Effects of deep seawater medium on growth and amino acid profile of a sterile Ulva pertusa Kjellman (Ulvaceae, Chlorophyta)

The potential use of deep seawater (DSW) for the cultivation of macroalgae was evaluated by analysis of the growth rate of thalli (by surface area), photosynthetic pigment content, and total free amino acid content of sterile Ulva pertusa cultured in the control medium (one-fifth strength Provasoli’s enriched seawater (PES) medium), surface seawater (SSW) medium and also seawater medium collected from 400 m depth (400-DSW) and 1,200 m depth (1,200-DSW). The growth rate of the sterile U. pertusa grown in 400-DSW and 1,200-DSW was similar to that of thalli cultured in the one-fifth strength PES medium that had previously been shown to be the optimum concentration for the cultivation of Ulva. Photosynthetic pigment content in the control medium, 400-DSW- and 1,200-DSW-cultured thalli was 1.7–2.0 times higher than that of the SSW-cultured thalli. The total free amino acids content of the control medium and SSW-cultivated thalli was 264?±?64 and 120?±?39 mg per 100 g dry weight, whereas the 400-DSW- and 1,200-DSW-cultivated thalli had significantly more amino acids (342?±?69–327?±?64 mg per 100 g dry weight). The results of this study indicate that DSW has a high potential for cultivation of edible macroalgae without any supply of extra nutrient until it grows to harvest size.     

SST broth,a new serum free germ tube induction medium for identification of Candida albicans

Three serum free media viz, sucrose solution, starch solution and SST broth have been formulated. The objective of the present study was to evaluate these three different serum free media for induction of germ tubes by Candida albicans and to compare their efficacy with the pooled human serum. Out of 50 C. albicans isolates 47 (94 %) and 49 (98 %) produced germ tubes in pooled human serum and SST broth, respectively. Germ tube production was positive in 40 (80 %) and 36 (72 %) isolates, respectively in sucrose solution and starch solution. This study reports SST broth as a new stable and less expensive germ tube induction medium, which requires less time for preparation and can be used without any safety concerns. SST broth is found to be more effective than pooled human serum for induction of germ tubes by C. albicans isolates.     

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.     

Free and immobilized Aspergillus niger epoxide hydrolase-catalyzed hydrolytic kinetic resolution of racemic p-chlorostyrene oxide in a neat organic solvent medium

An enantio- and regioselective hydrolytic kinetic resolution (HKR) of racemic p-chlorostyrene oxide (rac-pCSO) was achieved by epoxide hydrolase (EH) from recombinant Aspergillus niger in a selected neat organic solvent medium. The solid free EH was reused four times in repeated-batch reactors; however, the relative activity as well as the enantiomeric ratio (E-value) of this EH decreased from 100 to 20% and from 68 to 23, respectively. In order to overcome the diffusion hindrance, due to the accumulation of the hydrophilic diol in the enzyme micro-environment, and thereby to improve the operational stability of EH after recycling, strategies consisting of the immobilization of EH and the use of a binary organic solvent as the reaction medium were successfully applied. Although the highest protein immobilization yield (82%) and retention of EH activity (142%) in heptane were obtained upon the immobilization of EH on Accurel EP, the E-value and the operational stability of the resulted EH immobilizate after recycling were reduced as compared to the free EH. In contrast, the nonporous DEAE-cellulose improved the operational stability of EH by more than twofold. On the other hand, both the HKR efficiency and the operational stability of A. niger EH were found to be modest to excellent in various binary organic solvent mixtures of heptane and dioxane, depending on their ratio resulting in different Log P.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Continuous ethanol fermentation of concentrated sugar solutions coupled with membrane distillation using a PTFE module

Continuous ethanol fermentation of concentrated glucose and molasses solutions was coupled with membrane distillation using a PTFE ethanol stripping module. Experimental results indicated that the PTFE module can remove a high concentration of ethanol from the fermentation broth and thus maintain a low ethanol concentration in the broth, thereby alleviating the problem of product inhibition. Accordingly, the product yield and the specific ethanol production rate increased. During the continuous fermentation runs, long-time operation using the PTFE module was found to be possible (i.e. 430 h using the glucose medium and 695 h using the molasses medium) and no significant change in the ethanol separation performance of the PTFE module was observed. Although cell flocculation became undetectable when a concentrated molasses medium containing 316 g/l sugar solution was used, the ethanol separation performance of the ethanol stripper was not adversely affected by the presence of the free cells. This suggests that clogging of the membrane pores by cells or other particulates is not a major problem when using the PTFE module in continuous ethanol fermentation.     

Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media

Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [N-2-hydroxyethylpiperazine-N′ -2-ethanesulfonic acid−2-(N-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for Rhizobium fredii USDA 201. Arabinose-HEPES-MES was modified by addition of the chelator nitrilotriacetate to buffer the supply of free Cd²⁺ ion to maintain a constant Cd activity and by the use of only MES to buffer pH (buffered arabinose-MES medium [BAM]). Ca and Mg were supplied at the normal levels for soil solutions, and other trace elements were supplied at the levels required for normal growth of plants. The concentration of free Cd²⁺ ion was calculated by using the computer program GEOCHEM-PC with a corrected data base. The Cd MIC in BAM was 14.0 μM, while that in a tryptone-yeast extract medium was 107 μM. The results indicate that substantial free Cd²⁺ is removed from solution in most standard media, resulting in falsely high MICs. The new BAM medium allows for the precise determination of MICs, thus avoiding the uncertainties associated with other media.     

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23 days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Intervention Effects of Ganoderma Lucidum Spores on Epileptiform Discharge Hippocampal Neurons and Expression of Neurotrophin-4 and N-Cadherin

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg²⁺ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg²⁺ free medium for 3 hours), iii) GLS group I (incubated with Mg²⁺ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg²⁺ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.     

Enhanced activity of intracellular lipases from Rhizomucor miehei and Yarrowia lipolytica by immobilization on biomass support particles

The aim of this investigation was to obtain an efficiently immobilized intracellular lipase from Rhizomucor miehei and Yarrowia lipolytica. The activity of intracellular lipases from R. miehei and Y. lipolytica was enhanced by the addition of waste fats (beef tallow or poultry fat) to the medium and by cell immobilization on biomass support particles (BSPs, cubic particle of polypropylene or polyurethane foams). The highest intracellular activity of lipases was obtained after adding 20 and 50 BSPs to the medium of R. miehei (130.5 U) and Y. lipolytica (90.3 U), respectively. The best carrier for immobilizing intracellular lipases was polyurethane foam and the lipolytic activity of immobilized lipases was 2.1–4.3-times higher than the activity of lipases obtained from free biomass. The properties of the immobilized enzymes were very similar to the free enzymes but the immobilized intracellular lipases were more useful for the hydrolysis of waste fats. The highest reaction ratio (72%) and content of free fatty acids (68% (w/w)) in the reaction mixture was obtained after 72 h for beef tallow hydrolysis in a batch reaction with the immobilized lipases from R. miehei.     

Production of gibberellins and bikaverin by cells of Gibberella fujikuroi immobilized in carrageenan

The production of gibberellins and bikaverin by immobilized and free cells of Gibberella fujikuroi strains was followed. Both types of cells, free and immobilized, produced similar titers of the secondary metabolites during the normal growth cycle. The kinetics of nutrient use and product formation by the immobilized cells lagged behind that of the free cells and this was assumed to be the result of diffusional limitations imposed on the immobilized cells. A noticeable difference was that in the immobilized cells, all of the bikaverin was excreted into the medium for both strains of G. fujikuroi tested but in the free cell fermentation 44% was excreted for strain ACC 917 and only 10% for strain GF1a. Gibberellin and bikaverin could be produced in a semi-continuous fashion with both free and immobilized cells for a period of 16 d in a resuspension medium containing 0.12 mM or 0.60 mM ammonium chloride. No definite advantage, on a productivity basis, for using immobilized cells over free cells could be seen.     

Free-calcium and tension responses in single barnacle muscle fibres following the application of l-glutamate

The effect of the putative transmitter, l-glutamate, on free intracellular Ca²⁺, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of l-glutamate (0.1–10 mM) resulted in a transient increase in free intracellular Ca²⁺, monitored by the Ca²⁺-activated protein aequorin. This increase in free intracellular Ca²⁺ was associated with membrane depolarization and force development, and was followed by a period of ‘desensitization’ in which the preparation was unresponsive to l-glutamate. This could be reversed by removing l-glutamate from the external saline. External application of a number of closely related compounds, including d-glutamate and l-aspartate, were ineffective for initiating the transient light response. The l-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to l-glutamate were not reduced in Mg-free medium. The fibre's response to 1 mM l-glutamate was also inhibited by D-600 (10 μM) or by La³⁺ (1 mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.     

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate.     

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).     

Isolation, Identification, and Characterization of a Feather-Degrading Bacterium

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10⁹ cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10⁷ cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.     

Synthesis of betaine lipids: Dipropionylglyceryl(N,N,N-trimethyl)serine

1,2-Dipropionylglyceryl-3-O-3′-(N,N,N-trimethyl)serine was synthesized as a first example of a diglyceride containing an ether-linked hydroxyamino acid with betaine structure. The ether bond was formed by condensation of isopropylideneglycerol (Na alcoholate) and ethyl-2,3-dibromopropionate. Then, the quaternary ammonium group was introduced by replacing the secondary bromine (2-position) with trimethylamine. Finally, the isopropylidene group was split off in acidic medium and the free hydroxyl groups of the glycerol part esterified with propionic acid. The overall yield was 7.5%.