Induction of fern spore germination

Light-induced germination of Anemia spores can be inhibited by AMO-1618, a selective inhibitor of gibberellin biosynthesis. The inhibitor has no effect on gibberellin-induced dark germination and its inhibition of light-induced germination can be reversed by supplying gibberellin. Barley-endosperm bioassay of concentrates of medium in which spores are imbibed in light reveals the presence of substances with gibberellin-like activity; assay of medium from dark-imbibed spores does not. Simultaneous exposure of spores to sub-optimal levels of light and gibberellin leads to additivity of effect on germination level. Uptake of labeled gibberellin by spores in light is similar to that in darkness. The implications of these findings for the light-dependent synthesis of a gibberellin-like germination substance are discussed. The bearing of the observations upon understanding the interaction of light and gibberellins in seeds of higher plants is considered.     

Potential phytotoxicity associated with the use of soil urease inhibitors

Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH₂CONH₂ + H₂O → 2NH₃ + CO₂) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as N-(n-butyl)thiophosphoric triamide or phenylphosphorodiamidate. Continuation of this work showed that these inhibitors can induce leaf-tip necrosis in plants. Research to account for this phytotoxicity indicated that it resulted from an accumulation of toxic amounts of urea in plants through inhibition of urease activity by N-(n-butyl)thiophosphoric triamide and phenylphosphorodiamidate. Support for this conclusion was provided by experiments showing that these urease inhibitors increased both leaf-tip necrosis and urea concentrations in wheat (Triticum aestivum L.) and sorghum [Sorghum bicolor(L.) Moench] plants grown in soils treated with urea and that the necrotic areas of such plants had a much higher concentration of urea than did the nonnecrotic areas. The potential of urease inhibitors for inducing phytotoxicity should not preclude their use to eliminate the adverse effects of urea fertilizers on seed germination and seedling growth in soil because the ammonia produced through hydrolysis of urea fertilizer by urease is much more detrimental to plant growth than is the urea accumulation induced by urease inhibitors.     

Induction of cytokinin autonomy by N,N'-diphenylurea in tissue cultures of Phaseolus lunatus L

The ability of N,N′-diphenylurea (Ph₂urea) to substitute for cytokinin-active adenine derivatives in promoting callus growth of Phaseolus lunatus has been examined. In general, Ph₂urea stimulated callus growth at high concentrations, although the growth of most callus tissues was irregular. Variability in the sensitivity and uniformity of the growth response to Ph₂urea was found among different genotypes of P. lunatus. Most importantly, tissues cultured on Ph₂urea-containing medium for one passage had acquired the ability to proliferate in subsequent passages in the absence of either Ph₂urea or cytokinin-active adenine derivatives. Corresponding tissues maintained on kinetin-containing medium remained cytokinin-dependent. It appears that the effect of Ph₂urea in promoting the growth of P. lunatus callus tissue resides in its ability to induce cytokinin autonomy. This result suggests that the cytokinin activity of Ph₂urea may be due to promotion of endogenous cytokinin biosynthesis in the bioassay systems in which it is active.     

Isolation and characterization of Marine fungal metabolites against clinical pathogens

ObjectiveTo isolate and identify of marine fungal metabolites against clinical bacterial pathogens. To optimize the production medium for isolated fungus.MethodMarine fungus isolated from water and sediment samples from different places of Sundarbans mangrove, Muttukadu (Chennai) and Parangipettai in India. Antimicrobial substance from marine fungi was produced by agar plate method. The potent fungal were inoculated on production medium and extracted was done. The extracted compound was checked for anti bacterial activity. Suitable production medium were optimized.ResultTotally 30 fungal isolates were recovered and morphologically 10 different strains were belongs to the fungal genera such as Fusarium, Aspergillus, Mucor and Penicillium. Preliminary screening results showed 3 fungal isolates showed promising activity. After production of potent fungal SS2 crude extracts showed highest inhibition against the bacterial pathogens out of 3 fungal isolates. The results showed maximum zone in 20mm against E. coli and minimum 10 mm against Vibrio sp.ConclusionsThe present study identified Fusarium sp isolated from Sundarbans mangrove water as a potential source for bioactive compounds. Further isolation of active compound from potential fungal isolates will leads to the discovery of effective antimicrobials.     

