Sesquiterpene hydrocarbons from Lentinus lepideus

Several sesquiterpene hydrocarbons, mainly of the cadinane skeleton, have been identified in cultures of the brown rot fungus Lentinus lepideus. The main compounds are δ-cadinene, α- and γ-muurolene.     

Phenolic O-methyltransferase from Lentinus lepideus (basidiomycete)

The fungus, Lentinus lepideus, produces crystalline methyl p-methoxycinnamate in stationary cultures. O-methylation and methyl ester formation of hydroxycinnamic acids were examined with enzyme preparations of the fungus. Using S-adenosylmethionine-¹⁴CH3, it was found that only the methyl esters of the hydroxycinnamic acids are substrates for O-methylation and not the free acids. Benzoic acids and their methyl esters are not substrates. The activity of the enzyme is p-specific and its specific activity decreases with increasing number of hydroxyl groups in the substrate. The same enzyme preparations catalyze the formation of the methyl ester of cinnamic acid from the free acid.     

Immunomodulating activities of water-soluble exopolysaccharides obtained from submerged culture of Lentinus lepideus

Immunomodulating activities of water-soluble exopolysaccharides (LL-EX) obtained from submerged mycelial culture of Lentinus lepideus were studied and their effectiveness was compared with lipopolysaccharide (LPS). The influence of the LL-EX on macrophage cellular lysosomal enzyme activity was to stimulate up to 267%, 392%, and 464% at the level of 10, 50, and 100 mug/ml, respectively. When the LL-EX was further fractionated into LL-Fr.I and Fr.II by Sepharose CL-6B gel chromatography, the cellular lysosomal enzyme activity of LL-Fr.II (2.1- fold) was higher than Fr.I (1.2-fold). Moreover, both LL-Fr.I and Fr.II stimulated the cytokines IL-1beta, TNF-alpha, and IL-6 in macrophages. In mixed lymphocyte reaction, LL-Fr.I and Fr.II enhanced the splenocyte proliferation up to 1.2-fold and 1.4-fold (50 mg/ml), respectively, stimulating only T lymphocytes. The fractions of LL-EX not show any direct toxicity against human gastric adenocarcinoma cell (AGS). The molecular masses of LL-Fr.I and Fr.II were estimated to be about 1,986 kDa and 21 kDa, respectively. The total sugar and protein contents of the two fractions were 84.97% and 69.88% and 15.03% and 30.12%, respectively. The sugar and amino acid compositions of the LL-Fr.I and Fr.II were also analyzed in detail.     

洁丽香菇胞外粗多糖对小鼠移植性S180肉瘤的抑制作用和免疫功能调节

对洁丽香菇胞外粗多糖中多糖和蛋白含量进行测定,其中多糖含量为21.87%,蛋白含量为7.14%。通过体内实验研究该多糖对S180肉瘤的生长抑制作用。结果表明洁丽香菇胞外粗多糖高、中、低剂量组均能不同程度抑制S180 肉瘤的生长,其中高剂量组（500mg/kg）抑瘤率最高（为39.44%）,同时能显著增加胸腺指数,降低脾指数,有效改善免疫器官受损现象;中、低剂量（250mg/kg,125mg/kg）亦能抑制肿瘤生长,抑瘤率分别为30.43%和23.40%。实验中采用ELISA法对血清中各种细胞因子含量进行测定,结果表明洁丽香菇胞外粗多糖高剂量组可明显诱导血清中TNF-α、IL-12的分泌,低剂量组可诱导IFN-γ、IL-10的分泌。由此认为洁丽香菇胞外粗多糖通过恢复免疫器官功能,增加血清中TNF-α、IL-12、IFN-γ和IL-10的含量,从而达到增强细胞免疫的作用,发挥抗肿瘤活性。     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Removal of 2,4,6-trinitrotoluence and 2,4-dinitrotoluene by fungi (Ceratocystis coerulescens, Lentinus lepideus and Trichoderma harxianum)

At an initial concentration of 50 mg/l in liquid culture, 2,4,6-trinitrotoluene (TNT) was totally transformed by the three following fungi: Ceratocystis coerulescens, Lentinus lepideus and Trichoderma harzianum. 2,4-Dinitrotoluene (DNT) was also degraded by C. coerulescens and T. harzianum but not by L. lepideus. Composition of the culture medium in terms of carbon and nitrogen affected the transformation of TNT and DNT into amino-intermediates. These metabolites accounted for less than one quarter of the initial concentration of aromatics. In some instances, these transient intermediates disappeared at the end of the incubation period. © Rapid Science Ltd. 1998     

Plant regeneration and protoplast culture of Browollia speciosa

Explants from hypcotyls and cotyledons of Browalia speciosa were shown to regenerate plantlets.Protoplasts were isolated from etiolated cotyledon material, and, although callus was readily obtained, plantlet regeneration was not observed using numerous hormone regimes.Abbreviations M Mannitol - 2,4-D Dichlorophenoxyacetic acid - NAA Naphthalene-acetic acid - BAP Benzylaminopurine - MS medium Murashige and Skoog (1962) medium - UM medium Uchimiya and Murashige (1974) medium - COT cotyledon - SH shoot - R root     

