The lurlenes, a new class of plastoquinone-related mating pheromones from Chlamydomonas allensworthii (Chlorophyceae).

Chlamydomonas allensworthii is a recently described species, which differs phenotypically and in its reproductive behaviour from other members of this genus of flagellated green protists. Its female cells temporarily or constantly excrete a luring signal into the medium to attract the male cells for mating. The sperm is susceptible to the pheromone either all the time or only after a maturation process. Maturation in this species is accompanied with a colour shift from green to khaki (strain from Lemon cove CA) or to olive (strain from Catarina TX), apparently due to degradation of chloroplast material. In the bioassay with polyacrylamide beads (plain or DEAE-substituted) to which the lures are bound, acting as mock females, Catarina-strain males are only attracted to Catarina strain lures, whereas Lemon-Cove-strain males are attracted to Lemon-Cove-strain lures and to a lesser extent, to Catarina-strain lures. Being amphiphilic acid, Lemon-Cove-strain lure is more tightly bound to DEAE beads. Catarina-strain lure is an uncharged amphiphile. The two signal compounds have been isolated and identified by chemical analysis and mass and NMR spectroscopies as O(5)-beta-D-xylosylated degradation products of the chloroplast electron transporter hydroplastoquinone, its polyprenoid side chain being oxidatively cleaved at the fourth double bond. Lurlenic acid (lurlene L) is the resulting acid and lurlenol (lurlene C) the corresponding alcohol. The pheromonal activities of both compounds have a threshold at 1 pM, and are destroyed by deglycosylation and subsequent oxidation to the quinonoid aglycon. This class of amphiphilic lures, having the same basic glycosylated hydroquinone structure, is named lurlenes.

Purification and characterization of a cAMP-binding protein of Volvox carteri f. nagariensis Iyengar.

Two cAMP-binding proteins, cbp1 and cbp2, were purified from the cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contains a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiesterases of V. carteri with respect to charge, molecular mass and binding affinity to N6-cAMP-agarose. Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5' AMP. The binding specificity of purified cbp1 and cbp2 was probed by a set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic O atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methylxyanthines are not. At present it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.

Chemoattraction of male gametes by a pheromone produced by female gametes of Chlamydomonas.

In isogamous species of Chlamydomonas, such as Chlamydomonas reinhardtii and Chlamydomonas eugametos, the sexual process involves the use of flagella agglutinins by which the gametes of compatible strains adhere through chance encounter and ultimately pair and fuse to form zygotes. In a newly described heterogamous species, Chlamydomonas allensworthii, the sexual process is initiated by the chemoattraction of small sperm to a sexually competent female gamete, which continues to secrete the pheromone until it has fused with one of the sperm so attracted. From bacteria-free female strains of C. allensworthii, the chemoat-tractant has been isolated and identified as a pentosylated hydroquinone (Mr = 532) whose spectral, chemical, and physical properties are in accord with the structure of a 2,3-dimethyl-5-(triprenylcarboxymethyl)-1,4-benzohydroquinone-1-(beta-xyloside). A rapid bioassay of the pheromone uses DEAE-Toyopearl 650M beads to which the pheromone adsorbs. When such activated beads are placed in a suspension of sperm, they act as surrogate females and attract the small motile sperm. The purified pheromone shows activity at a concentration as low as 1 pM.

In-vitro efficacy of an antibiotic releasing silicone ventricle catheter to prevent shunt infection.

Infection due to implanted polymeric devices is a major problem in modern medicine. Microbial colonization of implants in neurosurgery, e.g. cerebrospinal fluid (CSF) shunts is the main reason for their failure, and often results in the consequent removal of the infected implants. In this paper we report on new approaches in the prevention of bacterial infections by incorporation of an antibiotic (rifampicin) into the polymer devices (silicone). Drug release characteristics are investigated, and the physico-chemical mechanism of the delivery is discussed. Measurements of killing kinetics and the bacterial adhesion to the antibiotic-loaded silicone in a static adhesion assay reveal that only the liberation of high antibiotic doses over a period of weeks can prevent the bacterial colonization of the polymeric surface.

Evidence for radical species as intermediates in cadmium/zinc-metallothionein-dependent DNA damage in vitro.

Toxicologic data on cadmium (Cd) indicate that intracellular metallothionein (MT) is protective for Cd exposure, whereas extracellular Cd-containing MT might be toxic. Moreover, Cd is suspected to be a carcinogen though the underlying mechanism is not known. Here we report on the genotoxic activity of cadmium/zinc-metallothionein (Cd/Zn-MT) in a cell-free test system: a concentration-dependent increase in DNA strand breaks was detected with increasing doses of Cd/Zn-MT, whereas no DNA strand breaks were observed in the presence of heat-denatured MT or Cd or Zn ions alone. Modifications of native Cd/Zn-MT by the metal ion-chelating agent EDTA or the sulfhydryl group alkylating agents N-ethylmaleimide and iodoacetamide suggest that the various cysteine residues of MT, together with the attached heavy metal ions, may be involved in the DNA cleavage reaction. Furthermore, DNA strand breaks caused by Cd/Zn-MT seem more likely to be random than sequence- or base-specific. Results from experiments with radical scavengers and electron spin resonance spectroscopy point to radical species formed by Cd/Zn-MT as mediators of the DNA damage. Thus, the actual activity of Cd/Zn-MT--whether protective or damaging--appears to depend on various parameters governed by the extra- and intracellular environment.

