Formation of organic acids by the fungus Cladosporium resinae in media containing n-alkanes]

The so-called fungus Cladosporium resinae that often occurs in oil fuels ane increases their acidity grows well at the expense of n-alkanes from C11 to C16. On the n-alkane containing media the fungus grows slowly and only under the stationary conditions. During the fungal cultivation on the media containing n-dodecane or kerosene the culture liquid shows acetic acid and other fatty acids, ketoacids (pyruvic and alpha-ketoglutaric) as well as citric and isocitric acids that dominate among nonvolatile acids. Upon nitrogen deficiency in the medium and comparatively good aeration the content of citric acids increases. The culture liquid of the fungus devoid from the mycelium and nonutilized n-alkanes can be used a a nutrient medium for different microorganisms.     

添加氧载体及表面活性剂对番茄红素发酵的影响

通过添加氧载体（正十二烷、正己烷、过氧化氰）,有效改善发酵体系中的氧传递速率,从而促进了三孢布拉氏霉菌合成番茄红素的能力。实验结果表明,在第0d加入1．0％的正已烷、正十二烷时番茄红素的生成量分别提高了25．32％、72．84％,在第1d,添加50μL/100mL过氧化氢时番茄红素的生成量提高了40．35％,在加入正十二烷的同时,再加入表面活性剂Trilorl—x100,Tween20,Tween80,Span-20等,可使番茄红素的产量最多提高114．83％．     

The effects of N-dodecane pretreatment on the metabolism and distribution of benzo(A) pyrene in the isolated perfused rabbit lung

B(a)P is an ubiquitous potent carcinogen that has been associated with the increased incidence of human bronchiogenic carcinoma in occupational and urban populations. An isolated perfused rabbit lung preparation (IPL) was used to study the influence of pretreatment with a cocarcinogen, n-dodecane, on the metabolism of benzo (a)-pyrene (B(a)P). ¹⁴C-B(a)P was administered intratracheally to the IPL following biweekly inhalation exposure of the rabbits to n-dodecane. Results from n-dodecane pretreatment group were compared to those from the no-pretreatment group and an intratracheally administered B(a)P pretreatment group (B(a)P IT). Metabolites were isolated from serial blood samples up to 3 hours after administration of the ¹⁴C-B(a)P to the IPL. Patterns of B(a)P metabolites were determined using thin layer and high performance liquid chromatography, and liquid scintillation counting. The rates of appearance of B(a)P metabolites in the blood and the B(a)P metabolic pattern for n-dodecane groups were compared to B(a)P IT and no-pretreatment groups. The rates of appearance of metabolites in the blood (ng/hr/g of lung) were similar for B(a)P IT and n-dodecane pretreatment groups, but were five and four times greater, respectively, when compared to rates of appearance of metabolites for the no-pretreatment group. The B(a)P metabolic pattern of n-dodecane pretreatment showed an increase in the dihydrodiol and nonextractable formation and a decrease in the monohydroxy and diones compared to the no-pretreatment group. The metabolite pattern for B(a)P IT pretreatment group was similar to the metabolite pattern for the n-dodecane pretreatment group. The significance of the findings is that a known cocarcinogen, n-dodecane, appears to be as good an enzyme inducer as B(a)P in stimulating metabolism of B(a)P in lung. Therefore, man's risk in developing cancer may not be solely a result of exposure to a carcinogen, but may be dependent upon other constituents in his environment.     

Development of 1-cell embryos from different strains of mice in CZB medium

One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB medium. CZB medium can be used to culture CF1 x B6SJLF1/J 1-cell embryos to the blastocyst stage provided glucose is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range of 5.5 to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65-85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CF1 and DBA/2J (both x B6SJLF1/J) mice, which exhibit a "2-cell block" to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2F1/J and CD1 female mice (both x B6SJLF1/J males), which do not exhibit a "2-cell block" to in vitro development, optimum development to morula and blastocyst stages (95% and 50%) was in CZB medium containing both glutamine and glucose from the start of culture.     

The role of the cytoskeletal elements during the return to the cell center of their nuclei displaced by centrifugation]

Living cells of the monolayer cultures of embryonal pig kidney epithelium (PKE-cells) and of embryonal bovine tracheal cells (FBT-cells) were ultracentrifuged at 20,000g. The centrifugal force was directed parallel to the surface of the culture slides. Just after centrifugation, the cellular nuclei were displaced to the centrifugal parts of cells. Centrifuged slides with cells were returned to the normal culture conditions, and 22 h later the nuclei were seen to restore their central position in the cells. The motion of the nuclei to the cell center was rather chaotic both in direction and speed. The speed of this motion never exceeded several microns per hour. After nocodasole treatment (0.1-10 mkg/ml) of the cells or in a hypotonic medium, the distance of nuclear dislocation during centrifugation was longer, and the nuclei returned to the cell centers faster than in the control ones. After cytochalasin B treatment (2 mkg/ml), the nuclei moved to the cell centers somewhat more slowly than they did in the control cells. Thus, the establishment of the central position of nuclei in the cells takes place in the absence of microtubules or intermediate filaments. Probably, the central position of nuclei depends mainly on the action part of the cytoskeleton.     

