Electronic Journal of Biotechnology

Publications
Recombinant bacterial proteins expressed in transgenic tobacco. 
Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms, because it is easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. Many bacterial proteins involved in the synthesis of commercial products are currently engineered for production in tobacco. Bacterial enzymes synthesized in tobacco can enhance protection against abiotic stresses and diseases, and provide a system to test applied strategies such as phytoremediation. Examples of bacterial gene expression in tobacco include production of antigen proteins from several human bacterial pathogens as vaccines, bacterial proteins for enhancing resistance against insects, pathogens and herbicides, and bacterial enzymes for the production of polymers, sugars, and bioethanol. Further improvements in the expression of recombinant proteins and their recovery from tobacco will enhance production and commercial use of these proteins. This review highlights the dynamic use of tobacco in bacterial protein production by examining the most relevant research in this field.
 
High-density cultures of mammalian neurons offer a model system for studies of brain development, but the morphological features of individual neurons is difficult to ascertain. We show that a herpes virus vector expressing a bioluminescent protein allows detailed morphometric analyses of living neurons in complex culture environments. Expression of enhanced green fluorescent protein (eGFP) was constitutively driven in neurons using the herpes simplex virus amplicon system. This system allowed us to make novel observations regarding development in high-density cultures from rat hippocampus and cerebellum. After the phase of initial neurite outgrowth, maturing neurons continue to show rapid remodeling of the neurite branches (0.79 +/- 0.11 mum/h per neurite; mean +/- SEM, n=8), and displacement of the soma within the neurite arbor (1.35 +/- 0.74 mum/h). These results demonstrate that a substantial capacity for morphological plasticity persists in maturing mammalian CNS neurons after cessation of net neurite outgrowth in early development.
 
Surface tension versus concentration of the purified biosurfactant.
Growth, pH and surface active profile of C. lipolytica grown in mineral medium with 6% industrial residue as substrate and 1% glutamic acid.
Background Biotechnological processes are costly, especially for the production of biosurfactants. The successful production of a biosurfactant is dependent on the development of processes using low cost raw materials. Considering the importance of the characteristics of a biosurfactant to facilitate its industrial application, the properties of the biosurfactant produced by Candida lipolytica through previously optimized medium have been established. Results The yeast was grown for 72 h to determine the kinetics of growth and production. The surface tension of the cell-free broth was reduced from 55 to 25 mN/m. The yield of biosurfactant was 8.0 g/l with a CMC of 0.03%. The biosurfactant was characterized as an anionic lipopeptide composed of 50% protein, 20% lipids, and 8% of carbohydrates. Conclusions The isolated biosurfactant showed no toxicity against different vegetable seeds: Brassica oleracea, Solanum gilo and Lactuca sativa L. and the micro-crustacean Artemia salina. The properties of the biosurfactant produced suggest its potential application in industries that require the use of effective compounds at low cost.
 
Effects of pH (a), temperature (b), catechol concentration (c), and inhibition by substrate (d) on activity of immobilized catechol 1,2-dioxygenase in Stenotrophomonas maltophilia KB2 cell extracts. The data points represent the average of 3 independent experiments.
Storage stabilities of free and immobilized catechol 1,2-dioxygenase from Stenotrophomonas maltophilia KB2. Data shown represent the average of three independent trials.
Background In biodegradation processes free enzymes often undergo deactivation. Thus, it is very important to obtain highly stable enzymes by different methods. Immobilization allows for successful stabilization of many multimeric enzymes by increasing the rigidity of the enzyme structure. This study aimed to evaluate some environmental factors that affect catechol 1,2-dioxygenase from Stenotrophomonas maltophilia KB2 immobilized in alginate hydrogel. The goal of the present work was to improve the functional stability of the enzyme by increasing its structural rigidity. Results Immobilization yield and expressed activity were 100% and 56%, respectively. Under the same storage conditions, the activity of the immobilized enzyme was still observed on the 28th d of incubation at 4°C, whereas the free enzyme lost its activity after 14 d. The immobilized enzyme required approximately 10°C lower temperature for its optimal activity than the free enzyme. Immobilization shifted the optimal pH from 8 for the soluble enzyme to 7 for the immobilized enzyme. The immobilized catechol 1,2-dioxygenase showed activity against 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, and 3,5-dichlorocatechol. The immobilization of the enzyme promoted its stabilization against any distorting agents: aliphatic alcohols, phenols, and chelators. Conclusions The entrapment of the catechol 1,2-dioxygenase from S. maltophilia KB2 has been shown to be an effective method for improving the functional properties of the enzyme. Increased resistance to inactivation by higher substrate concentration and other factors affecting enzyme activity as well as broadened substrate specificity compared to the soluble enzyme, makes the immobilized catechol 1,2-dioxygenase suitable for the bioremediation and detoxification of xenobiotic-contaminated environments.
 
Experimental results of C. butyricum DSP 1 during batch cultivation in 2-L bioreactor, at various initial crude glycerol concentrations without biomass recycling.
The changes in proteins profile of C. butyricum DSP1 in different variants of the synthesis process 1,3-PD from the crude glycerol. Culture conditions: T=37°C, pH7.0, growth in a 2L bioreactor, a) initial glycerol concentration 20±1.0g/L, without biomass recycling; b) initial glycerol concentration 120±1.0g/L, without biomass recycling; c) initial glycerol concentration 20±1.0g/L, with recycling biomass, glycerol concentration after biomass recycling 120±1.0g/L.
The block diagram of the fermentation process with filtration module.
Kinetics of glycerol consumption and biomass, 1,3-PD production during the growth of C. butyricum DSP1 on crude glycerol in batch bioreactor experiments with biomass recycling.
Background: 1,3-Propanodiol (1,3-PD), is used in the production of polytrimethylene terephthalate (PTT), an aromatic polyester that exhibits high elastic recoveries. It is also employed as a supplement with low solidification properties, a solvent and a lubricant in the formof propylene glycol. 1,3-PD is effectively synthesized by a microbiological way from crude glycerol. The main problem of this technology is using a high concentration of glycerol, which is a limiting factor for bacteria cells growth (especially in batch fermentation). Results: In this work, the influence of different glycerol concentration in batch fermentation on Clostridium butyricum DSP1 metabolism was investigated. The biomass was concentrated for two times with the use of membrane module (in case of increasing kinetic parameters). Increased optical density of bacteria cells six times increased the productivity of 1,3-PD in cultivation with 20 g/L of glycerol at the beginning of the process, and more than two times in cultivation with 60–80 g/L. Also the possibility of complete attenuation of 140 g/L of crude glycerol in the batch fermentation was investigated. During the cultivation, changes of protein profiles were analyzed. The most significant changes were observed in the cultivation in the medium supplemented with 80 g/L of glycerol. They related mainly to the DNA protein reconstructive systems, protective proteins (HSP), and also the enzymatic catalysts connected with glycerol metabolic pathway. Conclusions: The application of filtration module in batch fermentation of crude glycerol by C. butyricum DSP1 significantly increased the productivity of the process.
 
