Effect of hydroxylated fullerene C60(OH)25 on macrophage plasma membrane integrity

Our study has shown that the damaging effect of hydroxylated fullerene C60(OH)25 on mouse peritoneal macrophage plasma membranes increased when we enlarged the concentration of fullerene in the incubation media (from 0.005 to 0.5 mg/ml), the incubation temperature (from 22 degrees C to 37 degrees C) and the time of incubation (from 30 to 90 min). In conditions of the H2O2-induced membrane damage, fullerene was observed to intensify the H2O2-induced damaging effect at a concentration of 0.05 mg/ml and reduce it at a concentration of 0.5 mg/ml. In conditions of the UV-induced membrane damage, it was discovered that the damaging effect of UV increased when C60(OH)25 nanoparticles were added to the incubation media before irradiation and decreased when they were added after irradiation. Eventual participation of ROS in damaging effects of C60(OH)25 was discussed.     

Production of an antifungal antibiotic by Streptomyces aburaviensis 1DA-28

A broad-spectrum antifungal Streptomyces isolate, 1DA-28, from Indian soil has been characterized and identified as Streptomyces aburaviensis var. ablastmyceticus (MTCC 2469). Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been determined. Antibiotic production in synthetic medium reached the maximum on the 5th day of incubation at 30 degreesC. Glucose and starch were found to be the best carbon sources while NH4NO3 was preferred as nitrogen source. Optimum temperature and pH for antibiotic production were 32 degreesC and 7.4, respectively. Phosphate at a concentration sub-optimal for growth enhanced antibiotic production. Supplementation of medium with casein hydrolysate improved both growth and antibiotic titre but yeast extract exhibited marked inhibition.     

Optimization of alpha-galactosidase production in Streptomyces erythrus

Physiological studies on Streptomyces erythrus NRRL ISP 5517 grown on fourteen different media have revealed that the enzyme was formed and released in the medium with different levels depending upon the type of the medium and the carbon source used. The results indicate that S. erythrus produced the highest level of extracellular and endocellular enzyme when grown in modified Czapek-Dox's medium (containing 2% D-galactose as the only carbon source). The highest levels of enzyme formation was obtained upon using D-galactose (9.94 Units/ml and 2.92 Units/ml), raffinose (8.87 Units/ml and 2.69 Units/ml) or melibiose (8.14 Units/ml and 2.52 Units/ml) at a final concentration of 2% as inducers for extra- and endocellular enzyme, respectively. With respect to nitrogen sources tested, sodium nitrate produced the highest level of alpha-galactosidase in both fractions optimally at 2.0 g/l. Studies revealed that the extracellular enzyme levels were produced optimally at initial pH in culture of 7.0 and air:medium ratio in flasks corresponding to 1:5 and after 5 days of incubation at 30 degrees C. On testing the effect of the addition of eight leguminous seeds powders (at a final concentration of 2%), it was found that soybean powder gave the highest induction effect. The addition of sodium nitrate at a concentration of 2 g/l to Dox's soybean medium, the adjustment of initial pH value of the medium to 7.0 and the air:medium ratio in flasks to 1:5 for an incubation period of 4 days produced the highest level of extracellular alpha-galactosidase.     

"Stationary phase aging" of cell culture: an attempt of evaluation of growth medium "age" effect

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-FAF28 line) have been determined in growth medium conditioned by the same cells for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. The cells from all 3 flasks were collected by trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different "age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26,600 cells/cm2) and grown at 37degreesC, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later (i. e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i. e. in 2 days after seeding) 3H-thymidine was added to every well to final concentration 1.85 x 10(4) Bq/ml. After next 24 h (i. e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as log2 (N3/N1), where N1 - cell density on the first day after seeding, and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related" diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).     

The fate of ACTH released from rat anterior pituitary into the incubation medium in vitro: enzymatic degradation and acid activation.

The bioactivity of ACTH released from isolated rat anterior pituitary glands into the incubation medium was determined. After the pituitaries were removed, ACTH activity in the medium decreased exponentially during further incubation at 37degreesC. The loss of ACTH activity was temperature- and pH-dependent and inhibited both by protease inhibitor (trasylol) and by preheating. Crude tissue extracts from median eminence, cerebral cortex and liver similarly inhibited the loss of ACTH activity. These results indicate that ACTH released into the medium may be destroyed by proteolytic enzyme(s) from the rat anterior pituitary. ACTH activity in the incubation medium was increased promptly by acidification of the medium to pH 1.5-2.5 with HC1, and reduced to the initial level by NaOH reneutralization of the medium (pH 6.8-7.8). These phenomena were not observed after the incubation medium had been heated at 100degreesC for 5 min.     

