Preparation and use of the poly-l-lysine-gold probe: a differential marker of glomerular anionic sites

Summary The conditions required for the production of a polylysine-coated gold (PL-G) complex, which shows optimal sensitivity for the demonstration of tissue anionic sites, expressed under different conditions of pH have been investigated. Problems encountered with this complex have been compared with those found with other methods of conjugation of polylysine to colloidal gold. The performance of a bovine serum albumin (BSA)-stabilized PL-G complex was examined against other PL-G conjugates, including complexes that are commercially available, for the detection of heterogeneous glomerular anionic site populations, expressed at pH 2.5 and pH 7.0.     

The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.     

Protein kinase C levels and protein phosphorylation associated with inhibition of proliferation in a murine macrophage tumor

Treatment of M5076 tumor cells with the phorbol estes 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13 dibutyrate (PdBu) inhibited cellular proliferation, whereas 1,2-dioctanoyl-glycerol (DiC8) and 1-oleoyl2-acetyl-glycerol (OAG) did not affect cell growth. Inhibition of cellular proliferation in this cell line appears to be a consequence of protein kinase C (PKC) down-regulation since phorbol esters, but not a single application of diacylglycerols (DGs) down-regulated cellular PKC levels. By repeated application of DGs, PKC down-regulation was achieved and correlated with inhibition of proliferation. Phorbol ester-induced PKC down-regulation was reversible, upon removal of the phorbol ester, and the reappearance of PKC was associated with resumption of proliferation. The mitogenic responsiveness of these cells to added serum depended upon cellular PKC levels. Phorbol esters also caused the phosphorylation of two proteins which were not phosphorylated in response to DG treatment. Inhibition of growth of M5076 cells appears to be associated with phosphorylation of two novel proteins and/or PKC down-regulation.     

Characterization of soybean choline kinase cDNAs and their expression in yeast and Escherichia coli.

An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase. A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length. Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity. In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity. Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME. DME effectively inhibited only the GmCK2-encoded choline kinase activity. Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme. These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Proposal for adoption of the base of the Undulograptus austrodentatus Biozone as a global Ordovician stage and series boundary level

The base of the Undulograptus austrodentatus Biozone appears to be a synchronous event that is widely recognizable within graptolitic facies around the world. It occurs within an interval in which graptolite species ranges are now well known and in which there is a rapid turnover in the composition of graptolite faunas. This turnover reflects the rapid evolutionary radiation of the Diplograptacea simultaneously with the appearance of several distinctive pseudisograptid and glossograptid species. These events provide the basis for the recognition of two thin but widely applicable subzones; a lower Arienigraptus zhejiangensis Subzone and an upper U. sinicus Subzone. The occurrence of the lower boundary of the U. austrodentatus Biozone within a succession of first appearances also permits accurate and reliable identification of the boundary as well as assessment of stratigraphic completeness across the boundary interval in correlated sections. Diverse graptolite faunas of late Yapeenian and early Darriwilian age occur in association with the Histiodella altifrons Biozone of the North American midcontinent conodont zonation and the Paroistodus originalis and Microzarkodina parva biozones of the North Atlantic conodont zonation. They also occur in association with the shelly-fossil zonations developed for several different continents. These features of the base of the U. austrodentatus Biozone make it a suitable level for use as the boundary level for a global stage. Its stratigraphic position within the Ordovician System relative to other likely global stages as well as its coincidence with one of the major events in graptolite evolutionary history suggest that this level also may be a suitable level for the base of a global Middle Ordovician Series.Ordovician System, Ordovician stages, graptolite zonation, chronostratigraphy, international correlation. Charles E. Mitchell and Jörg Maletz, Department of Geology, State University of New York at Buffalo, Buffalo, New York 14260-1550, USA; 13th July, 1994; revised 22nd May, 1995.     

The milk of the fur-seal, Arctocephalus tropicatis gazella; in particular the composition of the aqueous phase

Milk and plasma samples from the Southern fur-seal (Arctocephalus tropicalis gazella) , collected from animals shot between 0 and 26 days post-partum , have been analysed. The aqueous phase of milk (ultracentrifugate) was hyperosmotic to plasma by about 35 m-osmol/kg water but contained no lactose. The osmolality of milk could be accounted for by the ionic content, of which sodium and potassium (in approximately equal amounts) were the main cations. The fat content of the milk was very high (approximately 40%), except in very early lactation (0 and 1 day post-partum). The concentrations of chloride, calcium, magnesium, phosphate and citrate in the aqueous phase are given. Major differences between the composition of fur-seal milk and that of other mammals are pointed out, and discussed in relation to water conservation, energy and fat supply, and prolonged milk storage in this snecies.