A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of Lycopersicon peruvianum leaf explants and streptomycin-resistant shoots on medium containing streptomycin

The effect of streptomycin on morphogenic explants of Lycopersicon peruvianum Mill. was examined microscopically at both the light and ultrastructural level. Early stages in shoot regeneration from leaf explants were distinguished as meristematic tissue at both levels. Small starch grains were observed in the plastids in this tissue but not in plastids in regenerated shoots. In the presence of streptomycin, adventitious shoot regeneration from sensitive leaf strips was inhibited. Large layered bodies were observed within the plastids of sensitive leaf tissue, suggesting the disruption of thylakoid membrane formation. Streptomycin resistant L. peruvianum lines, as well as a chlorophyll-deficient line, were also examined microscopically. The chloroplasts of newly regenerated streptomycin resistant shoots contained well developed internal membranes and conspicuous starch grains. Cells containing a mixture of resistant and sensitive plastids were not observed. The plastids in chlorophyll-deficient tissue completely lacked thylakoid membranes, although small vesicles and intraplastid bodies were seen within the stroma.Abbreviations NMU N-methyl-N-nitrosourea     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques.

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.     

Dichlorvos effects on estrous cycle onset in the rat.

Continuous exposure to dichlorvos (DDVP) vapors from birth in rats caused a statistically significant mean delay of 10 da in the onset of the first estrous cycle. DDVP, BUT NOT ITS PRIMARY METABOLITE (DES-METHYL DDVP), could be readily isolated from blood and ovarian tissue, and occassionally in minute quantities from the kidney and adrenal tissues. DDVP was recovered only once in 40 brain tissue samples examined.     

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

On the synthesis of the 2,6-dideoxysugar L-digitoxose

As deoxysugars are integral components of many natural products, the development of efficient chemical and enzymatic routes to prepare these compounds is of particular interest. Herein, we report a comparison of several synthetic methodologies used to prepare protected derivatives of the 2,6-dideoxysugar l-digitoxose. A novel, stereoselective synthetic route to efficiently access methyl 4-O-tert-butyldimethylsilyl-2,6-dideoxy-3-O-trimethylsilyl-alpha-l-ribo-hexopyranoside in 35% yield over nine facile steps is described.