Morphological Observations and Fatty Acid Composition of Indoor-Cultivated Cordyceps sinensis at a High-Altitude Laboratory on Sejila Mountain,Tibet

Cordyceps sinensis, a caterpillar entomopathogenic fungus-host larva complex, is a rare medicinal herb found in the Qinghai-Tibetan Plateau and its surrounding high-altitude areas. The alternation of generations in the life cycle, whatever the fungus or its host insect, requires special growth conditions. However, it is difficult to simulate the growth conditions of C. sinensis, which hinders its artificial cultivation. In this work, the life cycle from the host larva to C. sinensis was observed in an indoor-cultivation laboratory at 4,200 m a.s.l. on Sejila Mountain, Tibet. Comparative examinations between indoor-cultivated and wild C. sinensis demonstrated that the indoor-cultivated C. sinensis preferred to germinate multiple long, slim stromata at diverse positions on dead larvae, including but not limited to their heads. Their fatty acid composition shows a significant difference in the levels of polyunsaturated fatty acids (PUFAs). In indoor-cultivated C. sinensis, PUFAs constituted 24.59% and 49.43%, respectively, of neutral and polar lipids; meanwhile, in wild C. sinensis, PUFAs represented 34.34% and 61.25% of neutral and polar lipids, respectively. These observations and fatty acid data suggest that environmental factors, particularly temperature, soil pressure and light intensity, strongly affect the growth of C. sinensis. Our new findings may provide important information for improving techniques for the large-scale artificial cultivation of C. sinensis.     

Neutral lipid production in <Emphasis Type="Italic">Dunaliella salina</Emphasis> during osmotic stress and adaptation

The salt-tolerant green microalga Dunaliella salina can survive both hyper- and hypo-osmotic shock. Upon osmotic shock, the cells transiently and rapidly decreased or increased in size within minutes and slowly over hours acquired their original cell size and volume. Cell size distribution differs significantly in the cultures grown in the salinity range from 1.5 to 15 % NaCl. By using Nile Red fluorescence to detect neutral lipids, it became clear that only hyper-osmotic shock on cells induced transient neutral lipid appearance in D. salina, while those transferred from 9 to 15 % NaCl stimulated the most neutral lipid accumulation. These cells grew well in 9 % NaCl, but they cannot recover a shift to 15 % NaCl and cell division is accordingly slowed down. The transient appearance of neutral lipid could be dependent on the inhibition of cell division experiencing the NaCl shift. Moreover, the effect of nutrient limitation slows down cell division and photosynthesis as a secondary result, which triggers the cells to accumulate neutral storage lipids when they entered the stationary phase, which is seen in all the batch cultures of D. salina grown in the salinity range of 3–15 %. The changes in salt concentration did not significantly influence the overall fatty acid composition in D. salina cells. Although there shows both increased amounts of total lipids and neutral lipids in the cells grown in salinity higher than 9 % NaCl, lipid productivity is however compromised by the slower cell growth rate and lower cell density under this condition.     

Amoeba-bacteria relationship: Factors in the bacterial lipids supporting the growth of axenicEntamoeba histolytica

Live bacteria in modifiedDiamond’s axenic medium did not support growth ofEntamoeba histolytica. Cysteine hydrochloride, required for the multiplication of amoeba, was broken down by live bacteria and toxic substances were produced which were lethal for amoebae. Monoxenic and xenic cultures ofaxenically grownE. histolytica could be established in Boeck and Drbohlav medium with bacteria and rice starch. Bacterial lipids prepared from 15 human intestinal bacteria supported growth and multiplication ofE. histolytica in axenic medium. In a pilot experiment using lipids ofStreptococcus faecalis, free fatty acids did stimulate the multiplication of amoebae. When total lipids of this bacteria were fractionated into neutral lipids and phospholipids by chromatography and used, neither fraction was found to stimulate growth. Free fatty acids prepared by chemical hydrolysis of the total lipids, neutral lipids and phospholipids stimulated growth ofE. histolytica, The sterols present in the bacterial lipids (neutral lipids or non-saponifiable fractions) stimulated growth of amoebae. It was found thatE. histolytica is incapable of liberating fatty acids from di- or triglyceridesof phospholipids and the multiplication of the organism is stimulated by the presence of free fatty acids and sterols (cholesterol).     