Chemical structure of 1,8-cineole and the biotransformation products.
Influence of the inoculum's size on the biotransformation yield and product composition.
Evolution of the biotransformation product profile as a function of time. 
The forest industry in Uruguay has grown considerably during the last decade. Eucalyptus plantations account for 74% of the forested land, with Eucalyptus globulus being the most widely distributed species. This industry is dedicated exclusively to the production of wood without exploiting the by-products (leaves and small branches). Eucalyptus leaves are known to contain important amounts of essential oils composed primarily of 1,8-cineole (1,3,3-trymethyl-2-oxabicyclo[2.2.2]octane). In this work, the biotransformation of 1,8-cineole, is achieved using a native bacterium (Rhodococcus sp.) which was isolated from the soil of Eucalyptus forest. A 98% of bioconversion was achieved. Three different optically pure compounds were obtained, and they were identified as 2-endo-hydroxy-1,8-cineole, 2-exo-hydroxy-1,8-cineole and 2-oxo-1,8-cineole.
 
Time courses of growth, culture pH and emulsifications of n-hexadecane and cotton seed oil by Candida glabrata grown on mineral medium supplemented with 7.5% cotton seed oil plus 5.0% glucose. 
Surface tension reduction of distilled water by the cell-free broth of Candida glabrata grown on mineral medium supplemented with 7.5% cotton seed oil plus 5.0% glucose. 
Efficiency of emulsifying activity of cell-free broth of Candida glabrata grown on mineral medium supplemented with 7.5% cotton seed oil plus 5.0% glucose as a function of volumesvariation between the cell-free broth (aqueous phase) and the substrate (oil phase), respectively. (A = 3.0 ml and 2.5 ml; B = 2.5 ml and 3.0 ml; C = 2.0 ml and 3.5 ml; D = 1.5 ml and 4.0 ml; E = 1.0 ml and 4.5 ml). 
Effect of different sodium chloride concentrations on the emulsifying activity of cell-free broth of Candida glabrata grown on mineral medium supplemented with 7.5% cotton seed oil plus 5.0% glucose. 
Evaluation of both tenso-active and emulsifying activities indicated that a biosurfactant was produced by the newly isolated and promising strain Candida glabrata isolated from mangrove sediments. The extracellular water-soluble emulsifying agent was isolated and identified as a heteropolymer. The maximum of bioemulsifier production was observed when the strain was grown on soluble and insoluble substrates cotton seed oil plus glucose, reaching values of 10.0 g/l after 144 hrs at 200 rpm. The cell-free culture broth containing the examined agent lowered the surface tension of the medium to 31 mN/m. Stable and compact emulsions with emulsifying activity of 75% of cotton seed oil were detected. The emulsification capacity remained practically unaltered within a wide pH (2-12), temperature (4-80°C) ranges and under NaCl concentrations up to 10%.
 
Schematic representation of generating 100bp ladder. ® Figure 1a. Schematic representation of primers located on pGEM -T vector system. Arrow indicates primers and its distance in the vector backbone while the bp indicates the corresponding fragment sizes that are generated using the corresponding primers. Figure 1b. Agarose gel picture showing the amplified PCR products of the in house ladder. Figure 1c. Agarose gel picture showing the commercial ladder Lane M (1kb plus ladder Invitrogen) lane M1 (100bp ladder NEB) and lane M*- (In-house 100bp ladder). 
Primer sequences used in the study.
Developing countries are facing severe bottlenecks in the technological advancement in biotechnology, due to restrictions imposed by patent protected products and protocols. This calls for designing of simple and cost-effective alternatives for the indispensable products like DNA molecular weight markers. We demonstrate a novel, rapid and cost-effective method of making in-house 100bp ladder for routine use. In our method we use a single forward primer and five reverse primers designed on the backbone sequence of a commonly used vector template. These primers are used at a universal annealing temperature to amplify ten DNA fragments of accurate size ranging from 100bp to 1000bp. Our PCR-based method can provide size standards for an endless usage.
 
SPECT images of animals before (Week 4) and after (Week 9) treatment with pVEGF 121 or saline. Non ischemic control corresponds to the first gammagraphic study (week 0). White, pink and red colours indicate normal perfusion. Brown, yellow and green denote defective perfusion. 
Characterization of myocardial perfusion.
Effect of intramyocardial VEGF 121 gene transfer on 
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is angiogenic in vitro and in vivo. Several studies report on gene transfer of VEGF121 to promote angiogenesis in the ischemic myocardium of animals and patients. We hypothesized that intramyocardial administration of naked plasmid DNA encoding VEGF121 could improve myocardial perfusion and function in a porcine model of myocardial ischemia. Yorkshire swine underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, pVEGF121 plasmid was administered into the ischemic myocardium. Four weeks after gene transfer, SPECT imaging demonstrated significant reduction in the ischemic area in pVEGF121-treated animals compared with controls. In the pVEGF121 group, most of the animals evolved from light ischemia to a normal perfusion. In contrast, control animals exhibited similar or impaired ischemic conditions. Our results indicate that intramyocardial gene transfer of VEGF121 as naked plasmid DNA results in significant improvement in myocardial perfusion and function.
 
SPECT images of animals before (Week 4) and after (Week 9) treatment with pVEGF 121 or saline. Non ischemic control corresponds to the first gammagraphic study (week 0). White, pink and red colours indicate normal perfusion. Brown, yellow and green denote defective perfusion. 
Characterization of myocardial perfusion.
Effect of intramyocardial VEGF 121 gene transfer on 
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is angiogenic in vitro and in vivo. Several studies report on gene transfer of VEGF121 to promote angiogenesis in the ischemic myocardium of animals and patients. We hypothesized that intramyocardial administration of naked plasmid DNA encoding VEGF121 could improve myocardial perfusion and function in a porcine model of myocardial ischemia. Yorkshire swine underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, pVEGF121 plasmid was administered into the ischemic myocardium. Four weeks after gene transfer, SPECT imaging demonstrated significant reduction in the ischemic area in pVEGF121-treated animals compared with controls. In the pVEGF121 group, most of the animals evolved from light ischemia to a normal perfusion. In contrast, control animals exhibited similar or impaired ischemic conditions. Our results indicate that intramyocardial gene transfer of VEGF121 as naked plasmid DNA results in significant improvement in myocardial perfusion and function.
 
VEGF 121 -induced in vitro angiogenesis . Supernatants from cells transfected with pVEGF 121 , and controls were added to HMECs cultured in three-dimensional Matrigel. Cells were grown in the presence of (A) supernatant from pAEC- ∆ 2-transfected cells; (B) 0.5 ng/mL VEGF 165 protein; and VEGF 121 -containing supernatant at (C) 1:2 dilution, and (D) not diluted. Photographs were taken 16-20 h after treatment with 40X magnification. 
Angiographic images of control animal at (A) day 10 (immediately before gene transfer), and (B) day 40 (30 days after transfection); and pVEGF 121 -treated animal at (C) day 10 and (D) day 40. In contrast to control, collateral vessel development becomes evident in the pVEGF 121 -transfected animal. 
Effect of intramuscular VEGF 121 gene transfer on calf blood pressure ratio at days 10, 25 and 40. ** P<0.01 versus control and baseline. 
Effect of intramuscular VEGF 121 gene transfer on vasomotor reserve at days 10, 25 and 40. Animals were classified according to their response to an intravenous infusion of 2 mg nitroglycerine. 
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, has been shown to promote therapeutic angiogenesis in animal models of critical limb ischemia. Ischemic skeletal muscle is advantageous for taking up and expressing foreign genes transferred as naked plasmid DNA. Accordingly, we investigated the hypothesis that intramuscular administration of naked plasmid DNA encoding the 121-amino acid isoform of VEGF could augment collateral development and tissue perfusion in a dog hindlimb ischemia model. Unilateral hindlimb ischemia was surgically induced in Beagle dogs. Ten days later, animals received intramuscular injections of pVEGF121 plasmid directly in the ischemic muscles. Angiogenic effects were evaluated by angiography, calf blood pressure ratio and vasomotor reserve analyses. Thirty days after gene transfer, angiographically recognizable collateral vessels were increased in pVEGF121-treated animals compared with controls. Improvement in perfusion to the ischemic limb was documented by a significantly higher calf blood pressure ratio for pVEGF121 (0.79 ± 0.05) versus controls (0.56 ± 0.14, P<0.01). Vasomotor reserve assay suggested amelioration in blood availability at the microcirculation level in pVEGF121-treated animals. Hematological variables showed no significant modification due to the treatment. Our results suggest that intramuscular gene transfer of VEGF121 may promote therapeutic angiogenesis in critical limb vascular insufficiency.
 
