatty alcohols revealed that avocado wax contains a high content of very long chains. Waxes from nonhost plants containing an even higher content of the very long-chain alcohols did not induce appressorium formation. Waxes from nonhost plants strongly inhibited appressorium induction by avocado wax. Thus, a favorable balance between appressorium-inducing very long-chain fatty alcohols and the absence of inhibitors allows the fungus to use the host surface wax to trigger germination and differentiation of infection structures in the pathogen.

Cloning, sequencing and characterization of a fatty acid synthase-encoding gene from Mycobacterium tuberculosis var. bovis BCG.

Mycobacterial cell walls contain unique lipids such as mycolic acids, very long chain fatty acids and multimethyl-branched fatty acids. A multifunctional fatty acid synthase (Fas) with the unique capability of catalyzing both de novo synthesis and chain elongation of fatty acids has been purified and characterized from Mycobacterium tuberculosis var. bovis BCG (Bacillus Calmette-Geurin) [Kikuchi et al., Arch. Biochem. Biophys. 295 (1992) 318-326]. To understand how the various domains that catalyze the reactions involved in both de novo synthesis and elongation are organized in the mycobacteria, a fas gene was cloned from a cosmid library of genomic DNA from M. bovis BCG. Sequencing of the cosmid clone revealed a contiguous sequence of 11 577 bp of mycobacterial genome containing a 8389-bp open reading frame that could code for a protein of 2797 amino acids (301 kDa). By comparing the Fas aa sequence with the sequences in the active site regions of known fas and polyketide synthase-encoding genes, the functional catalytic domains in Fas were identified. This analysis revealed that the domains are organized in the following order: acyltransferase, enoyl reductase, dehydratase, malonyl/palmitoyl transferase, acyl carrier protein, beta-keto reductase, beta-ketoacyl synthase. This domain organization is like a head to tail fusion of the two yeast fas gene subunits. The results obtained constitute the first report of the cloning, sequencing and structural elucidation of a fas from the Mycobacteria.

Cloning and expression of cDNA encoding a mitogen-activated protein kinase from a phytopathogenic filamentous fungus.

We have cloned a mitogen-activated protein kinase (MAPK) designated Fusarium solani f. sp. pisi mitogen-activated protein kinase (FsMAPK) from the phytopathogenic filamentous fungus F. solani f. sp. pisi T8 strain. A single open reading frame (ORF) of 1068 bp encoding a polypeptide of 355 amino acids (aa) with a predicted molecular mass of 41,194 Da was found in the cloned 1583-bp cDNA insert. FsMAPK is highly homologous to SPK1 of fission yeast, FUS3 of budding yeast, MsERK1 of alfalfa, Sur-1 of nematode, and hERK1 of human. That this gene is expressed in F. solani f. sp. pisi was shown by the finding that immunoblot of the fungal extracts with anti-FsMAPK antibodies (Ab) raised in a rabbit against the FsMAPK, expressed in Escherichia coli (E. coli), detected the corresponding protein. DNA blot analysis indicated that Fsmapk is present as a single copy in the fungal genome.

Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus.

Aspergillus fumigatus (Afu) and A. flavus (Afl), two causative agents of invasive aspergillosis, produce highly homologous serine proteinases. In addition, the former produces a 42-kDa metalloproteinase (MEP), whereas the latter produces a 23-kDa MEP. The cDNA and the gene encoding the 42-kDa MEP were cloned and sequenced. Here, we report the cloning of the cDNA and the gene encoding the 23-kDa MEP from Afl and a homologous gene from the Afu. Using degenerate primers based on the amino acid (aa) sequence of A. oryzae (Ao) MEP and thermolysin-like proteinases, a 282-bp fragment of the 23-kDa MEP-encoding gene of Afl was cloned by PCR. A 6.5-kb KpnI fragment of Afl genomic DNA containing the complete gene was cloned. The open reading frame (ORF) in this gene encodes a protein of 381 aa. Since the mature enzyme from this and other aspergilli would have a theoretical molecular mass of about 20 kDa, this MEP-encoding gene is designated mep20. A Western blot of the protein in the culture filtrate of Afl with polyclonal antibodies prepared against the MEP showed a single band at 23 kDa. The N-terminal sequence of the extracellular MEP20, TKVAS, was found at aa 194-198 within the ORF. Thus, the primary translation product has a putative 19-aa signal and a pro region of 174 aa. A homologous gene cloned from a genomic DNA library of Afu showed an ORF encoding 365 aa. Comparison of the nucleotide (nt) sequences of the cDNAs cloned by RT-PCR with their respective genes showed that there are no introns in the ORF of mep20 in Afl, but there is a 59-bp intron in the gene from Afu. The MEP20 of Afl and Afu have 68% identity and show weak immunological cross reactivity. MEP20 from both these fungi share about 60% sequence identity with the penicillolysin of Penicillium citrinum and the neutral protease II of Ao. MEP20 of Afl and Afu show only the conserved sequence, HEFTHA, but not the two other conserved sequences seen in thermolysins and similar MEP.

Solubilization and purification of aldehyde-generating fatty acyl-CoA reductase from green alga Botryococcus braunii.

Membrane-bound fatty acyl-CoA reductase from the green alga Botryococcus braunii has been solubilized from the microsomal preparation by 0.1% octyl beta-glucoside and purified to near homogeneity by Blue A agarose and palmitoyl-CoA agarose affinity column chromatography. The molecular mass of the enzyme was estimated by SDS-PAGE to be 35 kDa. The enzyme generates fatty aldehyde by reduction of fatty acyl-CoA with NADH as the reductant. The N-terminal amino acid sequence of this protein that represents the first eucaryotic aldehyde-generating reductase to be purified shows high homology with the N-terminus of fatty acid reductase from bacteria.