Production of feather hydrolysate by <Emphasis Type="Italic">Elizabethkingia meningoseptica</Emphasis> KB042 (MTCC 8360) in submerged fermentation

A keratinolytic bacterium Elizabethkingia meningoseptica KB042 was isolated from dropped off feathers. The bacterium showed 82.50 ± 0.3% feather degradation when grown on medium containing 10 g/l chicken feathers with initial pH 7.0 at 37°C, 150 rpm in 6 days. The pH of the medium was increased up to 10.02 ± 0.10 during 6 days of incubation. Soluble protein and amino acids concentration in the culture fluid was also found increased until the end of incubation. During the cultivation of strain KB042 on feather as sole source of carbon and nitrogen, the maximum cysteine release was noted on the 3rd day. Varying feather concentration 1.0–2.0% in basal medium resulted in soluble protein release between 1814.42 and 1954.61 μg/ml. The amino acid concentration was found to be maximum, i.e. 937.85 ± 11.9 μg/ml in the cultures grown with 2% feather. The hydrolysate was also found rich in essential amino acids valine, tryptophan, threonine, leucine and cysteine and contains minor amount of methionine and arginine. These data indicate a potential biotechnology for biotransformation and utilization of feather keratin as a source of protein which can be used as animal feed after successful animal trials.     

Damaging effect of digitonin on macrophage plasma membranes exposed to UV-irradiation

It was shown that macrophage irradiation in 4.6 J/cm2 (lambda(max) = 306 nm) dose leads to small quantity of damaged cells in cell population, which doesn't change substantially during 60 min of incubation in darkness. So as detergent digitonin treatment (without irradiation) in 3 mkg/ml concentration doesn't lead to substantial cell damage. Also the result of combined influence of UV-irradiation and digitonin added after irradiation, 15 min before the damaged cells counting, has been got. It was shown that macrophage incubation for 15 minutes leads to cell damaging twice as much sum of UV (4.6 J/cm2) and digitonin (3 mkg/ml) damaging. However the level of cell damaging obtained 30 minutes later after finishing of irradiation doesn't exceed the sum of separate effects of this factors. Further increase of postradiation time leads to synergic effect again.     

Suppression of the damaging effect of amphotericin B on dog kidney lysosomes by amigluracyl

The effect of amphotericin B and its combination with amigluracyl on the dog kidney lyzosomes was studied in vitro. It was found that on incubation of the lyzosomes with the antibiotic in a concentration of 1 gamma/ml the latter stimulated liberation of proteases from them. At the same time, when the lyzosomes were exposed to amphotericin B in combination with amigluracyl, a significant decrease in the rise of the proteolytic activity in the incubation medium due to the antibiotic was observed. It was found that the combined use of amphotericin B with amigluracyl resulted in an intensive inhibition of the enzyme activity; The data are indicative of the fact that amigluracyl decreases the damaging effect of the antibiotic on the dog kidney lyzosomes.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Cytochemical detection of mannose-specific receptors for glycoproteins with horseradish peroxidase as a ligand

Summary Horseradish peroxidase (HRP), a glycoprotein rich in mannose groups, was used as a ligand to detect receptors for glycoproteins in formalinfixed, frozen sections of rat liver. Specific binding of HRP occurred to surface membranes of sinusoidal cells but not to those of parenchymal cells. The binding sites were visualized after the peroxidatic reaction in erythrocytes had been suppressed by methanol-H₂O₂ and phenylhydrazine, the latter reagent also decreasing the nonspecific background adsorption of HRP. Several factors influencing the reaction were studied systematically. The specific binding of HRP to sinusoidal cells was greatly decreased or abolished when tissue blocks were fixed for longer than 1–2 h in a cold 4% formaldehyde solution and the frozen sections subsequently treated for 30 min in cold methanol. The specific binding of HRP increased when the concentration of HRP in the medium was increased from 10 g/ml to 40 g/ml, when the time of incubation with HRP was increased from 1 h to 4 h, or when the temperature of incubation with HRP was increased from 4°C to 22°C, or from 22°C to 37°C. The specific binding of HRP also increased when the pH of the incubation medium was increased from 7.0 to 10.0. Little or no specific binding of HRP was observed in the absence of added Ca⁺⁺. The binding of HRP was suppressed by 10 mM mannose or 0.004% mannan whereas the suppression of the binding reaction by galactose or galactan required 30–40 times higher concentrations.This work was supported by the Morris A. Kaplan Fund     

Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius.