Growth and production of cellulase and ß-glucosidase (cellobiase) by Trichoderma aureoviride wild type ( o ) and mutant 7-121 ( • ). Cultures were carried out in Mandels salts solution with the addition of 5 g microcrystalline cellulose/L, at 28oC in an orbital shaker incubator. 
Endochitinases secreted by T. aureoviride mutant 7-121 grown in liquid Mandels medium which contained cell walls of different phytopathogenic fungi: B. cinerea (lane 1); A alternata (lane 2) or F. oxysporum (lane 3). Native PAGE was carried out in 15% polyacrylamide gels at pH 8.8. 100 µL of supernatants of cell wall-grown cultures were loaded into each well.
Proteases secreted by wt (lane 2) and mutant 7-121 of T. aureoviride (lane 3) and by isolate P6 of T. aureoviride (lane 4). Cultures were grown on chitin as carbon source. Native PAGE was carried out in 15%, polyacrylamide gels pH 8.8 in the presence of PMSF. Positive control is proteinase K (lane 1).
A mutant of the native fungus Trichoderma aureoviride , 7-121, selected for its overproduction of extracellular cellulase and β-glucosidase (cellobiase) was obtained. In shake flask cultures, production of endoglucanase, filter paper activity and cellobiase increased two to four- fold as compared with the wild type strain. The mutant strain is stable and grows rapidly in liquid as well as in solid culture media. Enzyme yields were best when pH was controlled so that it did not fall bellow pH 3.5. Cellobiase production by this mutant is particularly high (approximately 5 U/ml) as compared to other Trichoderma , strains, which makes it a suitable candidate for waste cellulose degradation. In addition, the mutant strain showed enhanced production of fungal cell wall degrading enzymes: chitinases, β-1,3-glucanases and proteases. This improvement in extracellular enzyme production by the mutant T. aureoviride 7-121 suggests that it is a suitable strain to be used in biological control.
 
Effect of different nitrogen sources on β-xylosidase production by K. marxianus PPY125 (light squares) and its mutant M125 (dark squares). Organisms were grown on xylose (20 g/l, w/v) containing different nitrogen sources (1 = ammonium nitrate; 2 = ammonium sulphate; 3 = corn steep liquor; 4 = soybean meal; 5 = sodium glutamate; 6 = sodium nitrate; 7 = urea; and 8 = peptone) at a rate of 1.1 g/l equivalent to that contained in ammonium sulphate in triplicate. Flasks were harvested and processed as described in Materials and Methods. Each value is a mean of three independent experiments.Error bars represent standard deviation among three replicates.
Effect of addition of glucose (5, 10, 15, 20 and 25 g/l) in the xylose-corn steep liquor medium on volumetric productivity of β-xylosidase produced by the parental (light squares) and mutant (dark squares) cultures. Each value is a mean of three independent experiments. Error bars represent standard deviation among three replicates
Effect of temperature on volumetric productivity (IU/lh) of β-xylosidase during growth of K. marxianus PPY125and its mutant (M125) cultures on xylose (2%) medium (pH 5.5). The organisms were grown as described in Methods. Each value is a mean of three independent experiments. Error bars represent standard deviation among three replicates.
Arrhenius plots for calculating enthalpy and entropy for reversible inactivation of β-xylosidase. They were calculated by applying the equation 2 in Materials and Methods (Aiba et al. 1973). The value of ∆H * was calculated from slope of the straight line between ln(k d /T) and 1/T while ln(k B /h)+ ∆S * /R=Intercept on ordinate. k B , h, and R are 1.38 x 10-23 JK-1 , 6.63 x10-34 J.S and 8.314 JK-1 mol-1 respectively (Rahsid and Siddiqui, 1998).
Production of β-xylosidaseby a cycloheximide and 2-deoxy-D-glucose-resistant mutant of Kluyveromyces marxianus PPY125 was studied when cultured on growth media containing galactose, glucose, xylose, cellobiose, sucrose and lactose as carbon sources. Xylose, cellobiose, lactose and sucrose were the key substrates. Both K. marxianus PPY125 and its mutant (M 125) supported maximum β-xylosidase specific product yield (YP/X) following growth on xylose. Basal level of activity was observed in non-induced cultures grown on glucose. The mutant produced 1.5 to 2-fold more β-xylosidase than that produced by the wild cells. Synthesis of β-xylosidase was regulated by an induction mechanism in both wild and mutant cells. Addition of glucose did not inhibit the synthesis of β-xylosidase in both parental and mutant cultures in the presence of corn steep liquor. Partially purified enzyme showed good stability when incubated at 60°C and was quite stable at pH 5.0-7.0. Thermodynamic studies revealed that the enzyme derived by the mutant M125 was more thermostable as evidenced by higher midpoint inactivation temperature, lower activation energy demand for β-xyloside hydrolysis, as well as lower enthalpy and entropy demand for reversible denaturation of enzyme.
 
Time course of growth of N. tabacum hairy root lines. Each point represents the mean of three replicates ± SD. Time of culture: 30 days. 
Time course of 14D9 expression by clones AcK 6 and Ac 2 in: 
Growth index (GI), antibody yield in the biomass (μg FW-1),total amount of 14D9 in the culture medium (μg 14D9 per flask) and in the biomass (μg 14Dp per flask) in 22 days-old N. tabacum AcK 6 hairy root clone with the addition of gelatine (1.0, 5.0 and 9.0 g l -1 ), and PVP (1.0, 1.5 and 2.0 g l -1 ) . Gelatine and PVP were added to 2 days-old cultures. As control AcK 6 hairy root without any addition was used. Each point represents the mean of three replicates ± SD. 
Time course of 14D9 expression in: 
Western-blot of hairy root biomass. The lines were loaded with equal volumes of samples. Line 1: Standard IgG Antibody; line 2: N. tabacum wild type; line 3: N. tabacum wild type + 5% DMSO; line 4: Ac 2 + 2.8% DMSO; line 5: Ac 2 + 5% DMSO; line 6: Ac 2 control; line 7: AcK 6 + 2.8% DMSO; line 8: AcK 6 +5%; line 9: AcK 6 control. Coating: gaot anti gamma mouse chain conjugated with peroxidase (1:1000) in 1% (P/V) non fat milk. Antibody binding was detected after incubation with Supersignal West Pico Chemiluminiscent Substrate 
Nicotiana tabacum hairy roots that express the antibody 14D9 were established. The 14D9 antibody yield obtained after 20 days of culture was 5.95 μg 14D9ml-1. The addition of the reticulum endoplasmic retention sequence KDEL demonstrated a positive effect over the intracellular 14D9 amounts with a yield increase up to 20.82 μg ml-1. DMSO increased the antibody amount in the biomass from 20.00 to 64.03 μg ml-1 while PVP (at 1.5 gl-1) and gelatine (at 5.0 gl-1) increased total 14D9 amounts in the culture medium to 25 μg and 14 μg respectively.
 