Lipid synthesis in germinating Tradescantia pollen

Synthesis of neutral and polar lipids in pollen of Tradescantia paludosa during germination and tube growth was studied by the incorporation of acetate-[1-¹⁴C] into lipids in the presence and absence of inhibitors of RNA and protein synthesis. The proteins required for the synthesis of both neutral lipids and phospholipids are not made de novo during germination but are already present in the mature ungerminated pollen grain and they are functionally stable during the first 2 hr of pollen growth.     

Secretion of human growth hormone in Bacillus subtilis using prepropeptide coding region of Bacillus amyloliquefaciens neutral protease gene

Using a secretion vector plasmid pES150, which contained a region coding for promoter and prepropeptide of Bacillus amyloliquefaciens neutral protease gene (Honjo et al. (1985) J. Biotechnol. 2, 73–84), a hybrid plasmid pES150GH was constructed. The plasmid pES150GH possesses a mature human growth hormone gene following the prepropeptide coding region. Bacillus subtilis N325 harboring pES150GH produced growth hormone in the medium. The secreted hormone was indistinguishable immunologically from human pituitary growth hormone on double immunodiffusion test. The level of secretory production of growth hormone by B. subtilis reached 40–50 mg l⁻¹ of the culture. The size of the hormone secreted by B. subtilis was approximately 23 K on SDS-PAGE. This value was rather large in comparison with that of natural growth hormone (22 K). It was observed that 99% of synthesized growth hormone was excreted into the medium and that the mode of secretion was co-translational. The secreted growth hormone (23 K) is suggested to be degraded to a 15 K form in the culture medium by proteolysis.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Negative allelopathic effects of rutin and quercetin on fourteen soil and enteric microbes

The flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial activity againts 14 soil and enteric microbes. Microbial inhibition was observed when rutin and quercetin (pH 5.7 and 5.8 respectively) solutions were not adjusted to pH 7.0. At neutral pH no antimicrobial activity was observed. The addition of flavonoids at neutral pH did not effect the bacterial growth curve of Agrobacterium tumefaciens or the exponential growth of Escherichia coli. Both bacteria grew to turbidity from a loop-inoculum in medium containing rutin or quercetin as the sole carbon source. Furthermore, medium containing rutin and/or quercetin did not decrease either the number or relative frequency of several microbes from open field and forest floor soil samples. It is concluded that microbial inhibition has not played a major evolutionary role in the production of flavonoids in plants.     

Emulsifying properties of a glycoprotein extract produced by a marine Flexibacter species strain TG382

We report, for the first time, on the production of an emulsifying polymer produced by a Flexibacter species (designated strain TG382). This polymer, E-382, was produced extracellularly during growth of the organism in a marine broth amended with glucose. After cold ethanol precipitation, extensive dialysis and lyophilization, a chemical analysis of the resultant dried polymer revealed it to be a glycoprotein composed of 10.9% protein, 23.3% carbohydrate and a 5.5% uronic acid content. At relatively low concentrations (0.02%, w/v), E-382 was found to form oil-in-water emulsions against hydrocarbon and food oils under neutral pH and acidic conditions. The most stable emulsions were formed against the oils sunflower, vegetable and ground nut under neutral pH conditions. Aqueous solutions of the polymer were viscous, and its reduced viscosity (η_red) was determined to be 0.54 m³/kg. Although proteins and uronic acids may possess surface-active properties, the viscosifying effect of this polymer, which is a typical feature of some commercial hydrocolloids, is more likely to confer its high emulsion-stabilizing qualities.     