Ethylene and ethane release during tobacco protoplast isolation and protoplast survival potential in vitro

Studies on stress ethylene and ethane during protoplast isolation from water-stressed and waterlogged donor plants Nicotiana tabacum L. xanthi-nc, show a correlation between ethane, but not ethylene, release and protoplast survival in vitro. Ethane release shows a high negative correlation with protoplast survival potential from donor plants subjected to both stresses. Ethylene showed a high negative correlation with protoplast survival potential in tissues from water-stressed but not from long-term waterlogged plants. The absence of correlation in the latter may be related to decreased ability to produce ethylene in hyperstressed plants.
The results are discussed in relation to the use of stress ethane release as a parameter of the physiological status of the plant.     

Factors influencing the production of water-soluble endopolysaccharides and exopolysaccharides from Lentinus lepideus and their effects on immune cytokine production

An efficient method to produce water-soluble polysaccharides from Lentinus lepideus is described. The productivity of both endopolysaccharides (PPS) and exopolysaccharides (EPS) was compared under various culture conditions. The effect of treating their own PPS and EPS on immune cytokine production was also studied in relation to culture factors. High yield production of EPS required moderate culture temperature (25 degrees ) as well as long culture period (16-20 days). In contrast, PPS production required high culture temperature (30 degrees ) and short culture period ( days). Most of the carbon sources did not affect polysaccharides and mycelial production except for sucrose. Immune cytokine levels in the EPS treatment varied among carbon sources or culture periods. PPS did not appear to affect much on the production of cytokines, regardless of the culturing factors, except for the culture period. These results suggest that the optimal culture conditions for L. lepideus vary according to culture purposes, and different culture conditions should be used for different targets including mycelial biomass, EPS, and PPS. Whereas the immunomodulating activity of EPS appeared to be affected by culture conditions in L. lepideus, that of PPS did not.     

Beauveria bassiana protoplast regeneration and transformation using electroporation

Summary Regeneration of Beauveria bassiana GK2016 protoplasts demonstrated three phases: (1) enlargement, (2) formation of a chain of budding cells, and (3) development of a true germ tube from the original protoplast. Regeneration frequencies of up to 80% were obtained when using the stabilizer ammonium sulphate. Using electroporation, protoplasts were transformed to methyl 1,2-benzimidazole carbamate (MBC) resistance with the Neurospora crassa MBC-resistant -tubulin gene. A transformation efficiency of one of three transformants per 5 g vector DNA was obtained. The MBC phenotype was stable and transformants grew in the presence of 5 g MBC/ml. Southern DNA-DNA hybridization analysis demonstrated that integration of the vector into the chromosomal DNA had occurred. Correspondence to: G. G. Khachatourians     

Changes in lipid composition during protoplast isolation

During the isolation of mesophyll protoplast from leaves of Arabidopsis thaliana (L.) Heynh. the neutral lipids increased from 8% to 17% of the total lipids, while each of the polar lipids decreased by 10–20%. This treatment also induced acyl group migration which was indicated by the transfer of 16:3 from monogalactosyldiacyglycerol to other lipid. Similar results were obtained when leaf slices or vacuum infiltrated leaves were incubated in an aqueous solution containing 0.5 M sorbitol, indicating that the effect is not specifically related to cell wall removal. These observations suggest that the possible deleterious consequences of such lipid changes on the properties of membranes should be considered whenever protoplasts or protoplast-derived organelles are used for physiological studies.     

Plant protoplast isolation - a practical approach

A method for the isolation of plant protoplasts is presented that uses inexpensive sources of enzymes. It is suitable for student use in Biotechnology.     

Properties ofSaccharopolyspora erythraea strains after protoplast regeneration

Protoplast formation, stabilization and regeneration was improved for 4 strains (erythromycin producers) of Saccharopolyspora erythraea. A modified medium was developed for protoplast regeneration. Parental and protoplast-regenerated strains exhibited changes in morphology, ultrastructure, and antibiotic production.     

Efficient protoplast regeneration ofBacillus thuringiensis andAgrobacterium tumefaciens

Summary Treatment ofBacillus thuringiensis andAgrobacterium tumefaciens taken from the early growth phase (8 h) with lysozyme at 1 mg/ml gave 90–99% protoplast formation and 10–12% protoplast regeneration on the minimal medium in absence of plasma expander (Bovine serum albumin). Enhanced fusion frequency was obtained when protoplasts from 8 h grown cells were used for fusion experiments.     

植物叶片原生质体分离的可能机制

分析了植物叶片在分离液环境中形成原生质体的过程,文中提出,分离液配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁进入分离液,继而又进一步发生质膜的酸性降解,使细胞核和细胞器进入分离液中,最终分离液中的细胞器以细胞核为中心进行细胞器重组,最后产生外貌形态一致的新的原生质体。植物细胞壁和质膜是植物细胞的包被系统。植物细胞包被系统的酸性降解使植物细胞器重组并产生新的原生质体成为可能。