Callus induction and regeneration for MC 169 and MC 182 explants derived from donor plants cultivated in growth chamber, field or greenhouse.
Although it is generally accepted that plant in vitro culture response is influenced by the donor genotype, the genetic and molecular bases of this phenomenon are barely known. As a consequence, the optimization of tissue culture protocols is mainly empirically done. Researchers of the IGEAF studied the genetic basis of the in vitro regeneration of various plant species, including the tissue culture response of artificially induced barley mutants. One barley mutant, MC 169, carries a nuclear gene, recently described controlling the root growth in hydroponic cultivation. Under this condition, the roots of MC 169 mutant plants were longer than those of the original wild type line MC 182, a fact that was associated with a reduced ethylene biosynthesis. On the other hand, it is known that ethylene accumulation is inhibitory for in vitro regeneration of several plant species. In this study, we compared the in vitro culture response of mutant MC 169 with that of its mother line MC 182. The data about induction and regeneration of calli as well as those of habituated calli formation demonstrated that mutant MC 169 and its mother line MC 182 show a similar in vitro behaviour.
 
An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37°C. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79% and 0.53%, respectively.
 
Proteolytic degradation of MB-1TrpHis and mutants by E. coli proteases. At variance with results in Figure 2, proteolysis was performed after separating MB-1TrpHis mutants from its fusion partner MBP.
CD spectra of MB-1Trp and MB-1TrpHis evolved mutants. All spectra have the usual features observed for helical proteins. Symbols: ♦ MB-1TrpHis; ∆ E44V45; E44M45.
Denaturation curves calculated from CD data obtained at various temperatures.
Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.
 
This long-term study demonstrates for the first time that it is possible to propagate embryogenic cultures in pelargoniums and to subsequently initiate the differentiation of embryos using the cultivar Madame Layal ( Pelargonium x domesticum ). Propagation of callus was only possible with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP), which gave rise to embryos from the primary culture stage on. However, the propagation of cells, as well as the differentiation of embryos, was inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in pelargoniums are discussed.
 
The BIO 2005 international convention is the largest gathering of the biotech industry in the world. This year it was held on June 19-22 inside the behemoth Convention Center in downtown Philadelphia, bringing together 18,730 executives, investors, consultants, lawyers, politicians, scientists, and dreamers from 56 countries. More than 500 media representatives covered the event. Biotechnology research and findings presented by countries outside the USA and Western Europe has begun to make a significant impact on these annual BIO gatherings. The achievements of some of these countries are briefly reviewed.
 
The pH influence on continuous citric acid secretion was investigated in Candida oleophila ATCC 20177 (var.) under NH4+ limiting state steady conditions, using glucose. Highest citric acid concentration of 57.8 g/l, citrate/isocitrate ratio of 15.6, space-time yield of 0.96 g/(l x hr) and biomass specific productivity of 0.041 g/(g x hr) were obtained at pH 5 and 60 hrs residence time. Only 22.8 g/l (39.4%) and a ratio of 9.9 were achieved at pH 6 pH and 12.4 g/l (21.5%) and a ratio of 3.7 at pH 3. Under non producing conditions, in excess of nitrogen, biomass concentration increased at raising pH. An iron concentration of 200 ppm was determined in biomass of C. oleophila at pH 5, compared with only 26 ppm found at pH 3 (factor 7.7). Intra- and extracellular concentrations of citrates and glucose confirmed the existence of a high specific, pH dependent active transport system for citrate secretion, while isocitrate isn't a high-affine substrate, displaying a strong correlation with ATP/ADP ratio. Differences between extra- and intracellular concentration of citrate higher than 1 and up to about 60 were determined. The active transport systemfor citrate excretion appears to be the main speed-determining factor in citrate overproduction by yeasts.
 
Initial batch fermentation of citric acid by Candida oleophila. (a) Glucose, citric acid and biomass as a function of fermentation time. (b) Formation rate of the generic product (Rj), specific citric acid productivity (m p ) and glucose consumption rate (Rs) and specific glucose consumption rate (r s ); (30ºC, pH 4.5).
Repeated batch fermentation (RB 1 ) of citric acid with Candida oleophila at 80% air saturation. (a) Glucose, citric acid and biomass as a function of fermentation time. 
Repeated batch fermentation (RB 1 ) of citric acid with Candida oleophila at 20% air saturation. (a) Glucose, citric acid and biomass as a function of fermentation time. (b) Formation rate of the generic product (Rj), specific citric acid productivity (m p ), glucose consumption rate (Rs) and specific glucose consumption rate (r s ); (30oC and pH 5). 
Repeated batch fermentation (RB 1 ) of citric acid with Candida oleophila at 80% air saturation and increased phosphate concentration. (a) Glucose, citric acid and biomass as a function of fermentation time. (b) Formation rate of the generic product (Rj), specific citric acid productivity (m p ), glucose consumption rate (Rs) and specific glucose consumption rate (r s ); (2.1 g/l KH 2 PO 4 , 30ºC and pH 5).
The effect of air saturation on citric acid production in batch, repeated batch and chemostat cultures has been studied. It was shown that, under continuous fermentation (chemostat mode), the highest concentration of citric acid equal of 98 g/l was produced at 20% of air saturation. In contrary to continuous fermentation, displaying an optimum at 20%, 80% air saturation yielded higher values in repeated batch fermentation process. 167 g/l citric acid were produced continuously with the fill and drain technique at 4.85 days, at 80% air saturation, compared with 157.6 g/l achieved within 5.4 days at 20%. Under repeated batch fermentation, the formation rate of the generic product (Rj) as well as the specific citric acid productivity (mp) reached a maximum of 1.283 g/(l x hr) at 4.01 days and of 0.0375 g/(g x hr) at 4.58 days, respectively. The glucose consumption rate (Rx) reached a maximum value of 3.33 g/(l x hr) entering stationary phase after 2.56 days at a glucose concentration of 131.2 g/l.
 