Functional Effects of Pgi Allozymes in ESCHERICHIA COLI

Five alleles representing three electromorphs of phosphoglucose isomerase (PGI) have been transferred from natural isolates of E. coli into the genetic background of E. coli K12 and examined for their effect on growth rate in chemostats limited for glucose or fructose. With glucose limitation, all alleles are selectively neutral or nearly neutral within the limit of resolution of the technique, whether the genetic background is nonmutant or whether it contains a deletion of the locus of glucose-6-phosphate dehydrogenase, the enzyme that provides an alternative metabolic pathway for the substrate of PGI. With fructose limitation, one of the naturally occurring alleles has a small but reproducible detrimental effect on growth rate. A kinetic difference in this detrimental allozyme, apparently relating to an inhibition constant, has been observed in some, but not all, lots of substrate, and a similar difference has also been noted in one of the rare electromorphs that could not be transferred into E. coli K12. These results support a model of genetic variation in which the alleles are neutral or nearly neutral in the prevailing environment but have a potential for selection that can be expressed under the appropriate conditions of environment or genetic background. This hypothesis is discussed in the context of allozyme polymorphisms observed in other organisms.     

Presence of two neutral disaccharides containing N-acetylhexosamine in bovine colostrum as free forms

Two neutral disaccharides which comprise 74.0% of the neutral oligosaccharides other than lactose were isolated from bovine colostrum taken 6 h after parturition. The chemical structures were revealed to be galactosyl-β-1,4-N-acetylglucosamine (N-acetyllactosamine, 70.3%) and N-acetylgalactosaminyl-β-1,4-glucose (3.7%). The two carbohydrates were the newly found oligosaccharides from mammalian milk in the free forms. 7 days after parturition, they had completely disappeared from bovine milk.     

The utilization of sterols and other lipids by insect cells grown in vitro

Cells derived from Antheraea eucalypti were grown in vitro in a medium containing triglycerides, diglycerides, free fatty acids, and sterols as the main ‘neutral’ lipids. The sterol content of the medium was derived chiefly from the haemolymph component. The ‘neutral’ lipids in the cells were triglycerides, free fatty acids, and sterols. During growth over 6 days there was a quantitative balance between cholesterol and β-sitosterol gained by the cells and those sterols removed from the medium when allowance was made for losses from sterile medium. Cells metabolized more triglycerides and free fatty acids than they incorporated.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

The regulation of Streptococcus mutans glucan-binding protein A expression

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.     

Growth and neutral lipid synthesis in green microalgae: A mathematical model

Many green microalgae significantly increased their cellular neutral lipid content when cultured in nitrogen limited or high light conditions. Due to their lipid production potential, these algae have been suggested as promising feedstocks for biofuel production. However, no models for algal lipid synthesis with respect to nutrient and light have been developed to predict lipid production and to help improve the production process. A mathematical model is derived describing the growth dynamics and neutral lipid production of green microalgae grown in batch cultures. The model assumed that as the nitrogen was depleted, photosynthesis became uncoupled from growth, resulting in the synthesis and accumulation of neutral lipids. Simulation results were compared with experimental data for the green microalgae Pseudochlorococcum sp. For growth media with low nitrogen concentration, the model agreed closely with the data; however, with high nitrogen concentration the model overestimated the biomass. It is likely that additional limiting factors besides nitrogen could be responsible for this discrepancy.     

Isolation of portulaca oleracea (regla) mucilage and identification of its structure

Portulaca oleracea leaves were found to contain 0.42% of a mucilage mixture. The mucilage was fractionated into an acidic and a neutral fraction. The acidic fraction consists of galacturonic acid residues joined by α-(1→4)-linkages; 60% of these residues are present as the calcium salt, and esterified galacturonic acid residues are absent. The neutral fraction is composed of 41% of arabinose and 43% of galactose residues, besides traces of rhamnose residues.     