Correlation between haemolymph juvenile hormone titre,corpus allatum volume,and corpus allatum in vivo and in vitro activity during oocyte maturation in a cockroach (Nauphoeta cinerea)

Characteristic changes in haemolymph juvenile hormone (JH) titre, corpus allatum (c.a.) volume, and c.a. in vivo and in vitro activity occur during the oocyte maturation phase of the ovoviviparous cockroach Nauphoeta cinerea. When oocyte growth proceeds at its highest rate the c.a. volume and in vitro activity as well as the JH titre in haemolymph are highest, indicating a positive correlation between these parameters. However, no correlation between c.a. in vitro activity and haemolymph JH titre is detectable if individual females in a stage of rapid oocyte growth are investigated. This suggests that JH is possibly synthesized in pulses and not continuously. The in vivo activity of transplanted c.a. is different from their in vitro activity and it seems that the in vitro activity reflects the rate of JH production at the time of dissection while the in vivo activity is indicative of the JH-producing potential under certain influences of the surrounding medium.     

Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin-probe techniques

Sucrose is accumulated in response to NaCl-induced stress in the cyanobacterium Synechococcus 6311. Internal cell volume was measured by ESR spectra with 2,2,6,6-tetramethyl-4-oxopiperidinoxy free radical (TEMPONE) as a spin probe in order to calculate sucrose concentrations inside the cell. This method is rapid and reliable and provides an unambiguous measurement of absolute volumes in different osmotic environments. Because the osmolar concentration of sucrose does not counter-balance the osmolar concentrations of ions in the growth medium, we suggest that sucrose accumulation is one of the mechanisms involved in the process of adaptation to salt of Synechococcus 6311. The accumulation of sucrose in non-N₂-fixing cyanobacteria such as Synechococcus 6311 and in N₂-fixing cyanobacteria such as Nostoc muscorum suggests a common mechanism of osmoregulation of fresh water cyanobacteria in response to increasing NaCl concentrations in the growth medium.     

Blood and pituitary prolactin levels in tilapia (Sarotherodon mossambicus; teleostei) from different salinities as measured by a homologous radioimmunoassay

Highly purified prolactin (PRL) of the tilapia (t), Sarotherodon mossambicus, was injected into rats to produce antibodies. A satisfactory antiserum to the tPRL was used to develop a homologous radioimmunoassay (RIA) in which ¹²⁵I-tPRL was used as the label. The RIA sensitivity (minimal detectable dose) was 0.8 ng of tPRL standard per milliliter. Specificity of the assay was established by demonstrating negligible cross-reactivity of the antiserum with highly purified tilapia growth hormone, a gonadotropin fraction from tilapia pituitaries, and serum from hypophysectomized Sarotherodon. Furthermore, the RIA disclosed that tPRL is highly concentrated in the rostral pars distalis of tilapia kept in either fresh water (FW) or artificial seawater (ASW). The content of immunoreactive tPRL was 6–10 times greater in pituitaries of FW-adapted fish than in glands of those kept in ASW. Serum levels of tPRL were low in fish kept in 100% ASW and they increased 7- to 12-fold with adaptation to FW. Lowering the environmental salinity from 100 to 30% ASW increased the serum level of tPRL only slightly; the major increase in serum PRL concentration in response to hypotonic medium occurs when the environmental salinity is lowered below 10% ASW. Fish adapted to 100% ASW lacking either CaCl₂ or MgCl₂ did not show elevated serum PRL levels. These results indicated that in tilapia a major stimulus for prolactin secretion is reduced environmental NaCl and/or osmolarity.     

Quorum sensing in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium strains grown in Luria–Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication. The factor is not produced when the strains are grown in Luria–Bertani medium in the absence of glucose. Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase. Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E. coli and S. typhimurium is critical for regulating behavior in the prestationary phase of growth. Our results further suggest that the signaling factor produced by E. coli and S. typhimurium is used to communicate both the cell density and the metabolic potential of the environment. Several laboratory and clinical strains of E. coli and S. typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E. coli AB1157 and S. typhimurium LT2. However, we also show that E. coli strain DH5α does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance. Implications for the involvement of quorum sensing in pathogenesis are discussed.     

Solid Peroxy Compounds as Additives to Organic Waste for Reclamation of Post-Industrial Contaminated Soils

Solid peroxy compounds have been increasingly applied for the removal of organic pollution from contaminated groundwater and soil due to their ability to release oxygen and hydrogen peroxide. The influence of two solid peroxy compounds (sodium percarbonate, 2Na₂CO₃·3H₂O₂ and calcium peroxide, CaO₂) with poultry manure (PM) added to contaminated soil on the growth of the tested plants (Sinapis alba, Lepidium sativum L. and Sorghum bicolor L. Moench) and the quality of soil water leachates was investigated. A series of experiments involving the addition of CaO₂ and 2Na₂CO₃·3H₂O₂ at the dose of 0.075 g/g PM improved the growth of tested plants. The conducted study indicated that the use of peroxy compounds not only removed pathogens from livestock waste, but also improved the quality of plant growth. The calculated factors for the growth of roots (GFR) and growth of shoots (GFS) in soils treated with a mixture of peroxy compounds and PM were higher than in soils treated only with PM. The physicochemical analysis of soil water leachates indicated that solid peroxy compounds may be a promising alternative compared to the currently used hygienizing agent such as calcium hydroxide (Ca(OH)₂). Solid peroxy compounds increased the bioavailability of components necessary for proper seed germination and plant growth (N, P, K, Ca, Mg and S). In most of the studied cases, the obtained plant shoot and root growth rates were higher for soil mixtures containing organic waste deactivated by biocidal compounds, compared to soils that contained only poultry manure.     