Time course of macroscopic yields during A. niger idiophase. A. Citric acid (Φp/s), CO 2 (Φco 2 /s) and Biomass (Φx/s) substrate yields. Values from Röhr et al. (1987) B. Polyol (Φpolyol/s) and Oxygen (Φo 2 /s) substrate yields. The substrate-polyol yields were determined by carbon balance among the cellular input (glucose) and outputs (citric acid, biomass and CO 2 ). The negative values are indicative of net polyol consumption. The oxygen-substate yields was calculated by reductance grade balance among the all compounds interchanged between the cell and the medium. In all these calculations an average polyol composition [CH 2.54 O] 3.72 was assumed. 
The purpose of this study was to test the applicability of the API 20E and API Rapid NFT systems for the identification of some predominant gram-negative and gram-positive bacteria isolated from lab-scale activated sludge treatment systems. In this study, one lab-scale sequencing batch reactor (SBR) and one lab-scale continuous-flow stirred tank reactor (CFSTR) were setup. After both reactors had reached equilibrium, many pure cultures isolated from the activated sludge in both systems were obtained and many morphological, biochemical, physiological tests were conducted to identify each pure culture. The API 20E system is a standardized, miniaturized version of conventional procedures for rapid identification of Enterobacteriaceae and other gram-negative bacteria, and the Rapid NFT kit is used for the identification of the gram-negative, non-fermentative bacteria. Also, a Phillips 300 Transmission Electron Microscope and a Phillips 301 Transmission Electron Microscope were applied to further verify the identification of some genera. According to the results of this study, it has been concluded that some commercial products, such as API 20E system and API Rapid NFT system, can be applied for the identification of microorganisms only at the genus level. Many other additional morphological, biochemical, and physiological tests are always needed to obtain the exact identification of each microorganism at the species level. More advanced technologies such as 16S rRNA may be necessary, however, for a rapid identification of the total bacterial population. In this study, it has also been found that Brevibacterium acetylicum and Pseudomonas vesicularis are two of the most dominant species in the activated sludge of CFSTR system. Gram-positive bacteria such as members of the genus Arthrobacter have shown to be very significant and predominant in the SBR system.
 
We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.
 
In a world where population growth is outstripping food supply agricultural -and especially plant-biotechnology, needs to be swiftly implemented in all walks of life. Achievements today in plant biotechnology have already surpassed all previous expectations, and the future is even more promising. The full realisation of the agricultural biotechnology revolution depends on both continued successful and innovative research and development activities and on a favourable regulatory climate and public acceptance. Biotechnology should be fully integrated with classical physiology and breeding: (1) as an aid to classical breeding, (2) for generation of engineered organisms, (3) for integration of microorganisms into agricultural production systems. Biotechnology is nowadays changing the agricultural and plant scene in three major areas: (1) growth and development control (vegetative, generative and reproduction/propagation), (2) protecting plants against the ever-increasing threats of abiotic and biotic stress, (3) expanding the horizons by producing specialty foods, biochemicals and pharmaceuticals.
 
Meiotic configurations of maize x Tripsacum F1 hybrid plants. (a) Diakinesis with 2n = 56. (b) Chromosomes of maize (external chromosomes in diakinesis) and Tripsacum (internal chromosomes in pachytene) disposed in two groups. (c) Anaphase I showing 7 and 8 univalent chromosomes in each pole. (d) Anaphase I showing 2 univalent chromosomes (arrowed) and bridges.  
Maize (2n = 40) x Tripsacum dactyloides (2n = 72) F1 hybrid plants (2n = 56) were obtained by embryo rescue and induction of somatic embryogenesis/organogenesis. Hybrid plants showed Tripsacum-like phenotypes, tolerance to stresses such as NaCl salinity and low temperatures. The more frequent meiotic configurations were 28 II (24%), 24 II + 2 IV (19%) and 26 II + 1 IV (12%), with an average per cell of 0,55 I + 25,18 II + 1,19 IV. Significant differences between plants were not observed. Pollen fertility ranged from 0% to 50%. After pollination with maize or Tripsacum, 20% of F1 plants have developed viable seeds, which originated the progeny. Thirty five percent of the progeny showed 2n = 56 chromosomes and F1 like-phenotypes, which suggests they have apomictic origin. The remaining plants were fertile and they showed maize-like phenotypes and different chromosome numbers (2n = 22, 24, 26, 28 and 30), because they kept the complete maize chromosome complement and some of the Tripsacum chromosomes. Meiotic cells showed pairing between chromosomes from both parental species, which suggests the possibility of genetic recombination between them.
 
Dendrogram showing genetic relationship between full-sib 2x and 4x Musa hybrids, and  
The triploid plantain landrace Obino l'Ewai (Musa spp., AAB genome) has been crossed with a wild diploid banana (M. acuminata subsp. burmannica var. 'Calcutta 4', AA genome) to generate full-sib diploid and tetraploid hybrids combining good agronomic performance and disease resistance. Microsatellite marker analysis of the parental genotypes confirmed the highly heterozygous nature of both parental genotypes. Comparative analysis of 2x and 4x full-sib hybrids with their parental genotypes indicated that tetraploid hybrids are generally more closely related to Obino l'Ewai than their diploid full-sibs. Based on VNTR analysis it is possible to identify those hybrids, which may be most useful in subsequent breeding of secondary triploid hybrids. There was a significant (P<0.05) negative association between the VNTR-based genetic similarity of hybrids to Obino l'Ewai and a phenotypic distance index based on eight agronomic descriptors. However, there was no association between the molecular genetic similarity of hybrids to Calcutta 4 and the respective phenotypic distance index. Many microsatellite markers generated an unexpectedly high number of amplification products from AA and AAB genotypes plus their progeny which may suggest the presence of a high frequency of loci duplication in both A and B genomes, in addition to the detection of heterozygous and/or homoeologous loci.
 
Gene cloning and transformation analysis. 
Detection of the A and B gene transcripts in the transformed tomato leaf tissues by primer extension. Lane 1 and 3 represent the reactions from plant 1 and 3 carrying pPCV701-AB. Primer P14 was used for the detection of the A gene transcripts; primer P21 was used for the detection of the B gene transcripts. Lane C was the reaction from a non-transformed plant. All the reactions contained 5 μg of total RNA isolated from the plant leaf tissues. 
Immuno detection of the alpha and beta polypeptides from the transformed plants using polyclonal anti-beta antibody. 
Cold tolerance test of the transformed tomato plants. 
Figures
L-DOPA is a useful drug for Parkinson's disease. This investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae . There was a marked difference between the mycelial morphology and pellet type of the parental and UV-irradiated mutant cultures. The mutant strain of Aspergillus oryzae UV-6 showed pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA since tyrosinase is an intracellular enzyme. The mutant was found to give 3.72 folds higher production of L-DOPA than the parental strain. The comparison of kinetic parameters was also done which showed greater ability of the mutant to yield L-DOPA (i.e. Yp/x 32.73 mg/mg with parent and 95.71 mg/mg with mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and qp, there was significant enhancement (p<0.0025-0.005) in these variables by the mutant strain of Aspergillus oryzae UV-7 over GCB-6 on all the rates.
 
"Shilajit" is a panacea in Ayurveda, the Indian traditional system of medicine. The major bioactives of "shilajit" have been identified as dibenzo-α-pyrones (DBPs), its oligomers and aminoacyl conjugated derivatives. These bioactive compounds play a crucial role in energy metabolism in all animal cells including those of man. 3-hydroxydibenzo-α-pyrone (3-OH-DBP), a key DBP component of "shilajit" is converted, among other products, to another active DBP derivative, viz. 3,8-hydroxydibenzo-α-pyrone, 3,8(OH)2-DBP, in vivo, when its precursor is ingested. 3,8(OH)2-DBP is then involved in energy synthesis in the mitochondria in the reduction and stabilization of coenzyme Q10 in the electron transport chain. As the chemical synthesis of 3,8(OH)2-DBP is a complex, multi-step process and economically not readily viable, we envisioned the development of a process using microorganisms for bioconversion of 3-OH-DBP to 3,8(OH)2-DBP. In this study, the biotransformation of 3-OH-DBP is achieved using Aspergillus niger, which was involved in the humification process on sedimentary rocks leading to "shilajit" formation. A 60% bioconversion of 3-OH-DBP to 3,8(OH)2-DBP and to its aminoacyl derivatives was achieved. The products were characterized and estimated by high performance liquid chromatography (HPLC), high performance flash chromatography (HPFC) and gas chromatography-mass spectrometry (GC-MS) analyses. Among the Aspergillus species isolated and identified from native "shilajit", A. niger was found to be the most efficient for this bioconversion.
 