Regulation of carbon metabolic fluxes in response to CO<Subscript>2</Subscript> supplementation in phototrophic <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>: a cytomic and biochemical study

As one of the promising species of microalgae for biofuel production, Chlorella vulgaris CS-42 was cultivated phototrophically in two cylindrical photobioreactors with aeration of 5 % (v/v) CO₂ or air for 13 days to evaluate the effects of CO₂ supplementation on biomass, CO₂ fixation performance, and biochemical content. Significant increases of specific growth rate and total carbon content in biomass resulting in a higher CO₂ fixation rate were found with 5 % CO₂. The maximum biomass concentration, carbohydrate and fatty acid contents with 5 % CO₂ were significantly higher than those with air, while carbohydrate biosynthesis was most affected as compared to other biochemical components. Cytomic analysis revealed a rapid accumulation of neutral lipid in the late growth phase with more lipid bodies visualized by confocal laser scanning microscopy (CLSM), when nitrate consumption was accelerated with CO₂ supplementation. Gas chromatography mass spectrometry (GC-MS) analysis indicated that 5 % CO₂ favored the formation of C18:2, which led to a decrease in the degree of lipid unsaturation (DLU). These results proved that CO₂ supplementation was one of the most efficient methods to significantly prompt the growth of microalgae and increase the C/N ratio in the medium, which in turn regulated the carbon metabolic flux to enhance neutral lipid and fatty acid production in C. vulgaris.     

Influence of forearm orientation on biceps brachii tendon mechanics and elbow flexor force steadiness

Achilles tendon mechanics influence plantar flexion force steadiness (FS) and balance. In the upper limb, elbow flexor FS is greater in supinated and neutral forearm orientations compared to pronated, with contributions of tendon mechanics remaining unknown in position-dependent FS. This study investigated whether distal biceps brachii (BB) tendon mechanics across supinated, neutral and pronated forearm orientations influence position-dependent FS of the elbow flexors. Eleven males (23 ± 3 years) performed submaximal isometric elbow flexion tasks at low (5, 10% maximal voluntary contraction (MVC)) and high (25, 50, 75% MVC) force levels in supinated, neutral and pronated forearm orientations. Distal BB tendon elongation and CSA were recorded on ultrasound to calculate mechanics of tendon stress, strain and stiffness. Relationships between FS, calculated as coefficient of variation (CV) of force, and tendon mechanics were evaluated with multiple regressions. Supinated and neutral were ∼50% stronger and ∼60% steadier than pronated (p < 0.05). Tendon stress was ∼52% greater in supinated and neutral compared to pronated, tendon strain was ∼36% greater in neutral than pronated (p < 0.05), while tendon stiffness (267.4 ± 78.9 N/mm) did not differ across orientations (p > 0.05). At low forces, CV of force was predicted by MVC (r²: 0.52) in supinated, and MVC and stress in neutral and pronated (r²: 0.65–0.81). At high force levels, CV of force was predicted by MVC and stress in supinated (r²: 0.49), and MVC in neutral (r²: 0.53). Absolute strength and tendon mechanics influence the ability of the BB tendon to distribute forces, and thus are key factors in position-dependent FS.     

Phenotypic divergence of the common toad (Bufo bufo) along an altitudinal gradient: evidence for local adaptation