Bacillus acidocaldarius Agnano 101 produces an inducible thermoacidophilic alpha-amylase. The enzyme production occurs during the stationary phase of growth in the presence of compounds with alpha-1,4-glucosidic linkages. The enzymatic activity is both present in the culture medium and associated with the cells; the enzymes purified from both sources show identical molecular and catalytic properties. The purified amylase has a single polypeptide chain of molecular weight 68,000 and behaves like an alpha-amylase with affinity constants for starch and related substances of 0.8 to 0.9 mg/ml. The pH and temperature optima for activity are 3.5 and 75degreesC, respectively. The amylase is stable at acidic pH (below 4.5). Its thermal stability is strictly dependent upon protein concentration; the half-life at 60degreesC of the amylase in a 70-mug/ml solution is about 5 days.     

Effect of Temperature and pH on Growth of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Co-Culture with <Emphasis Type="Italic">Lactococcus garvieae</Emphasis>

Staphylococcus aureus growth and enterotoxin production in co-culture with Lactococcus garvieae were studied in laboratory medium as a function of incubation temperature and pH values. Doehlert experimental design was used to study the effect of L. garvieae concentration, temperature, and pH on S. aureus growth in laboratory medium. The mathematical model obtained was validated in cheeses. The inhibition of S. aureus growth by L. garvieae was more important during the first 6 hours of incubation, and its effect increased when its concentration increased. After 24 and 48 hours, the effect of L. garvieae decreased, and the growth of S. aureus was positively influenced by higher temperature and pH values. Staphylococcal enterotoxins were detected in only one experimental set after 48 hours of incubation at 30°C at pH 6.8. Our results argue in favor of adding antagonist strain early in the cheese-making process.     

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: biological activity

L-HGP is a highly glycosylated protein from Bufo arenarum egg-jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L-HGP was purified from egg water and the estimated percentage of L-HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L-HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris-HCl, pH 7.6, during 60 min, a condition that resembles egg-jelly osmolarity. The addition of purified L-HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L-HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L-HGP binds to B. arenarum spermatozoan membranes.     

Detection, purification and efficacy of warnerin produced by Staphylococcus warneri

Summary Three strains of Staphylococcus warneri (FM10, FM20 and FM30) isolated from meat samples were investigated for their ability to synthesize bacteriocin. All the tested strains produced warnerin, a new peptide bacteriocin; which inhibits the growth of a large number of Gram-positive and Gram-negative bacteria. The inhibitory effect of warnerin produced by the FM20 isolate was high when compared to the other isolates. The results on the effect of carbon sources, nitrogen sources, pH, temperature, incubation time and surfactant (tween 80) inferred that the bacteriocin production was high in medium supplemented with 1% glucose (12,800 AU/ml), 1% urea (6800 AU/ml), and 0.5% Tween 80 (25,600 AU/ml). The higher productivity of bacteriocin was registered during 12 h of incubation in the medium pH 6.5 at 37 °C temperature. Among the various indicator strains tested, Staphylococcus aureus was more sensitive to the bacteriocin activity. Partially purified warnerin exhibited a single band on SDS-PAGE with an apparent molecular weight of 2500 Da. Warnerin, the antibacterial compound was determined as a proteinaceous substance, since it lost its activity when pepsin was added.     

Proton-stimulated opening of stomata in relation to chloride uptake by guard cells

