POLLEN REMOVAL AND POLLEN DEPOSITION AFFECT THE DURATION OF THE STAMINATE AND PISTILLATE PHASES IN CAMPANULA RAPUNCULOIDES

We examined factors affecting the duration of the staminate and pistillate phases in the protandrous flowers of Campanula rapunculoides L. (Campanulaceae). Under conditions of natural pollinator visitation, flowers experiencing low rates of pollen removal lasted significantly longer than flowers that had faster rates of pollen removal. Experimental manipulations showed that low levels of pollen removal resulted in extension of the staminate phase. Hand-pollinations in which we varied the amount and source of pollen showed that when the number of fertilized ovules within an ovary is low, senescence of the flower is delayed, resulting in extension of the pistillate phase. We also report on pollinator foraging patterns within the vertical inflorescences of C. rapunculoides and the limiting factor for seed set in this population. The results are relevent to recent suggestions that floral characters often serve to reduce interference between the sexual functions in cosexual plants.     

Prostaglandin H Synthetase-Mediated Metabolism of Dopamine: Implication for Parkinson's Disease

Abstract: Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and post-mortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E₂ (PGE₂, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE₂ synthesis. Dopamine stimulated PHS synthesis of PGE₂. [³H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H₂O₂-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H₂O₂-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. ³²P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/µg DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.     

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.     

The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea . In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an 'extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of 'self' coating (i.e. that with the same S -haplotype as the stigma) prevent the success of a compatible cross pollination, but a 'cross' coating (i.e. that with a different S -haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an M_r10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: P ollen C oat P roteins-class A)—one of which is known to bind to stigmatically-expressed components of the S -locus in Brassica . Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins.

The major non-glycosylated structural proteins of feline leukemia virus have been isolated, and competition immunoassays have been developed for each. These proteins include the 27,000- to 30,000-molecular-weight major internal antigen designated p30, a 15,000-molecular-weight protein (p15), an acidic protein of 12,000 molecular weight (p12), and a highly basic 10,000-molecular-weight protein (p10). Immunologically and biochemically corresponding proteins of feline and murine leukemia viruses have been identified. and, on the basis of analogy to the known sequence of a prototype type C virus of mouse origin, the map order of the gag region of the feline type C viral genome has been tentatively deduced as NH2-p15-p12-p10-COOH. The demonstration of two feline leukemia virus gag gene-coded proteins, p15 and p12, expressed in the form of an uncleaved precursor in a mink cell line nonproductively transformed by feline sarcoma virus provides indirect support for the proposed sequence.     

Translation of type C viral RNAs in Xenopus laevis oocytes: evidence that the 120,000-molecular-weight polyprotein expressed in Abelson leukemia virus-transformed cells is virus coded.

The genomic RNA of Abelson leukemia virus (AbLV) has been purified and translated in Xenopus laevis oocytes. The primary AbLV-specific protein synthesized is a polyprotein corresponding in molecular weight and immunological properties to a previously described p15 and p12 containing 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells. In contrast, translation of woolly monkey sarcoma virus genomic RNA resulted in symthesis of a 55,000-molecular-weight polyprotein consisting of woolly helper virus p30, p15, and p12. These findings demonstrate the value of the X. laevis oocyte in vitro system for studies of translational products of replication-defective transforming viruses and establish the virus-coded nature of the nonstructural component of the 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells.