An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.     

An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

A simple method for cloning leishmanial promastigotes

A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3 X 10(3) promastigotes per ml. Hanging-drop preparations made from 0.2-0.4 microliter volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22 degrees C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species of Leishmania, with up to 100 percent of the cloned organisms growing.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture.

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Effects of an azasteroid on growth, development and reproduction of the free-living nematodes Caenorhabditis briggsae and Panagrellus redivivus

The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth, development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus. The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 micrograms cholesterol per ml and/or 25 micrograms ASA-6 per ml medium. All cultures also contained 50 micrograms Tween 80 per ml medium. After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability. These effects were more apparent in the sterol-deficient medium containing ASA-6. In the presence of cholesterol and ASA-6, growth and reproduction of C. briggsae, but not of P. redivivus, was inhibited after five generations. Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium. These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species. In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.     

Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus]

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.     

Lysosomal enzyme release from human monocytes by particulate activators is mediated by beta-glucan inhibitable receptors

Human peripheral blood monocytes ingest particulate activators and generate leukotrienes via a trypsin-sensitive, beta-glucan-inhibitable receptor. The incubation of monolayers of monocytes with from 4 X 10(5) to 2 X 10(8) zymosan or glucan particles resulted in a dose-dependent release of up to 9% +/- 1.9 and 17.8% +/- 5.3 (mean +/- SD, n = 3) of the lysosomal enzyme, N-acetylglucosaminidase, into the culture medium. Lysosomal enzyme release occurred throughout the 2-hr period studied, with the greatest rate of N-acetyl-glucosaminidase release occurring during the first hour; the presence of 5 micrograms/ml of cytochalasin B accelerated this process when zymosan was the agonist. The preincubation of monocytes with from 0.5 to 500 micrograms/ml of soluble yeast beta-glucan inhibited N-acetylglucosaminidase release by 4 X 10(7) zymosan and glucan particles in a dose-dependent manner, with 50% inhibition occurring with 50 micrograms/ml of soluble yeast beta-glucan (mean +/- SD, n = 3). Preincubation with as much as 5 mg/ml of yeast mannan had no inhibitory effect on N-acetylglucosaminidase release. The pretreatment for 30 min of monolayers of monocytes with 50 micrograms/ml of affinity-purified trypsin, which selectively inactivates the monocyte-phagocytic response to particulate activators, also fully inhibited lysosomal enzyme release induced by zymosan and glucan particles. The inhibitory effects of a soluble ligand, yeast beta-glucan, and of trypsin pretreatment on lysosomal enzyme release correspond to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles and thus indicates ligand specificity for the beta-glucan receptor in the release of stored intracellular mediators.     

Effects of metabolic factors in the diabetic state on the in vitro development of preimplantation mouse embryos

The effects of insulin, glucagon, beta-hydroxybutyrate, and acetoacetate on the in vitro development of preimplantation mouse embryos were studied. In controls, 24% of blastocysts failed to develop successfully when grown for 72 h in Eagle's medium supplemented with 10% fetal calf serum. Insulin at concentrations of 1.0 and 2.0 IU/ml of culture medium interfered with development in 62-63% of the blastocysts. Preimplantation embryos showed a threshold pattern in their reaction to glucagon: its addition in concentrations of 0.0015 mM (5 micrograms/ml) did not significantly inhibit blastocyst development, while concentrations of 0.003 mM (10 micrograms/ml) inhibited 70% of blastocysts. The embryotoxic effects of ketone bodies were manifested only in relatively high doses. beta-hydroxybutyrate was embryotoxic at concentrations greater than 5 mg/ml, and its effects were dose dependent: 48 mM (6 mg/ml) inhibited 45% of blastocysts, while 80 mM (10 mg/ml) arrested 87% of embryos from further development. Acetoacetate at concentrations of 0.1 mM (10 micrograms/ml) inhibited the development of 50% of the blastocysts, and its effects were not dose dependent: concentrations of 1 mM (100 micrograms/ml) inhibited development in 63% of the embryos. The combination of the diabetic metabolic factors in relatively low concentrations was highly embryotoxic, especially when accompanied by hyperglycemia.     

Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 × 10⁷/ml after 10 days of incubation at 33 C, and 5 × 10⁷/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 × 10⁷/ml after 12 days of incubation.     

Characterization of Populations of Promastigotes of Leishmania donovani1

Promastigotes of Leismania donovani cultured for either 3 or 10 days in vitro and inoculated intracardially into golden hamsters with an equal number of organisms from either population showed a 7-fold difference in infectivity when compared at both 10 to 16 days post-infection. Reproducible histochemical staining for the promastigote enzymes glucose-6-phosphate dehydrogenase (G6PDH) and peptidase after polyacrylamide gel electrophoresis showed two isoelectric variants of G6PDH (Bands 1 and 2) that displayed a 45% decrease (Band 1) and a 60% increase (Band 2) in total activity when 3- and 10-day-old promastigores were compared. Peptidase activity, present in a single band, increased 7-fold in 10-day-old promastigotes. A decrease in the lectin-induced agglutination of promastigotes by castor bean agglutinin (RCA₆₀), specific for D-galactose and N-acetyl-D-galactosamine, was seen when 3- and 10-day-old promastigotes are compared. Antisera raised against sonicated 10-day-old promastigotes showed a unique precipitin band between the antiserum and sonicated 10-day-old promastigotes not found between the antiserum and sonicated 3-day-old promastigotes.     

The primary culture of mouse adipocyte precursor cells in defined medium.

1. A defined medium supporting the proliferation and differentiation of adipocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was developed. 2. It consists of a 1:1 mixture of DME and WAJC404A media supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 micrograms protein/ml). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differentiation. 5. Differentiation was enhanced by addition of dexamethasone (10(-9) M) but could be almost completely inhibited by transforming growth factor beta 1 (TGF-beta 1). 6. TGF-beta 1 was shown to be effective only when present in the early stages of differentiation.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.     

Growth Inhibition of Caenorhabditis elegans and Panagrellus redivivus by Selected Mammalian and Insect Hormones

Caenorhabditis elegans and Panagrellus redivivus were cultured in axenic medium in microwells. The addition of selected steroids and terpenoids to the medium caused quantitative inhibition of numbers of offspring produced per well. Three out of 14 vertebrate sex hormones and analogs, and seven out of 10 insect juvenile hormones and analogs inhibited growth at 25 or 50 micrograms per ml. In addition, two insecticide synergists which mimic juvenile hormones, propyl 2-propynyl phenyl phosphonate and piperonyl butoxide, inhibited growth at 7 μg/ml. Total lipids from Panagrellus and from Nematospiroides dubius were inhibitory. Separation by silicic acid column chromatography yielded active and inactive portions. We concluded that the inhibition observed was non-specific.     

Reversible inhibition of osteoclastic activity by bone-bound gallium (III).

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12 microM Ga(III) reduces calcium release from bone, but surprisingly, 10-100 microM Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-3H)-proline bone. 3H-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) greater than 100 microM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 microM gallium from 500 microliters of tissue culture medium, with steady state at greater than 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 micrograms of bone pre-incubated with 1 ml of 1 microM Ga3+, with 10 pmoles Ga3+/micrograms bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/micrograms bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15 microM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/micrograms bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations less than 15 microM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] greater than 50 microM.