The concentration of potassium chloride required in the incubation medium to open stomata in isolated epidermal tissues of Commelina communis L. and Vicia faba L. could be lowered from 100 mM to 10 mM if the proton concentration of the ambient solution was increased from pH 5.6 to pH 3.5. This acidification effect was formerly attributed to the destruction of epidermal and subsidiary cells resulting in a relief of back pressure upon guard cells. While guard cells remain viable at pH 3.5, as demonstrated by their susceptibility to inhibition by uptake of glucose or to uncoupling by DNP, incipient destruction of the cells surrounding them could first be observed 30 min after the onset of the incubation experiment. By this time, however, the stomata had already opened; the time course of stomatal opening at pH 3.5 did not show any lag phase corresponding to the time required for damaging epidermal cells and showed no difference to that at pH 5.6 Thus, the acid-stimulated opening of stomata appears to be a biphasic phenomenon consisting of a physiologic effect onto which the physical effect of the relief of back pressure is superimposed over longer periods of incubation. To interpret the physiologic role of an increased proton concentration in the ambient solution of isolated epidermal strips, it is suggested that guard cells take up protons and chloride ions in an electroneutral symport. While protons are extruded again to generate the negative membrane potential required for potassium influx, chloride ions are retained to maintain electroneutrality.     

Production of Polyalcohol by a Corynebacterium sp.

A gluconate-utilizing strain of Corynebacterium was found to be capable of utilizing aldopentoses and producing corresponding pentitols when pentoses were added to the medium containing gluconate as a carbon source during the cultivation of the organism.

Pentitols produced from d-xylose, l-arabinose, and d-ribose were isolated from the cultured medium and identified as xylitol, l-arabitol, and ribitol, respectively.

The pentitol production was significantly influenced by the concentration of gluconate in the initial medium and that of pentose added to the medium during the cultivation.

The amount of xylitol, l-arabitol, and ribitol reached 69 mg/ml, 60 mg/ml, and 32 mg/ml, respectively, after 14 days of incubation when pentoses were added to the medium containing 9.6% potassium gluconate to give a final concentration of 150 mg/ml.     

Production of α-galactosidase by a novel actinomycete <Emphasis Type="Italic">Streptomyces griseoloalbus</Emphasis> and its application in soymilk hydrolysis

α-Galactosidase production by a newly isolated actinomycete Streptomyces griseoloalbus under submerged fermentation was investigated. The influence of initial pH of medium, incubation temperature, inoculum age and inoculum size on α-galactosidase formation was studied. Various carbon sources were supplemented in the medium to study their effect on enzyme production. The influence of the concentration of locust bean gum on enzyme production also was optimized. Optimization of process parameters resulted in a highest α-galactosidase activity of 20.4 U/ml. The highest α-galactosidase activity was obtained when the fermentation medium with initial pH 6.0 and containing 1% locust bean gum as growth substrate was inoculated with 10% (v/v) of 72 h grown inoculum and incubated at 30°C. The hydrolysis of flatulence-causing oligosaccharides in soymilk by the enzyme was also investigated. Thin layer chromatographic analysis of enzyme-treated soymilk samples showed the complete hydrolysis of soy oligosaccharides liberating galactose, the final product.     

The inhibitory effect of hydrocortisone on the chicken embryo cartilage somatomedin assay.

In a study of the effects of hydrocortisone on the embryonic chicken cartilage somatomedin assay, in the absence and in the presence of normal human reference serum (NHRS), it was found that: (1) The basal uptake of 35S into chicken embryo pelvic cartilage was reduced when hydrocortisone hemisuccinate was added to the incubation medium in concentrations ranging from 1.5 to 1.5 X 10(5) ng/ml. There was a correlation between the inhibitory effect and the quantity of hydrocortisone added (r=-0.869; p less than 0.01). (2) The 35S uptake stimulated by 1.25 and 5% serum present in the incubation medium was reduced by hydrocortisone in a final concentration range of 150-1.5 X 10(5) ng/ml incubation medium. The minimal dose was 1,000 times that required to affect the basal 35S uptake. (3) When hydrocortisone was directly added to the NHRS, its interfering effect on the 35S uptake stimulated by 1.25, 5 and 20% of serum in the incubation medium was demonstrable with 5 X 10(5) ng hydrocortisone/ml serum. This concentration exceeded the physiological level of hydrocortisone by a factor of 5,000.     

灰黄霉素产生菌耐前体变株F-1012的选育及其发酵特性

以灰黄霉素产生菌D-756为出发菌株,经过三代的紫外线+氯化锂的复合诱变处理,采用快速筛选方法,获得了耐前体变株F-1012。对该变株的耐氯特性和发酵特性进行研究,结果表明,把发酵培养基中的氯化物浓度提高到2.0%,大米粉量提高到18%,该变株发酵单位最高。发酵最适条件,起始pH自然(约5.7);移种量为15%;装量20 ml/250 ml三角瓶;发酵周期为288小时。     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.