Expression of highly active sex-inducing pheromone of Volvox carteri f. nagariensis in a mammalian cell system.

A cDNA fragment coding for the sex-inducing glycoprotein of Volvox carteri f. nagariensis was expressed in a mammalian cell system (baby hamster kidney (BHK) cells). The transfection product exhibited a specific biological activity intermediate between the natural pheromone of the strains Volvox carteri f. nagariensis and Volvox carteri f. weismannia. Immunoblot analysis showed that the sex-inducing activity was expressed as a set of three iso-glycoproteins (35, 34 and 31 kDa).

The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product.

Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.

Mapping of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3',5'-monophosphate.

A series of systematically modified cyclic AMP (cAMP) analogues, including newly synthesized benzimidazole ribofuranosyl 3',5'-monophosphates was used to map the essential molecular interactions between cAMP and the monoclonal antibody 4/2C2 (mab 4/2C2) directed against 2'-O-succinoyl cAMP [Colling, Gilles, Nass, Moka & Jaenicke (1988) Second Messengers Phosphoproteins 12, 123-133]. Its paratope binds the purine base in syn conformation by dipole-dipole interactions and hydrophobic forces and/or stacking interactions. The ribose phosphate moiety is recognized by a combination of charge interactions and H-bonds to the exocyclic and the 5'-oxygen atoms and a hydrophobic interaction at the 2'-position. There is no regioselectivity for the exocyclic oxygen atoms. Compared with the known types of binding, mab 4/2C2 thus shows a new combination of molecular interactions which may be the basis of its strikingly specific recognition and binding of the cyclic adenylates. On this account mab 4/2C2 may become an important tool in studies on cAMP metabolism.

Short-chain dolichols of defined chain length as cofactors in reactions of the microsomal dolichyl-phosphate cycle and transglycosylations.

1) The biological cofactor and carrier activities of dolichyl phosphates of low isoprene multiplicity (n) and defined geometry, (synthesized according to L. Jaenicke and H.-U. Siegmund, Chem. Phys. Lipids 51 (1989) 159-170), were assayed in different transfer reactions of the microsomal dolichyl-phosphate cycle against natural pig liver dolichyl phosphate (n = 18 to 20). 2) The apparent Michaelis constants and maximal velocities were determined from initial reaction rates for the transfer from GDP-mannose, UDP-N-acetylglucosamine, and UDP-glucose to the synthetic truncated dolichyl phosphates. They afford quantitative comparison and show increasing biological activities from dolichyl-6 phosphate to dolichyl-11 phosphate, which is about as active as the natural mixture. This is in accord with previous findings on the starting reactions of the cycle. 3) Truncated dolichyl diphosphochitobioses, biosynthesized in vitro from synthetic dolichyl phosphates, were used as acceptors for nucleoside diphosphohexoses in solubilized membranes. All of them show about the same activity. The kinetics and yield were determined for each of the transfers. Activity is increased by adding UDP-glucose. The inactive very short-chain dolichol compounds do not interfere with the transfer to active longer chain dolichols. 4) The oligosaccharides produced by transfer of mannose and glucose to truncated dolichyl diphosphate-bound chitobiose were isolated and analysed for sugar multiplicity. The heptasaccharide and the un-decasaccharide are accumulated most, pointing to the transport across the endoplasmic membranes (ER) as the rate limiting reaction. 5) The truncated dolichyl-diphosphate-bound oligosaccharides are transferred to protein(s) by the crude, solubilized microsomal preparation independent of chain length of the cofactor/carrier, yet with increasing yield as shown by enzyme immunoblot analysis.

Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors.

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.

Cadmium/zinc-metallothionein induces DNA strand breaks in vitro.

The in vitro DNA strand breaking activity of metallothionein (MT) containing Cd2+ and Zn2+ in a molar ratio of 5:2 is described. Studies with radical scavengers and electron paramagnetic resonance spectroscopy indicate that the DNA damage might be caused by a radical species formed by the native protein (i.e., MT) charged with the heavy metal ions. No DNA strand breaks are detectable with the heat-denatured MT or with Cd2+ or Zn2+ alone. Inhibition studies using EDTA as a metal ion chelator or N-ethylmaleimide to alkylate sulfhydryl groups suggest that both the bound heavy metal ions as well as the SH groups of the various cysteine residues of MT may be involved in the MT-dependent DNA cleavage. Further characterization showed that the DNA cleavage is more likely random than sequence- or base-specific. These observations may provide a clue in the search for initial events in Cd-related carcinogenicity.

The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae).

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.

Measurement of 3':5'cyclic AMP in biological samples using a specific monoclonal antibody.

A fast and sensitive radioimmunoassay for 3':5'cyclic AMP based on a monoclonal antibody has been worked out. Mice were immunized with protein-conjugated 2'-O-succinyl-3':5'-cyclic AMP. The monoclonal antibody detects 0.1 and 1 pmole cAMP with succinyl cAMP (125I)iodotyrosine methyl ester and (3H) cAMP, respectively, as tracers. It shows no cross-reactivity to other adenosine nucleotides up to the millimolar range; cGMP interferes only if present at a 500 fold excess. Plant and animal tissue samples as well as adenylate cyclase activity were analysed directly or after appropriate purification in case of interfering substances. Cyclic AMP levels measured in various tissues by the antibody binding assay correspond to those obtained by HPLC determination using fluorescent etheno-cAMP.

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii.

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO4) and oxidizing agents (NaClO2) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)2 inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.