Variation in the environment can induce different patterns of genetic and phenotypic differentiation among populations. Both neutral processes and selection can influence phenotypic differentiation. Altitudinal phenotypic variation is of particular interest in disentangling the interplay between neutral processes and selection in the dynamics of local adaptation processes but remains little explored. We conducted a common garden experiment to study the phenotypic divergence in larval life-history traits among nine populations of the common toad (Bufo bufo) along an altitudinal gradient in France. We further used correlation among population pairwise estimates of quantitative trait (Q_ST) and neutral genetic divergence (F_ST from neutral microsatellite markers), as well as altitudinal difference, to estimate the relative role of divergent selection and neutral genetic processes in phenotypic divergence. We provided evidence for a neutral genetic differentiation resulting from both isolation by distance and difference in altitude. We found evidence for phenotypic divergence along the altitudinal gradient (faster development, lower growth rate and smaller metamorphic size). The correlation between pairwise Q_STs–F_STs and altitude differences suggested that this phenotypic differentiation was most likely driven by altitude-mediated selection rather than by neutral genetic processes. Moreover, we found different divergence patterns for larval traits, suggesting that different selective agents may act on these traits and/or selection on one trait may constrain the evolution on another through genetic correlation. Our study highlighted the need to design more integrative studies on the common toad to unravel the underlying processes of phenotypic divergence and its selective agents in the context of environmental clines.     

Morphological and spectrometric analyses of lipids accumulation in a novel oleaginous microalga, Eustigmatos cf. polyphem (Eustigmatophyceae)

Eustigmatos cf. polyphem is a yellow-green unicellular edaphic microalga belonging to the eustigmatophyte. The characteristics of lipids accumulation of E. cf. polyphem grown in a bubble cylindrical photobioreactor under nitrogen-limited conditions was dissected by morphological and spectrometric analyses. Total lipids accumulation rate increased rapidly at early growth phase, with the emergence of many small lipid bodies. Afterwards, lipid bodies became abundant and enlarged primarily because of the progressive accumulation of lipids and the fusion of nearby lipid bodies. Maximum total lipids and neutral lipids content reached up to 60.59 % and 53.08 % of cell dry weight, accompanied with a biomass dry weight 7.9 g/l. E. cf. polyphem is thus referred to as an oleaginous microalga for biodiesel production due to its high biomass and considerable production of oils.     

Validity of Eucaryote Inhibitors for Assessing Production and Grazing Mortality of Marine Bacterioplankton

Application of eucaryote inhibitors to the estimation of production and grazing mortality of bacterioplankton was evaluated. Exposure to a range of concentrations of thiram, cycloheximide, and neutral red (0.4 to 210, 36 to 1,777, 4 to 346 μM, respectively) was 98 to 100% effective at inhibiting growth of a chrysomonad in culture. Exposure to colchicine and griseofulvin (50 to 1,000 μM for both) yielded only 24 to 94 and 53 to 79% inhibition, respectively. Exposures to thiram, neutral red, and griseofulvin were 90 to 100% effective at inhibiting growth in culture of a ciliate, Cyclidium sp., and the responses to colchicine and cycloheximide were variable (64 to 100 and 0 to 100% inhibition, respectively). Thiram and neutral red inhibited field populations of nanozooplankton more effectively than cycloheximide and colchicine. Direct effects of eucaryote inhibitors on growing cultures of bacterioplankton varied with parameters measured and duration of exposure. After 3-day exposures, specific growth rates and “instantaneous” heterotrophic potential ([¹⁴C]glucose uptake) were not consistently affected, but biosynthetic activity (RNA and DNA syntheses) was depressed. The degree of inhibition of isolates and field populations of phytoplankton depended upon type of inhibitor and phytoplankton species. In field experiments, it was possible to calculate rates of bacterioplankton production and grazing mortality for only 16 of 29 inhibitor experiments and for 4 of 10 size fractionation experiments. Bacterioplankton production and mortality estimates varied greatly with the eucaryote inhibitor used, and those derived from inhibition techniques were substantially different from those derived from fractionation techniques. The poor performances of both techniques are attributed to the following: (i) effects of inhibitors on phytoplankton, (ii) indirect effects of the inhibitors on bacterioplankton, and (iii) insufficient separation of grazers from prey by filtration techniques. Because of the inconsistent results obtained in this investigation, we strongly recommend exercising caution in the application of inhibitor techniques to ecological problems, especially in phototrophically dominated systems.