Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro

Leaf and stem explants of Trillium grandiflorum and T. erectum produced mini-rhizomes (MRs) in vitro which gave rise to shoots and roots. The apical portion of the stem and the basal portion of the leaves were the most effective explants from these tissues, while stem tissue was more responsive than leaf tissue. The best response with both species was observed on half-strength MS basal medium supplemented with cytokinin and auxin. T. erectum was more responsive than T. grandiflorum overall, and in some cases produced MRs in the absence of growth regulators. Culture at 21°C appeared to stimulate growth from T. grandiflorum tissues, compared with controls at 27°C, whereas the outgrowth of shoots from MRs was inhibited in both species at 21°C. In vitro production of MRs could provide a more rapid, alternative propagation method for these species than traditional methods.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa-related epitope labeled mature tissue strongly; antibodies to an iota-related epitope and a lambda-related epitope labeled weakly, consistent with the kappa-enriched carrageenan produced by this alga. Kappa-related epitopes were distributed throughout the wall and matrix, whereas iota-related epitopes were concentrated in the middle lamella. Lambda-related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.     

Desiccation tolerance in developing soybean seeds: The role of stress proteins

The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of certain "late embryogenesis abundant" (LEA) proteins suggests that these proteins reduce desiccation-induced cellular damage. The aim of the present work was to test this hypothesis. Exogenous abscisic acid (ABA) was used to elevate the level of heal-soluble LEA-like proteins in axes from immature (30 days after flowering: mid-development) seeds of soybean ( Glycine max [L.] Merrill cv. Chippewa 64). As the LEA-like proteins accumulated in response to ABA, the leakage of all elements after desiccation and subsequent rehydration markedly declined. Both LEA-like protein accumulation and the decline in desiccation-induced electrolyte leakage were apparently dependent on the presence of ABA. Both effects of ABA were inhibited by cycloheximide. Light microscopy revealed a marked effect of the ABA on cellular integrity following desiccation. Osmotic stress also caused a decrease in desiccation-induced electrolyte leakage and stimulated the accumulation of LEA-like proteins. Our data are consistent with the hypothesis that the LEA-like proteins contribute to the increase in desiccation tolerance in response to ABA, and are consistent with a general protective role for these proteins in desiccation tolerance.     

Frequency-dependent interference by magnetic fields of nerve growth factor-induced neurite outgrowth in PC-12 cells

We have shown that 50 Hz sinusoidal magnetic fields within the 5-10 micro Tesla (μT) rms range cause an intensity-dependent reduction in nerve growth factor (NGF) stimulation of neurite outgrowth (NO) in PC-12 cells. Here we report on the frequency dependence of this response over the 15-70 Hz range at 5 Hz intervals. Primed PC-12 cells were plated in collagen-coated, 60 mm plastic petri dishes with or without 5 ng/ml NGF and were exposed to sinusoidal magnetic fields for 22 h in a CO₂ incubator at 37 °C. One 1,000-turn coil, 20 cm in diameter, generated vertically oriented magnetic fields. The dishes were stacked on the center axis of the coil to provide a range of intensities between 3.5 and 9.0 μT rms. The flux density of the ambient DC magnetic field was 37 μT vertical and 19 μT horizontal. The assay consisted of counting over 100 cells in the central portion (radius ≤0.3 cm) of each dish and scoring cells positive for NO. Sham exposure of cells treated identically with NGF demonstrated no difference in the percentage of cells with NO between exposed and magnetically shielded locations within the incubator. Analysis of variance demonstrated flux density-dependent reductions in NGF-stimulated NO over the 35-70 Hz frequency range, whereas frequencies between 15 Hz and 30 Hz produced no obvious reduction. The results also demonstrated a relative maximal sensitivity of cells at 40 Hz with a possible additional sensitivity region at or above 70 Hz. These findings suggest a biological influence of perpendicular AC/DC magnetic fields different from those identified by the ion parametric resonance model, which uses strictly parallel AC/DC fields. © 1995 Wiley-Liss, Inc.     

Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, we examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with ¹²⁵I-lipoproteins [human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)]. The media were then analyzed for lipoprotein protein coat degradation products (mainly ¹²⁵I-monoiodotyrosine) and progestin [mainly 20α-dihydroprogesterone (20α-DHP)]. In the absence of FSH and androgen, 2 × 10⁵ granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20α-DHP. The addition of 10^?7 M androstenedione (A), testosterone (T), or 5α-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20α-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20α-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20α-DHP production. The addition of a 10-fold excess cyproterone acetate (an anti-androgen) inhibited the effect of T, suggesting that the action of T was mediated by the granulosa cell androgen receptor. Androgen and FSH also synergistically stimulated the production of ³H-progestin when the granulosa cells were incubated with either ³H-cholesterol ester core labeled human HDL or similarly labeled human LDL. This report demonstrates that androgen, in combination with FSH, augments the steroidogenic pathway of the granulosa cell from the degradation of lipoprotein and utilization of the cholesterol ester core, to the production of progestin product.     

The relationship of male-male mounting to mate choice and sexual performance in male dairy goats

Rearing of male farm animals in unisexual groups has been implicated as a factor contributing to the failure of many males to breed as adults. The present study examines the relationship of male-male mounting in yearling dairy goats to subsequent mate preferences and sexual performance.Twenty-four sexually inexperienced male dairy goats, representing the Alpine, LaMancha, Saanen and Toggenburg breeds, were observed for male-male mounting in their home enclosure and then tested for mate choice and sexual performance when exposed to male and female (estrous and diestrous) stimulus animals. Their sexual behavior was compared with 7 adult goats with previous breeding experience.In the mate choice-sexual performance tests, 4 sexually inexperienced goats (17%) were sexually inactive, 6 (25%) mounted both male and female stimulus animals and 14 (58%) mounted only the female stimuli. Mate choice and sexual performance of the 20 sexually active males was not related to the number of male-male mounts initiated or the number of different males mounted in their home enclosure. However, the goats that received the greatest number of mounts in their home pen tended to be bisexual (would mount both male and female stimulus animals) in the mate choice tests. Males that were sexually inactive in mate choice-sexual performance tests repeatedly mounted the same male during home pen observations. Except for ejaculation frequency, the sexual performance of the sexually naive and experienced goats was similar. Goats of the Saanen breed were favored recipients of mounts from other males. There was no relationship between the number of male-male mounts performed and received.It was hypothesized that the reproductive failure of many male farm animals reared in all-male groups may be more closely related to the formation of specific sexual attachments to other males rather than the frequency with which they exhibit homosexual behaviors.     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Identification, physical map location and sequence of the denV gene from bacteriophage T4

The denV gene from bacteriophage T4, which codes for endonuclease V, a small DNA repair enzyme, has been cloned and identified by an approach combining DNA sequencing and genetics, independent of the phenotypic effect of the cloned gene. Appropriate DenV+ and DenV- deletion mutants were mapped physically to define precisely a region encompassing the denV gene. This region was sequenced in order to identify a protein-coding sequence of the correct size for the denV gene (400-500 bp). Finally, identification was confirmed by sequencing the corresponding fragments cloned from four genetically and phenotypically well-characterized denV mutants. The denV gene is located at 64 kb on the T4 genome, adjacent to the ipII gene, and codes for a basic protein of 138 amino acids with a deduced molecular weight of 16,078.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.