Factors affecting radiocaesium uptake by plant roots . LMW* =Low molecular weight. 
Scattergram of 
Mobile mean of 137 Cs activity concentration in 
The uptake of radionuclides by plant roots constitutes the main pathway for the migration of radiocaesium from soil to humans, via food chain. In this study we assessed radiocaesium uptake by plant in order to piece together information on factors affecting uptake processes, particularly K supply and differential uptake among different plant species. Soil contaminated by the Chernobyl accident and forage from a semi-natural alpine grassland, situated in Tarvisio, Italy, were sampled during 1999. Under field conditions, (137)Cs uptake for Graminaceae and Taraxacum officinale seem to behave in a comparable way. Higher (40)K soil activity concentration leads to a lower (137)Cs plant uptake, suggesting an inhibitory pattern of potassium on radiocaesium plants uptake. For forage samples, a similar tendency was observed. We analyzed the influence of the ratio of (137)Cs/(40)K in soil on (137)Cs plant uptake. Under field conditions, the ratio observed varied in a range of 0.5 to 1.3. For most of the species, at higher (40)K soil concentration a lower (137)Cs uptake was observed, a fact that reflects the resulting effect of the complexity of factors controlling ion absorption from soil.
 
Cunninghamella elegans (UCP 542) grown in different culture media, after 96 hrs of time cultivation, at 28ºC, 150 rpm. (a) Biomass production. (b) Yield of chitin. (c) Chitosan extracted of dry weight biomass. Legend: SS (Sabouraud Sucrose), A (Medium proposed by Andrade et al.), H&A (Hesseltine and Anderson medium), MG (Malte glucose) and YB (Yam Bean medium)
Curve of growth, glucose and nitrogen consumption of Cunninghamella elegans (UCP 542) grown in yam bean medium at 28oC, 150 rpm, during 96 hrs of cultivation. 
Chitin (mg/g) and chitosan (mg/g) production from biomass of Cunninghamella elegans (UCP 542) grown in yam bean medium, at 28oC, 150 rpm during 96 hrs of cultivation. 
shows chitin and chitosan yield extracted, to each 24 hrs, from C. elegans (UCP 542) grown in yam beam medium during 96 hrs of cultivation. The best yields of the polysaccharides (mg per gram of dry mycelia biomass) are obtained with 48 hrs of culture for chitosan (66 mg/g or 6.6%) and with 72 hrs for chitin (440 mg/g or 44%). Similar results were reported to Tan et al. (1996), which studied different Zygomycetes strains and observed that
Microbiological processes were used for chitin and chitosan productions by Cunninghamella elegans (UCP 542) grown in a new economic culture medium. The assay was carried out to evaluate the growth of C. elegans using yam bean ( Pachyrhizus erosus L. Urban) medium, in different times of growth (24, 48, 72 and 96 hrs), incubated at 28°C in an orbital shaker at 150 rpm. The lyophilized biomass was determined by gravimetry. The polysaccharides were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, titration and viscosity. C. elegans grown in the yam bean medium and produced higher yields of biomass (24.3 g/ mL) in 96 hrs. The high level was chitosan (66 mg/g), and chitin (440 mg/g) were produced at 48 and 72 hrs of growth, respectively. The polysaccharides showed degree of deacetilation and viscosimetric molecular weight as: 6.2% and 3.25 x104 g/mol for chitin, and 85% and 2.72 x 104 g/mol for chitosan, respectively. The results obtained suggest high biotechnological potential of yam bean as an economic source to produce chitin and chitosan by C. elegans. In addition, the new medium using yam bean for production of the chitin and chitosan may be used for many purposes to reduce the cost price of fermentation processes.
 
Subcellular fractionation of immature sunflower seeds.
Biochemical characterization of sunflower transport percoll vesicles (PV, part a) and sucrose vesicles (SV, part b) fractionated from immature seeds at stages I2 and I3. Presence of proglobulins was analyzed under non reducing conditions with a serum specific to sunflower 11S globulins (11S). Association with the chaperon BiP was analyzed with BiP specific antibody. Sera specific to tonoplast intrinsic proteins α and δ (α-TIP and δ-TIP) were used to characterized the nature of transport vesicles (vacuolar markers). Association of proglobulins with the chaperon BiP was evaluated with BiP specific sera. The presence of vacuolar sorting receptors in PV and SV was studied with sera specific to Arabidposis, pumpkin and pea vacuolar sorting receptors (At-ELP, PV 72 and BP-80 sera, respectively, Part c) Endosplasmic reticulum (microsomes, M) and protein storage vacuoles (PSV) isolated from mature seeds were used as controls. Molecular weight markers (kD) are shown on the left.
Ultrastructure of immature sunflower cotyledons and immunolocalization of storage proteins and vacuolar sorting receptors in the transport vesicles. Formation of electron-dense aggregates containing storage proteins began to be observed in the ER 
Storage proteins are transported to a special storage compartments in seeds by Golgi dependent or independent pathways depending on the plant species. The aim of this work was to study the sunflower storage protein transport pathway and identified component of the sorting machinery. Immature sunflower seeds were analyzed by subcellular fractionation (using percoll and sucrose gradients) and electron microscopy. The vesicles isolated with percoll, have precursors of 11S globulins, α-TIP, δ-TIP, BiP, and two proteins that have homology to the pumpkin vacuolar sorting receptor PV72. Sucrose isolated vesicles have the same composition than percoll ones, except for the lack of BiP and the presence of only one protein that has reactivity with pea VSR BP80. Electronic micrographies of developing seeds show that the formation of electron dense aggregates starts in the endoplasmic reticulum, and that these aggregates are very abundant in the trans-Golgi apparatus, where release of dense vesicles happens. These vesicles contain a homolog of PV72 in their membranes. Storage proteins are also detected in multivesicular bodies whose membranes have reactivity with PV72 serum. All these results indicated that sunflower storage proteins are transported to protein storage vacuoles by a Golgi dependent pathway in a process in which homologous of PV72 are involved.
 
Citric acid fermentation of cane-molasses by submerged fermentation in 15 L stirred fermentor (working volume 9 L) was carried out. A hyper mutant strain of Aspergillus niger GCMC-7 was used in the present study which was obtained from the culture collection of our own labs. Ferrocyanide treated molasses [K4Fe(CN)6 200 ppm] medium containing sugar 150 g/l was employed as the basal fermentation medium. Different cultural conditions such as incubation temperature (30ºC), initial pH (6.0), air supply (1.0 l-1l-1min), agitation intensity (200 rpm) and time profile (144 h after inoculation) were optimised for enhanced citric acid production. Maximum amount of anhydrous citric acid obtained during the course of study was 106.65 g/l, with a sugar consumption of 107 g/l. Final pH, ferrocyanide concentration and dry cell mass were 2.1, 60 ppm and 16.5 g/l, respectively.
 