Preliminary screening for in vitro anti-enteritic properties of a traditional herb Dillenia pentagyna Roxb. fruit extracts

ObjectiveTo explore anti-enteric properties of Dillenia pentagyna Roxb. (D. pentagyna) fruit extract fractions of different polarities by comparative antimicrobial activity against municipal sewage microflora and to assess its urease inhibition potential.MethodsDifferent polar fractions of D. pentagyna fruit extracts were studied by antimicrobial susceptibility test with several adjustments in this resource limited setup. Tests were done against municipal sewage microbes at various bacterial load (initially with 1.0 McFarland followed by 1.5 McFarland) using basal (nutrient agar) and selective medium (MacConkey's agar with and without supplementation of 5% NaCl). All assays statistically scaled with a gradient for uniformity and comparison with ciprofloxacin standard. Fraction with highest activity was studied for urease inhibition potential by kinetic method.ResultsDP-47 separated by 30% ethyl acetate (EtOAc) in CHCl₃ from CHCl₃ extract had slightly higher antimicrobial potency (y=0.758x+7.571) as dissolved in methanol than in dimethylsulfoxide (y=0.300x+6.000). EtOAc extract fractioned by 10% methanol in EtOAc (DP-43) was more potent antimicrobial (y=1.428x+8.392) and soluble in water. Butanol extract fractioned by water (DP-50) showed highest antimicrobial potency (y=3.384x+2.000) than both DP-47 and DP-43 in disk diffusion assays. In higher microbial load DP-50 was potent antimicrobial (y=1.538x+3.000) and completely inhibited any vegetative growth at lower load of 0.5 McFarland. In selective environment DP-50 was effective (y=1.076x+7.500 in MacConkey's, y=1.307x+6.000 in 5% NaCl supplemented). It was evident that enteric pathogens may not easily develop resistance against DP-50 and at high concentration it inhibited urease activity by 94.87%. The standard inhibition by thiourea (1%) is 33.914%.ConclusionsHighly polar fraction of D. pentagyna Roxb. fruit extract DP-50 have potential antimicrobial activity against sewage microflora in basal and selective culture indicating its potential against non-fastidious, coliforms and Vibrio pathogens. Urease inhibition indicates efficacy against Helicobactor pylory.     

Studies on a peptide with red pigment-concentrating and hyperglycemic activity from the cephalic endocrine system of the honeybee, Apis mellifera

Shrimp red pigment-concentrations hormone (RPCH), locust adipokinetic hormone (AKH), and locust Compound I all elevate hemolymph lipid in locusts and concentrate pigment in shrimp erythrophores. In addition, RPCH and AKH both elevate hemolymph carbohydrate in cockroaches. Material with comparable biological activity has been reported in many insects, but not in representatives of the order Hymenoptera. Distilled water head extracts of the honeybe, Apis, exhibited red pigment-concentrating activity in the shrimp Leander and hyperglycemic activity in the cockroach Periplaneta. Chemical characterization of the active substance in Apis extracts showed that like RPCH, AKH, and Compound II, it was heat stable, soluble in butanol and methanol, degraded by proteolytic enzymes, and localized in the cephalic neuroendocrine system. Similarly, like RPCH and Compound II, but unlike AKH, the Apis substance eluted at 1.2 V_t during Sephadex G-25 gel filtration. The data thus provide the initial demonstration that the endocrine system of at least one hymenopteran contains a peptide that is chemically and biologically comparable to the RPCH/AKH/Compound II group of hormonally active peptides. Furthermore, the results support the hypothesis that such peptides may be commonly used as hormones in holometabolic insects. In addition, pure Compound II was shonw for the first time to be hyperglycemic in the cockroach.     

Sodium-retaining bioassay of prolactin in the intact teleost Tilapia mossambica acclimated to sea water

A bioassay method for fish prolactin was developed based on the ability of prolactin to increase the plasma sodium concentration of intact Tilapia mossambica acclimated to sea water. The plasma sodium concentration is linearly related to log dose of ovine prolactin. Homogenates of whole pituitary gland from fresh water T. mossambica give a parallel dose response. A significant elevation of plasma sodium is elicited by 4 μg/g body wt of ovine prolactin. The same amount (wet wt) of whole pituitary gland from freshwater T. mossambica has equal potency, indicating that the assay is much more sensitive to at least some teleost prolactins than to mammalian prolactins. The assay is unaffected by ACTH, dexamethasone, bovine growth hormone, porcine prolactin, or human chorionic somatomammo-from Cichlasoma labiatum, Roccus saxatilis, Mugil cephalus, and Poecilia latipinna,In addition to Tilapia, sodium-retaining activity was detected in pituitaries from Cichlasoma labiatum, Roccuss saxatilis, Mugil cephalus, and Poecilia latipinna, but not in any of 13 other species of fish examined. This activity was restricted to the rostral pars distalis of Tilapia and Cichlasoma. It was localized in a single prominent band with a R_f of .47 after electrophoresis of Tilapia pituitaries. The sodium-retaining potency of seawater Tilapia pituitaries was about one-eighth that of freshwater Tilapia pituitaries.     

Chemical stability,biological activity and cellular uptake of a cisplatin analogue having a 1,2-diarylethyleneamine ligand in cultures of human breast cancer cells

The platinum(II) complex PtCl₂(meso-6), which has the estrogenic ligandmeso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (meso-6), has been reported to be an effective antitumor drug for estrogen-receptor(ER)-positive tumors in animal experiments. The goal of this study was to investigate whether the observed biological effects could be ascribed to the intact PtCl₂(meso-6). Cultures of the ER-positive human breast cancer cell line MCF-7 were used as the in vitro test system. In culture medium containing 10% fetal calf serum, PtCl₂(meso-6) had a half-life of about 2 h, as determined by HPLC analysis, and no PtCl₂(meso-6) was detectable after 10 h. The Pt complex bound irreversibly to serum protein. After 30 min, the diamine ligand was found released, with a maximum conversion of about 35% at 24 h. At this time the culture medium still had estrogenic activity, i. e. it induced ER processing in the MCF-7 cells. This indicates that the estrogenic effect was elicited by the released diamine ligand. In contrast, the growth-inhibitory activity of the medium preincubated with PtCl₂(meso-6) was lost at a rate similar to the rate of loss of PtCl₂(meso-6) from the medium. This accords with the platinum complex being the main cytotoxic entity. When MCF-7 cells were incubated with PtCl₂([³H]meso-6), no free Pt complex could be identified in cellular extracts, and most of the cell-associated radioactivity coeluted with meso-6 in HPLC analysis. After 12 h, only 1.4% of the total cellular platinum was bound to DNA, but no tritium label could be detected. In conclusion, diamine ligand is released from the Pt(II) complex and can account for the estrogenic effects so far ascribed to PtCl₂(meso-6).     

Synthesis,Structural Characterization,and Antitumor Activity of a Ca(II) Coordination Polymer Based on 1,6-Naphthalenedisulfonate and 4,4′-Bipyridyl

A novel Ca(II) coordination polymer, [CaL(4,4′-bipyridyl)(H₂O)₄]_n (L = 1,6-naphthalenedisulfonate), was synthesized by reaction of calcium perchlorate with 1,6-naphthalenedisulfonic acid disodium salt and 4,4′-bipyridyl in CH₃CH₂OH/H₂O. It was characterized by elemental analysis, IR, molar conductivity and thermogravimetric analysis. X-ray crystallography reveals that the Ca(II) coordination polymer belongs to the orthorhombic system, with space group P2₁2₁2₁. The geometry of the Ca(II) ion is a distorted CaNO₆ pengonal bipyramid, arising from its coordination by four water molecules, one nitrogen atom of 4,4′-bipyridyl molecule, and two oxygen atoms from two L ligands. The complex molecules form a helical chain by self-assembly. The antitumor activity of 1,6-naphthalenedisulfonic acid disodium salt and the Ca(II) coordination polymer against human hepatoma smmc-7721 cell line and human lung adenocarcinoma A549 cell line reveals that the Ca(II) coordination polymer inhibits cell growth of human lung adenocarcinoma A549 cell line with IC50 value of 27 μg/mL, and is more resistive to human lung adenocarcinoma A549 cell line as compared to 1,6-naphthalenedisulfonic acid disodium salt.     

