Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

HOST SPECIFICITY IN THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE,RHODOPHYTA)1

The determinants of host specificity, which are poorly understood in red algal parasites, were studied in the red algal parasites Bostrychiocolax australis Zuccarello et West and Dawsoniocolax bostrychiae (Joly et Yamaguishi-Tomita) Joly et Yamaguishi-Tomita. Culture studies were performed to determine host range, sites of host resistance, and genetics of transmission of resistance. Both species parasitize Bostrychia radicans (Montagne) Montagne, whereas Bostrychiocolax australis also parasitizes Bostrychia moritziana (Sonder ex Kützing) J. Agardh and Stictosiphonia kelanensis (Grunow ex Post) R. J. King et Puttock. Isolates of B. radicans resistant to both parasites were found worldwide, often within the same population as susceptible isolates. On resistant Bostrychia species and isolates, specificity was manifested at three stages: 1) host penetration, in which the spore germ peg failed to penetrate the host cuticle/wall; 2) parasite–host cell fusion, in which the fusion cell died and the parasite died; and 3) growth, in which parasites grew but soon died; parasites rarely reproduced and infections did not continue in culture. Resistance to parasite infection was usually transmitted as a dominant trait and did not segregate as a single locus during meiosis. In certain crosses, transmission of resistance was non-mendelian.     

Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.     

Oxidation and aromatic ring cleavage of 4-methoxy and 3,4-dimethoxycennamic acid by Lentinus edodes

Several products of metabolism and aromatic ring cleavage of 3-methoxy and 3,4-dimethoxycinnamic acid from ligninolytic cultures of Lentinus edodes were isolated and identified.     

Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.     

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Kinetics and stereochemistry of the microsomal epoxide hydrolase-catalyzed hydrolysis of cis-stilbene oxides

The microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of cis-4,4′–dimethylstilbene oxide ( 1a ), cis-4,4′-diethylstilbene oxide ( 1b ), cis-4,4′-diisopropylstilbene oxide ( 1c ), and cis-4,4′-dichlorostilbene oxide ( 1d ) have been investigated using rabbit liver microsomal preparations. The kinetic parameters, K_m and V_max, and the absolute stereochemistry of the reactions have been determined and compared with those of cis-stilbene oxide ( 1e ). All epoxides 1a – d are hydrolyzed by mEH with high product enantioselectivity to give (R,R)-(+)-diols with ee ≥ 90%. The presence of the substituents on the phenyl rings markedly reduces the rates of mEH catalyzed hydrolysis with respect to cis-stilbene oxide, by increasing K_m and reducing V_max in the cases of 1a , 1b , and 1d , or reducing only the V_max in the case of 1c . The very low V_max, together with a persistent ability to fit into the mEH active site, make all these epoxides, and particularly 1c , inhibitors of cis-stilbene oxide hydrolysis. The kinetic and stereochemical results are interpreted on the basis of the proposed topology of the mEH active site. © 1994 Wiley-Liss, Inc.