Effect of SAN-9785 on the growth of S. platensis and C. minutissima. Biomass of cultures growing in different concentration (mM) of SAN 9785 is shown as a function of time. 
Effect of SAN 9785 on the fatty acid profile of S. platensis.
Effect of SAN 9785 on the fatty acid profile of C. minutissima.
The accumulation of polyunsaturated fatty acids by algae Spirulina platensis and Chlorella minutissima was studied. Response of these organisms to the substituted pyridazinone, SAN 9785, an inhibitor of the long chain fatty acid desaturase, indicated that fatty acid synthesis and their desaturation were regulated differently in these organisms. While the pool of palmitic acid, the precursor for the unsaturated C18 fatty acids, was stringently maintained in the green alga C. minutissima, in the cyanobacterium S. platensis the level of palmitic acid was liberally maintained in spite of the enhanced accumulation of unsaturated C18 fatty acids.
 
PCR amplification of sample A, B, C, and D. The three first lanes of each sample are three repetitions of 15 ng of DNA each (A: lanes 1-3, B: 6-8, C: 15-17, D: 20-22) while the last two are repetitions of the sample with the addition of plasmid DNA (A: lanes 4 and 5, B: 9 and 10, C: 18 and 19, D: 23 and 24). Lane 11 is the plasmid DNA alone. Lanes 12 and 13 are the negative controls, GMO-free DNA and without DNA, respectively. Lanes 25 to 28 represent the calibration curve with 0.5, 1, 2, and 5% of GMO content, respectively. Lane 14 is the molecular size marker (100 bp DNA Ladder, Promega Biotech, USA).  
Dividing cells of leaves used as sources of explants from coffee plants (Coffea arabica cv. 'Catimor') and those of their derived calli were analyzed for mitotic aberrations. The studied tissues were prepared by squashing and stained with carbolfuchsin. A total of 1551 leaf and 4568 callus cells were surveyed. The majority (79%) of leaf and calli (75%) cells showed normal mitosis, however, cells with mitotic abnormalities were also found in both tissues. These included: polyploids, aneuploids, sticky chromosomes, double prophases and lagging chromosomes. Additionally, interphase cells with micronuclei or binucleated were also observed. The frequencies of these abnormalities were statistically different in calli and leaves. Calli showed a few other abnormalities such as c-mitosis, chained chromosomes, multipolar metaphases and chromosome bridges. Therefore, we conclude that these pre-existing abnormalities originate by errors in the process of normal mitosis in both leaves and in calli, and are therefore not caused by tissue culture conditions.
 
Biomass production in Erlenmeyer flask using RM with glucose and pectin in different proportions as CES. Total amount of CES was constant. 
Characterization of the growth of G. klebahnii cultivated in Erlenmeyers flasks using the RM with different CES.
Sugar extraction profile during enzymatic tissue maceration using PPase-SE. 
Time course of G. klebahnii culture in bioreactor before (a) and after (b) lemon peel addition. 
Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin.
 
Representations of the structural features of : (a) The ABA gene cluster in B. cinerea strain SAS56 (Siewers et al. 2006). (b) The ABA gene cluster in B. cinerea strain B05.10. (c) Protein GAG-POL of Boty-aba.
Analysis of the: (a) gag domain. (b) RT domain. (c) IN domain of the Boty-aba GAG-POL protein.
The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.
 
A strain of Absidia fusca was isolated from a pesticide-contaminated soil (Annaba, Algeria). The biotransformation capability of this strain towards two polycyclic aromatic hydrocarbons (PAHs): anthracene and fluoranthene was compared to that exhibited by another strain of A. fusca isolated from a non-contaminated milieu and considered as a control. The results obtained were statistically analyzed and showed that the strain isolated from the contaminated soil was more efficient than the control to remove anthracene from the medium, during all the kinetics (90% removed versus 45% after 24 hrs). Concerning fluoranthene, the amount removed by both strains was very high during the first 24 hrs however the control strain was slightly more efficient (94% versus 89%) while the results were similar for the two strains during the rest of the kinetics. This study reveals for the first time the potential interest of the species A. fusca for the bioremediation of PAHs.
 
Lanes 1 to 18 represent RAPD profiles of leaf DNA from 18 niger cultivars (Table 1) using primer OPM-6 (Table 2) Lane M, 1 kbp DNA ladder. 
Dendrogram showing genetic variability relationships between 18 niger cultivars. The Squared-Euclidean distance (horizontal axis) between objects is used as the distance measure; the clustering was performed following the method of Ward (1963). 
PCA of RAPD-generated DNA markers for 18 niger cultivars. 
Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 18 cultivars of niger from India. Total genomic DNA was extracted and subjected to RAPD analysis using 80 arbitrary 10-mer primers; 17 primers were selected, which yielded a total of 124 bands, 41.20% of them polymorphic. None of the primers produced unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidean Distance matrix which revealed a minimum genetic distance between cultivars JNC-6 and N-48 and a maximum distance between IGP-76 and JN-30. Based on the distance matrix, a cluster analysis was done using a minimum variance algorithm. The dendrogram generated, based on Ward’s method, grouped 18 niger cultivars into two major clusters. The first cluster consisted of early maturing cultivars (e.g. N-129 and N-134; 80-90 days), and the second of late maturing cultivars (e.g. GA-8 and GA-9; 135-145 days). The present study shows that there is high diversity among the niger cultivars tested and indicates the potential of RAPD markers for identification and maintenance of niger germplasm for crop improvement purposes.
 
Participants in the workshop for biosafety education in East Africa.
Barriers to biotechnology information transfer to non-academic stakeholders and possible solutions.
Development and deployment of genetically engineered crops requires effective environmental and food safety assessment capacity. In-country expertise is needed to make locally appropriate decisions. In April 2007, biosafety and biotechnology scientists, regulators, educators, and communicators from Kenya, Tanzania, and Uganda, met to examine the status and needs of biosafety training and educational programs in East Africa. Workshop participants emphasized the importance of developing biosafety capacity within their countries and regionally. Key recommendations included identification of key biosafety curricular components for university students; collaboration among institutions and countries; development of informational materials for non-academic stakeholders and media; and organization of study tours for decision makers. It was emphasized that biosafety knowledge is important for all aspects of environmental health, food safety, and human and animal hygiene. Thus, development of biosafety expertise, policies and procedures can be a stepping stone to facilitate improved biosafety for all aspects of society and the environment.
 
High seed quality is essential for optimum stand establishment in lettuce. As a result, it is necessary to have seed vigour tests that permit rapid, objective and accurate evaluation of seed quality. This study evaluated physical and physiological seed quality components of four seed lots of six lettuce varieties obtained from a commercial company. Seeds were evaluated for seedling emergence under greenhouse conditions, standard germination, seed physical aspects, the Saturated Salt Accelerated Aging (SSAA) test and the Seed Vigour Imaging System (SVIS). Results indicated that large-seeded lettuce varieties had higher percentage germination, higher SSAA values, higher SVIS index and more rapid and uniform greenhouse emergence. Black-seeded lettuce varieties possessed higher seed quality and less fungal invasion when evaluated by the SSAA test. The SVIS index significantly correlated with SSAA values and seedling emergence under greenhouse conditions suggesting it can be used as a measure of seed vigour. It is concluded that the SSAA and SVIS tests are practical and accurate determinants of lettuce seed quality and distinguish between high and poor quality lettuce seed lots.
 
