A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses

Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.     

Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l⁻¹ h⁻¹; 0.8 to 2.3 g l⁻¹ h⁻¹). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol d-lactate (90 g) per 100 g sugar (glucose or sucrose). Revisions requested 17 January 2006; Revisions received 7 February 2006     

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 μg g⁻¹ fresh weight) and high quality (A _260/280 ratio 1.96 ± 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible.     

Simultaneous and selective production of levan and poly(γ-glutamic acid) by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis(natto) Takahashi, used to prepare the fermented soybean product natto, was grown in a basal medium containing 5% (w/w) sucrose and 1.5% (w/w) l-glutamate and produced 58% (w/w) poly(-glutamic acid) and 42% (w/w) levan simultaneously. After 21 h, 40–50 mg levan ml^-1had been produced in medium containing 20% (w/w) sucrose but without l-glutamate. In medium containing l-glutamic acid but without sucrose, mainly poly(-glutamic acid) was produced. Revisions requested 28 August 2004/14 October 2004; Revisions received 11 October 2004/22 November 2004     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Enhancement of poly-β-hydroxybutyrate accumulation in <Emphasis Type="Italic">Nostoc muscorum</Emphasis> under mixotrophy,chemoheterotrophy and limitations of gas-exchange

Nostoc muscorum, a heterocystous cyanobacterium, produced poly--hydroxybutyrate ( PHB) up to 8% (w/w) dry cells when grown photoautotrophically but 35% when grown mixotrophically with 0.4% (w/v) glucose and acetate after 21 d. Gas-exchange limitations under mixotrophy and chemoheterotrophy with 0.4% (w/v) acetate enhanced the accumulation up to 40–43% (w/w) dry cells, the value almost 5-fold higher with respect to photoautotrophic condition.Revisions requested/10 September 2004; Revisions received 9 September 2004/11 November 2004     

Synthesis of (<Emphasis Type="Italic">R</Emphasis>)-2-trimethylsilyl-2-hydroxyl-ethylcyanide catalyzed with (<Emphasis Type="Italic">R</Emphasis>)-oxynitrilase from loquat seed meal

The synthesis of optically active (R)-2-trimethylsilyl-2-hydroxyl-ethylcyanide by asymmetric trans-cyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system was achieved using (R)-oxynitrilase from loquat seed meal. Diisopropyl ether was the most suitable organic phase among the organic solvents examined. The optimal concentration of acetyltrimethylsilane, concentration of crude enzyme, volume ratio of the aqueous to the organic phase, temperature and the buffer pH value were 14 mM, 61.4 U ml^-1, 13% (v/v), 30 °C and 4, respectively. The substrate conversion and the product enantiomeric excess were 95% and 98% under the optimized conditions. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon counterpart. Revisions requested 24 August 2004; Revisions received 12 November 2004     

Adventitious shoot regeneration of scotch spearmint (menthaxgracilis Sole)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Menthaxgracilis Sole (scoth spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 μM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, at TDZ 4.5 μM and 10% (v/v) coconut water and solidified with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% sucrose, and 0.054 μM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the first report describing adventitious shoot regeneration of scotch spearmint.     

Evidence of fusion and artificially initialized parthenogenesis in isogametes of Endarachne binghamiae (Scytosiphonaceae, Phaeophyta) from Taiwan

Approximately 120–140 minutes after Endarachne binghamiae isogametes are released and kept in an incubator (50 μmol photonm^-2s^-1, 18 ^°C, 10/14 h L/D), the isogametes from single or mixed cultures undergo isogamete fusion to mate sexually. It can therefore be inferred that E. binghamiae is monoecious. However, when these newly released isogametes are keptin similar conditions, but treated with low dosages of various kinds of solution (0.012% (v/v) formaldehyde,0.105% w/v EDTA, 0.198% w/v EGTA, in filtered seawater) that can paralyze flagellar movement to initiate parthenogenesis, the isogametes in one of these, 0.012% formaldehyde, develop parthenogenetically into filament, precrustose (early, middle and late stage), crustose and finally erect thalli. SEM and TEM show that the mastigoneme of the anterior flagellum in this group is broken, which results in the isogametes being non-motile. In comparison, the development of the isogametes in the other two groups stops somewhere before the stage where they grow into erect thalli. In other words parthenogenetic reproduction occurs under our culture conditions and we have identified clearly the morphology and parthenogenetic development of E. binghamiae. It is suggested tha the findings of this study could be applied to increase the biomass of E. binghamiae, an edible species of very high economic value in north-east Asia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.     

The isolation of RNA from raspberry (Rubus idaeus) fruit

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.     

A Simple and Green Procedure for the Microbial Effective Synthesis of 1-phenylethyl Alcohol in Both Enantiomeric Forms

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40–80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker’s yeast. Revisions requested 29 November 2005; Revisions received 9 January 2006     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006