Phytotoxicity of foliar-applied urea

Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH₂CONH₂ + H₂O → 2NH₃ + CO₂) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as phenylphosphorodiamidate. Because the leaf-tip necrosis often observed after foliar fertilization of plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease, we studied the possibility that this necrosis could be eliminated or reduced by adding phenylphosphorodiamidate to the urea fertilizer. We found that, although addition of this urease inhibitor to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean [Glycine max. (L.) Merr.] leaves fertilized with urea, it increased the leaf-tip necrosis observed after fertilization. We conclude that this necrosis resulted from accumulation of toxic amounts of urea rather than from formation of toxic amounts of ammonia. This conclusion was supported by our finding that the necrotic areas of soybean leaves treated with urea or with urea and phenylphosphorodiamidate contained much higher concentrations of urea than did the nonnecrotic areas.     

NO x generation by cultured small intestinal epithelial cells

The effect of cytokines, growth factors, mitogens, and bacterial products on nitric oxide (NO) generation by monolayers of small intestinal epithelial cells-6 (IEC-6) cells was evaluated. Subconfluent IEC-6 cells were maintained in DMEM containing 5% fetal calf serum and after 16–24 hr of incubation, the medium was replaced with fresh medium in the presence or absence of calcium ionophore (CaI),l-NAME,l-NNA, individual growth factors, cytokines, or mitogens. After 72 hr of culture, the media supernatant was collected and NO _x generation was determined. NO synthase activity was determined in sonicated supernatants of IEC-6 cells by [¹⁴C] arginine conversion to citrulline. NO _x generation in subconfluent cultures was greater than in fully confluent cultures, suggesting contact inhibition. NO _x generation by IEC-6 cells was significantly increased by CaI and inhibited byl-NAME andl-NNA. LPS, IL-1β, IL-2, IL-8, IFN-8, TFN-α, EGF, TGF-α, bFGF, and PHA significantly increased NO _x generation. NO synthase activity in IEC-6 cells (4.2±1.7 pmol/min/10⁶ cells) was NADPH dependent. These results suggest that stimulation of NO _x generation by intestinal epithelial cells through cytokine bacterial products and mitogens may be one of the mechanisms responsible for their effects in the intestinal tract.     

Changes in the activity and properties of trehalase during early germination of yeast ascospores: Correlation with trehalose breakdown as studied by in vivo13C NMR

The regulation of trehalose breakdown during dormancy and the induction of germination in yeast ascospores was studied both by in vivo high-resolution NMR spectroscopy and in vitro assays of trehalase activity. Natural-abundance ¹³C NMR spectra taken during the induction of germination with glucose and phosphate showed a rapid breakdown of part of the trehalose content. The presence of both glucose and phosphate was important for maximal trehalose breakdown. The ¹³C NMR spectra showed that the externally added glucose and the internal trehalose were metabolized mainly to glycerol and ethanol. Under these conditions of nitrogen deprivation, full germination is not possible and trehalose breakdown stopped after ≈1 hr. At this moment resynthesis of trehalose occurred while glycerol and ethanol production from the exogenous glucose continued. In complex media where full spore germination can occur, trehalose breakdown was more pronounced. Measurements of trehalase activity in spore extracts made after addition of varying amounts of glucose and phosphate to the spores revealed a sudden 10-fold increase in the activity of trehalase, within the first minutes of spore germination. The activation was transient: after reaching a maximum between 5 and 10 min, the activity declined back to low values during the next hours. The increase in trehalase activity was not inhibited by cycloheximide or by anaerobic conditions. The decline in trehalase activity that occurred after the initial activation could be correlated with the extent of trehalose breakdown as measured by ¹³C NMR. In addition to the increase in trehalase activity, differences in the control properties were found between the enzymes from dormant and germinating spores. Trehalase from dormant spores was strongly inhibited by ATP at a concentration of ≈0.5 mM, which corresponds with the ATP concentration found in dormant spores. On the other hand, trehalase from germinating spores was not inhibited by ATP up to the much higher ATP concentrations that are found in germinating spores. It is suggested that the low activity and the stringent ATP feedback inhibition of trehalase from dormant spores are responsible for the very slow mobilization of the huge amount of trehalose in dormant spores. Therefore, dormancy seems to be caused primarily by extreme curtailment of the energy production within the spore at one selective and primary point. The switch towards high activity and low ATP inhibition upon induction of germination is suggested to be responsible for the breaking of dormancy and for the rapid breakdown of trehalose that occurs during the initial phase of germination.     

Expression of hydrogenase activity in free-living Rhizobium japonicum

A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H₂ uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H₂ under conditions such that the cells did not exhibit nitrogenase activity. H₂ uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H₂ oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H₂ in air either showed no or low capacity to take up H₂ under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H₂. This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H₂. This mutant strain forms nodules that evolve H₂ but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.