Competing Lagrangians for incompressible and compressible viscous flow.

A recently proposed variational principle with a discontinuous Lagrangian for viscous flow is reinterpreted against the background of stochastic variational descriptions of dissipative systems, underpinning its physical basis from a different viewpoint. It is shown that additional non-classical contributions to the friction force occurring in the momentum balance vanish by time averaging. Accordingly, the discontinuous Lagrangian can alternatively be understood from the standpoint of an analogous deterministic model for irreversible processes of stochastic character. A comparison is made with established stochastic variational descriptions and an alternative deterministic approach based on a first integral of Navier-Stokes equations is undertaken. The applicability of the discontinuous Lagrangian approach for different Reynolds number regimes is discussed considering the Kolmogorov time scale. A generalization for compressible flow is elaborated and its use demonstrated for damped sound waves.

A non-conventional discontinuous Lagrangian for viscous flow.

Drawing an analogy with quantum mechanics, a new Lagrangian is proposed for a variational formulation of the Navier-Stokes equations which to-date has remained elusive. A key feature is that the resulting Lagrangian is discontinuous in nature, posing additional challenges apropos the mathematical treatment of the related variational problem, all of which are resolvable. In addition to extending Lagrange's formalism to problems involving discontinuous behaviour, it is demonstrated that the associated equations of motion can self-consistently be interpreted within the framework of thermodynamics beyond local equilibrium, with the limiting case recovering the classical Navier-Stokes equations. Perspectives for applying the new formalism to discontinuous physical phenomena such as phase and grain boundaries, shock waves and flame fronts are provided.

Iridals are a novel class of ligands for phorbol ester receptors with modest selectivity for the RasGRP receptor subfamily.

Since 1990, the National Cancer Institute has performed extensive in vitro screening of compounds for anticancer activity. To date, more than 70 000 compounds have been screened for their antiproliferation activities against a panel of 60 human cancer cell lines. We probed this database to identify novel structural classes with a pattern of biological activity on these cell lines similar to that of the phorbol esters. The iridals form such a structural class. Using the program Autodock, we show that the iridals dock to the same position on the C1b domain of protein kinase C delta as do the phorbol esters, with the primary hydroxyl group of the iridal at the C3 position forming two hydrogen bonds with the amide group of Thr12 and with the carbonyl group of Leu 21 and the aldehyde oxygen of the iridal forming a hydrogen bond with the amide group of Gly23. Biological analysis of two iridals, NSC 631939 and NSC 631941, revealed that they bound to protein kinase C alpha with K(i) values of 75.6 +/- 1.3 and 83.6 +/- 1.5 nM, respectively. Protein kinase C is now recognized to represent only one of five families of proteins with C1 domains capable of high-affinity binding of diacylglycerol and the phorbol esters. NSC 631939 and NSC 631941 bound to RasGRP3, a phorbol ester receptor that directly links diacylglycerol/phorbol ester signaling with Ras activation, with K(i) values of 15.5 +/- 2.3 and 41.7 +/- 6.5 nM, respectively. Relative to phorbol 12,13-dibutyrate, they showed 15- and 6-fold selectivity for RasGRP3. Both compounds caused translocation of green fluorescent protein tagged RasGRP3 expressed in HEK293 cells, and both compounds induced phosphorylation of ERK1/2, a downstream indicator of Ras activation, in a RasGRP3-dependent fashion. We conclude that the iridals represent a promising structural motif for design of ligands for phorbol ester receptor family members.

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.

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.).

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 +/- 0.5 and 6 +/- 1 mug/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus.

Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga.

Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue-light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11-cis- and all-trans-retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9-cis-retinal to bleached, isolated eyespot apparatuses.

The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways.

Resveratrol and chalcone synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of stilbenes and flavonoids, respectively. The stepwise condensing reactions correspond to those in other polyketide and fatty-acid synthases. This predicts that the two proteins also contain cysteines that are essential for enzyme activity because they bind the substrates. We exchanged, in both enzymes, all of the 6 conserved cysteines into alanine by site-directed mutagenesis and tested the mutants after expression of the proteins in the Escherichia coli heterologous system. Only cysteine 169 was essential in both enzymes, and inhibitor studies suggest that it is the main target of cerulenin, an antibiotic reacting with the cysteine in the active center of condensing enzymes. Most of the other exchanges led to reduced activities. In two cases, the enzymes responded differently, suggesting that the cysteines at positions 135 and 195 may be involved in the different product specificity of the two enzymes. The sequences surrounding the essential cysteine 169 revealed no similarity to the active sites of condensing enzymes in other polyketide synthases and in fatty acid biosynthesis. The available data indicate that resveratrol and chalcone synthases represent a group of enzymes that evolved independently of other condensing enzymes.

Tumour genes in plants: T-DNA encoded cytokinin biosynthesis.

Gene 4 from the T-region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl-transferase activity catalyzing the formation of N6-(delta2-isopentenyl)adenosine from 5'-AMP and delta2-isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin-autotrophic, T-DNA free tobacco cells. The results establish that gene 4 from the T-region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.

Comparative receptor study in gamete chemotaxis of the seaweeds Ectocarpus siliculosus and Cutleria multifida. An approach to interspecific communication of algal gametes.

Gamete fusion in phaeophytes is initiated by olefinic hydrocarbons as messenger substances. They are secreted by the female gametes and act on male gametes or gametangia. The sensitivity thresholds of the androgametes of Ectocarpus siliculosus and Cutleria multifida, two brown algae of different evolutionary stages, for ectocarpene, which is produced by either species, were found to be 0.89 nmol/l and 8.9 nmol/l sea water, respectively. The molecular recognition mechanism is shown to be identical for these two species and probably also for most other phaeophytes. The pheromones involved are structurally related and sometimes resemble each other in their overall pi-electron distribution (e.g. ectocarpene and multifidene). Knowing the specific messenger, the typical response behaviour of each receptor involved, the pheromone productivity of the considered species as well as other biological parameters (phytogeographic distribution, the natural benthic habitat and times of fertilization), a method is presented to uncover possible interspecific cross-reactions of such algal communication systems as a 'chemical warfare' device in their fight for living space.

The Sperm Attractant of the Marine Brown Alga Ascophyllum nodosum (Phaeophyceae).

Spermatozoids of the intertidal seaweed Ascophyllum nodosum (Fucales, Phaeophyceae) are attracted to eggs prior to fertilization. The attractant has been isolated and its structure identified as 1(3E, 5Z, 8Z)-undecatetraene (finavarrene). The relation of finavarrene to sex hormones in other brown algae is discussed.

Dictyota dichotoma (Phaeophyceae): Identification of the Sperm Attractant.

Freshly released eggs of the marine brown alga Dictyota dichotoma secrete a substance that attracts spermatozoids. This compound has been identified as n-butyl-cyclohepta-2,5-diene. It is closely related to attractants in several other brown algae and confirms that a relation exists between phylogeny and attractant compounds.

Seaweed dermatitis: structure of lyngbyatoxin A.

A highly inflammatory and vesicatory substance, lyngbyatoxin A, has been isolated from the lipid extract of a Hawaiian shallow-water variety of Lyngbya majuscula Gomont; its gross structure was determined from chemical and spectral data. Lyngbyatoxin A is closely related to teleocidin B, a poisonous substance associated with several strains of Streptomyces.