Gene transfer is economically important and model fish species has produced a great impact in modern biology and biotechnology. Transgenic zebrafish ( Danio rerio ) were generated through the co-injection of a GFP-expressing plasmid and an "all fish" transgene composed by the carp β-actin promoter and the chromosomal tilapia ( Oreochromis hornorum ) growth hormone gene. The GFP expression was a good indicator of stable transformation and allowed for high efficiency selection of transgenic fish. Transgenic F1 zebrafish grew 20% faster than full sibling non-transgenic controls.
 
The pattern of catalase activity in YPD medium at different hours of cultivation. Lane 1. Catalase marker enzyme (Merck KgaA, Darnstadt, Germany ); Lane 2. At 6 h of cultivation; Lane 3. At 12 h of cultivation, at 48 h of cultivation, at 72 h of cultivation. Lane 4. at 48h of cultivation Lane 5. at 72h of cultivation.
Determination of purity of mitochondrial fractions, obtained from Saccharomyces cerevisiae strains NBIMCC 582, NBIMCC 583 and NBIMCC 584 trough measurement specific activities of cytosolic 1 , peroxisomal 2 and mitochondrial 3 marker enzymes. Strains NBIMCC 582 NBIMCC 583 NBIMCC 584 NBIMCC 582 NBIMCC 583 NBIMCC 584
Activity of mitochondrial catalase, Mn SOD and Cu/Zn SOD of Saccharomyces cerevisiae during cultivation in YPD medium: a. strain NBIMCC 582; b. strain NBIMCC 583; c. strain NBIMCC 584.
Dependence of HRm on molecular weight of marker proteins (HMW Native, Amersham Pharmacia Biotech) on 10% PAGE and drawn equation, describing the curve.
Figures
Genetic variability fuels crop improvement and has sustained crop evolution and adaptation for thousands of years. It is a vital resource for humanity's future survival given that increases in crop production will most likely come from higher yields per unit area rather than from new crop lands. Intensive plant breeding using scientific methods during much of the 20th century has led to significant gains in productivity for most major world crops. However, the cost of these achievements has been narrower genetic variability within the elite gene pool, increased genetic uniformity and vulnerability in crops, and erosion of native genetic resources (Lee, 1998). The consequences of narrow genetic pools have been disastrous in the past. Examples abound: the infamous Irish famine of 1850 caused by a genetically uniform potato crop susceptible to blight; the famine triggered in India in 1943 by the brown spot disease of rice. More recently the Southern corn blight that struck the United States corn industry in 1970 caused over a billion dollars worth of damage (Horsfall, 1972). Future consequences will be harsh unless steps are taken to reverse the dangerous trend in genetic erosion of our main sources of food. Furthermore, it is suspected that reduced genetic variability in most major crops is responsible for the decrease in genetic gain for yield (Lee, 1998). Among the important causes for this narrow genetic base is that crop species have undergone genetic bottlenecks during the domestication and breeding processes (Tanksley and McCouch, 1997). In the past it has been difficult to use exotic germplasm because useful genes are often linked to genes controlling undesirable traits and there were no tools to go after specific genes or genomic regions. The development of densely populated genetic linkage maps, marker assisted selection, expressed sequence tags, chromosome walking and gene cloning, have changed the scenario and it is now possible to transfer specific genes or loci to cultivated plants. In developed countries, like the United States, major efforts are underway to use these tools to mine, capture and transfer useful genes from exotic germplasm to crop plants (Tanskely and Nelson, 1996; Tanksley and McCouch, 1997). Also the completion of the Arabidopsis genome sequencing project will result in a number of new techniques (Sommerville and Dangi, 2000) that will enhance our understanding of genomes and, therefore, our ability to exploit exotic genetic resources. Not surprisingly increased research and gene discovery will correspondingly increase the value of exotic germplasm, meaning land races and wild ancestors of cultivated plants.
 
RAPD marker profiles of 10 landraces of V. umbellata generated by primer OPBB1 in 1.5 per cent agarose gel. 
ISSR marker profiles of 10 landraces of V. umbellata generated by primer UBC864 in 1.5 per cent agarose gel.
ISSR primers used to detect polymorphism, number of bands for polymorphism between landraces per primer.
Dendrogram generated using UPGMA analysis, showing relationships between rice bean landraces, using RAPD, ISSR and combining RAPD and ISSR data. (a) RAPD. (b) ISSR. (c) RAPD and ISSR. 
Vigna umbellata (Thunb.) Ohwi and Ohashi commonly known as rice bean or climbing mountain bean is under-exploited tropical legume. Genetic variation between 10 landraces of rice bean was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. Among these markers, RAPD primers generated 987 amplification products of which 719 were polymorphic and ISSR markers produced 479 amplification products, out of which 296 were polymorphic. RAPD fingerprinting detected more polymorphic loci (70.30%) than the ISSR fingerprinting (61.79%). Mean PIC (polymorphic information content) for each of these marker systems (0.243 for RAPD and 0.203 for ISSR) suggested that both the marker systems were equally effective in determining polymorphisms. The dendrograms constructed using RAPD and ISSR marker systems were highly correlated with each other as revealed by high Mantel correlation (r = 0.95). Pair wise similarity index values ranged from 0.530 to 0.782 (RAPD), 0.608 and 0.862 (ISSR) and 0.559 to 0.777 (RAPD and ISSR) and mean similarity index value of 0.677, 0.729 and 0.694 for RAPD, ISSR and combined data respectively. RAPD and ISSR marker systems were found to be useful for the genetic diversity studies in V. umbellata andidentify variation within landraces.
 
In this study, a total of 14 agronomic traits, five AFLP primer combinations and ten SSR loci were used to describe and to classify a group of Tunisian olive genotypes into groups based on molecular profiles and agronomic traits. The analysis of variance of the agronomical data revealed significant differences among accessions for all measured traits. The mean phenotypic dissimilarity (0.34 with a range of 0.08-0.6) was low in comparison to dissimilarity calculated using AFLP (0.50 with a range of 0.16-0.70) and SSR markers (0.76 with a range 0.35-0.94). The correlation between the agronomical dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers was very weak: 0.156 (p = 0.05) and 0.185 (p = 0.05), respectively. The SSR-AFLP dendrogram based on unweighted pair-group cluster analysis using Jaccard’s index revealed that the genetic diversity was predominantly structured according to fruit size. A trend of clustering together of accessions originating from the same or adjacent regions was also observed. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Tunisia and providing also additional information that could form the basis for the rational design of breeding programs.
 
The persistence of CryIAb protein rhizosecreted in soil is important in the assessment of its environmental risk. Here we report that CryIAb protein from transgenic maize does not accumulate at high levels in soils. Levels of CryIAb protein rhizosecreted by three maize transgenic events (BT11, MON810 and 176) were studied in hydroponic cultures and found only in the MON810 and BT11 events but not in event 176 or control plants. Under field conditions, the cryIAb gene and a basal level of CryIAb protein was detected in soils from plots cultivated with transgenic and non-transgenic maize, possibly from Bacillus thuringiensis present in the soils.
 
Top-cited authors
Helena Freitas
  • University of Coimbra
Walter Maccheroni
  • University of São Paulo
Joao Lucio Azevedo
  • University of São Paulo
Jose Odair Pereira
  • Federal University of Amazonas
Welington Luiz Araújo
  • University of São Paulo