Peroxisome proliferator-activated receptor gamma1 (PPAR-gamma1) as a major PPAR in a tissue in which estrogen induces peroxisome proliferation.

Estradiol administration induces peroxisome proliferation and the production of 3-hydroxy fatty acid pheromones in the uropygial glands of the duck, but not in the goose gland, which does not produce such pheromones. We isolated a peroxisome proliferator-activated receptor (PPAR)gamma1 cDNA from a duck uropygial gland cDNA library. Northern blots revealed two transcripts, PPAR gamma1 and gamma2, and showed that PPAR gamma was expressed at higher levels than PPAR alpha in the uropygial gland of the duck. Although PPAR gamma2 was expressed in both duck and goose uropygial gland, PPAR gamma1 was expressed only in the duck gland, which responds to estrogen by peroxisome proliferation. In NIH 3T3 transfected cells, PPAR gamma1 was activated by peroxisome proliferators such as Wy-14643, clofibric acid and Ly-171883 causing induction of the target marker gene. By cotransfection with a plasmid containing alpha-cis-retinoic acid receptor RXR alpha, the induction increased up to 9-fold. These results suggest that PPAR gamma1 may be involved in peroxisome proliferation while PPAR gamma2 may be involved in lipid metabolism.

Molecular cloning of a new unc-33-like cDNA from rat brain and its relation to paraneoplastic neurological syndromes.

Anti-CV2-autoantibodies from patients with paraneoplastic neurological syndromes were used to purify protein(s) related to this disease. A novel cDNA, c-22, was obtained by PCR with primers based on amino-acid sequence of peptides obtained from this protein and rat brain cDNA as template. The deduced amino-acid sequence of c-22 shows homology to the Unc-33 gene from C. elegans in which mutations lead to defects in neuritic outgrowth and axonal guidance and cause uncoordinated movements of the nematode. Several consensus sites for putative protein kinase C phosphorylation were found, suggesting that the c-22 gene product may be a phosphoprotein. Northern hybridizations show that the apparently unique 3.8-kb mRNA of c-22 is present in rat brain tissue and its expression is developmentally regulated: the levels of C-22 mRNA, detectable in brain at embryonic day 17 (E17), increase up to post-natal day 7 (P7) and decline rapidly to an almost undetectable level in adult.

Nucleotide sequence and genomic structure of duck acyl-CoA binding protein/diazepam-binding inhibitor: co-localization with S-acyl fatty acid synthase thioesterase.

A computer-aided homology search of the GenBank nucleotide database using the amino acid sequence of human acyl CoA-binding protein (ACBP)/diazepam-binding inhibitor (DBI)-endozepine as a probe revealed that a genomic fragment containing the gene encoding the mallard duck (Anas platyrhynchos) S-acyl fatty acid synthase thioesterase also contains sequences which encode the duck homolog of ACBP/DBI. The duck ACBP/DBI gene is positioned downstream of the thioesterase gene in a tail-to-tail orientation separated from the 3' end of the thioesterase gene by only several hundred nucleotides. Three exons were identified that have strong homology to the published cDNA sequences of human and bovine ACBP/DBI. These exons define all of the coding region except for the amino-terminal domain, which was subsequently cloned by polymerase chain reaction (PCR) amplification. The encoded amino acid sequence of the duck ACBP/DBI is 62-68% homologous to mammalian ACBP/DBI sequences. While the mammalian ACBP/DBI is expressed mainly in the liver, with smaller amounts in the brain and heart, mRNA transcripts of duck ACBP/DBI were detected only in the brain with no evidence for expression in the liver or heart. The close proximity of the genes for ACBP/DBI and S-acyl fatty acid synthase thioesterase raises the possibility of co-regulation of expression.

The optical rotation of a major component of plant cutin.

The major component of cutin from the fruit of both tomato and papaya, dihydroxypalmitate, is shown to have plain positive rotation and is, therefore, assigned L configuration in analogy to other known hydroxy fatty acids.

Biosynthesis of lipids by bovine meibomian glands.

Isolated bovine meibomian glands incorporated exogenous [1-14C]acetate into lipids. Thin layer chromatographic analysis of the lipids showed that wax esters and sterol esters contained 61% of the total label. Radio gas liquid chromatographic analysis of the acid and alcohol moieties of both ester fractions showed the label was distributed equally between the two portions of the ester in both cases. Cholesterol and 5-alpha-cholest-7-en-3 beta-ol were the major labeled sterols, and anteiso-C25, anteiso-C27 and anteiso-C23 were the most highly labeled alcohols. The major labeled fatty acids in the wax esters were anteiso-C15, n-C16, anteiso-C17 and n-C18:1, whereas anteiso-C25 and anteiso-C27 were the major labeled acids in the sterol esters. The diester region with 6% of the total label contained labeled fatty acids and fatty alcohols each with anteiso-C25 as the major component and omega-hydroxy acids in which n-C32:1 was the major labeled component. The triglyceride fraction which contained 8% of the total lipids was composed of labeled fatty acids similar to those found in both sterol and wax ester fractions. Chromatographic analyses of the labeled lipids derived from exogenous labeled isoleucine showed that anteiso-branched products were preferentially labeled. The labeled triglyceride fraction derived from [U-14C] isoleucine also contained esterified C15, C13, C11, C9, C7 and possibly shorter anteiso-branched acids.