Manganese peroxidase production by immobilized Phanerochaete chrysosporium BKM-F-1767 in a cell bioreactor

Manganese-dependent peroxidase (MnP) production was performed in an immobilized cell bioreactor in which Phanerochaete chrysosporium BKM-F-1767 was immobilized on polystyrene foam. The immobilized cell culture yielded significantly greater MnP activity than the conventional stationary liquid culture. Cultivation was carried out in batch mode; the effect of glucose concentration was investigated and growth kinetics parameters were found as, micromax=0.59 day(-1), Ks=0.33 g/L and Kss=14.5. Batch operation led to maximum MnP (770.82 U/L) in the culture medium containing 0.05% Tween 80, 10 g/L glucose, and 174 microM Mn2+ at 37 degrees C and pH 4.5. Enzyme productivity was obtained as 110.12 U/day/L.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose     

Biosynthesis of corrinoids and other tetrapyrrole compounds by an acetogenic Clostridium

Biosynthesis of corrinoids and other tetrapyrrole pigments by the pure culture of the acetogenic Clostridium 99 was studied. When growing on media containing glucose or methanol, the physiological and biochemical characteristics of Clostridium 99 are very close to those of C. thermoautotrophicum. Methanol was shown to stimulate the corrinoid accumulation with the yield increasing from 154 micrograms/g dry biomass (glucose medium) up to 2250 micrograms/g dry biomass (methanol medium). According to the paper chromatography the corrinoid accumulated in Clostridium 99 cells differed both from vitamin B12 and Factor III. A study on the composition of extracellular tetrapyrroles, accumulated when the culture grows on the medium containing glucose and delta-aminolevulinic acid, revealed that they are represented both by uroporphyrin III and sirohydrochlorine-like pigments. The latters differ by a number of properties from sirohydrochlorine (corrifirine-2) of propione acidic bacteria. These pigments appear to be involved as intermediants in biosynthesis of corrinoids and other tetrapyrroles.     

Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.     

Avermectins: biotechnological features of the producing strain of Streptomyces avermitilis VKM As 1301]

The monosporic plating of the avermectin-producing strain Streptomyces avermitilis VKM Ac-1301 with low activity showed the heterogeneity of the population. By selection of natural mutants the authors obtained a strain synthesizing up to 60 micrograms avermectin B1 per ml of culture liquid. The maximum avermectin yield was observed in the medium containing 7% glucose after 100-120 h of culture growth.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Production of glucose 6-phosphate dehydrogenase from genetically modified Saccharomyces cerevisiae grown by batch fermentation process

In a 5-L fermentor (NBS-MF 105), Saccharomyces cerevisiae W303-181 (1.0 g dry matter/L) was inoculated into 3.0 L of liquid medium containing glucose (10 or 20 g/L), yeast nitrogen base (YNB, 3.7 or 7.4 g/L), l-histidine (0.02 g/L), l-tryptophan (0.02 g/L), uracil (0.02 g/L), and adenine (0.02 g/L). The culture was carried out batchwise for 12 or 24 h at 30 degrees C, pH 4.6 or 5.7, aeration of 0, 0.8, 1.7 or 2.2 vvm, and agitation of 400 rpm. The highest G6PDH productivity (10.5 U/L.h) and specific activity (320 U/mg of protein) occurred at aeration of 2.2 vvm, pH 5.7, 10 g/L of glucose, and 3.7 g/L of YNB. The G6PDH specific activity attained was comparable with those of commercial preparations, which are between 50 and 600 U/mg of protein.     

Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture

Among about 200 Rhizopus strains isolated in Thailand, Rhizopus sp. MB46 was selected as a producer of raw cassava starch-digestive glucoamylase. Rice bran was effective for the enzyme production in a solid culture as well as wheat bran. Addition of turpentine oil into the rice bran solid culture increased the productivity. Rhizopus sp. MB46 was found to produce glucoamylase in a liquid culture containing 1% rice bran but not in one consisting of 10% raw cassava starch of 2% glucose. The productivity per 1 g solids in the medium in liquid culture was finally improved 6-times by utilization of n-hexane-treated rice bran, supplement of 0.1% meat extract and addition of gauze as a support. The activity was superior to that in turpentine oil-supplemented solid culture.     

Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

Decolorization of Remazol Brilliant Blue (RBBR) and Poly R-478 dyes by <Emphasis Type="Italic">Bjerkandera adusta</Emphasis> CCBAS 930

An anamorphic Bjerkandera adusta CCBAS 930 strain isolated from soil was found to decolorize two anthraquinonic dyes: Remazol Brilliant Blue R and Poly R-478. The reduction in the level of phenolic compounds in liquid B. adusta cultures containing RBBR and Poly R-478 was correlated with decolorization of studied dyes, which suggested their biodegradation. It was shown that this process was coupled with induction of secondary metabolism (idiophase) and peak peroxidase activity in culture medium, and the appearance of aerial mycelium. Decolorization of dyes depended on the presence of glucose (cometabolism).     

Biosynthesis of vitamins B by the fungus Pleurotus ostreatus in a submerged culture

The intra- and extracellular contents of vitamins were studied in the course of submerged cultivation of the higher basidial mushroom Pleurotus ostreatus (Jacq.: Fr.) Kummer st. IMBF-1300 on liquid nutrient media. This strain was found to be autotrophic in respect of thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B5), pyridoxine (vitamin B6) and biotin (vitamin B7), but it failed to synthesize cyanocobalamin (vitamin B12). The composition and pH of the culture medium, containing such complex biostimulating supplements as maize extract and concentrated potato sap noticeably influence the contents of vitamins B1, B5 and B7 in the mycelium, and to a less degree they change the level of the intracellular biosynthesis of vitamins B2 and B6. Higher excretion of vitamins B5, B7 and especially B6 was observed on the semisynthetic media during the postexponential growth. Under experimental conditions vitamins B1 and B2 were accumulated only in the cells. The dry mycelium of P. ostreatus obtained by submerged cultivation on liquid media is a valuable source of B vitamins and, especially, of niacin. Thus the oyster mushroom and other edible mushrooms can be put at one of the top places among food-stuffs by the content of niacin.     

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.