Application of fluorescence-based resistance gene analog analysis for genotyping plant genetic resources

Conventional approaches for detecting disease resistance gene analogs (RGAs) in plants are based on agarose gels or on polyacrylamide gel electrophoresis (PAGE) in combination with silver staining or radioactive labeling. A modified method for RGA analysis has been developed by using fluorescence-labeled primers for PCR amplifications. The amplified fragments are detected by denaturing PAGE using an automated laser fluorescence DNA sequencer and analyzed by fragment analysis software. This technique is not limited to specific plant species and is suitable for high-throughput genotyping plant genetic resources. We demonstrate here the efficiency of this method for comparison of RGA patterns in diverse plant species and for genotyping of natural populations of the wheat progenitor, Triticum dicoccoides. Revisions requested 8 October 2004; Revisions received 15 November 2004     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.     

A flow-cytometric gram-staining technique for milk-associated bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

Use of plant protein hydrolysates for varicella virus production in Serum-Free Medium

Serum-free media containing no animal-derived components were assessed for their efficacy to produce live attenuated varicella virus. Serum-free medium containing an ultrafiltrate of soy protein acid hydrolysate and lipid resulted in a viral production yield comparable to media containing fetal bovine serum, indicating that varicella virus can be produced without the risk of contamination associated with the use of bovine serum. Revisions requested; Revisions requested 20 December 2004; Accepted 21 December 2004     

Mediated electrochemical measurement of the inhibitory effects of furfural and acetic acid on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Candida shehatae</Emphasis>

The toxic effects of furfural and acetic acid on two yeasts, Saccharomyces cerevisiae and Candida shehatae, were evaluated using an electrochemical method. Intracellular redox activities were lowered by 40% and 78% for S. cerevisiae and C. shehatae, respectively, by 8 g furfural l^–1, and by 46% and 67%, respectively, by 8 g acetic acid l^–1. The proposed method can accurately measure the effects of inhibitors on cell cultures.Revisions requested 27 September 2004/17 November 2004; Revisions received 15 November 2004/10 December 2004     

A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

High-yield conversion of (<Emphasis Type="Italic">R</Emphasis>)-2-octanol from the corresponding racemate by stereoinversion using <Emphasis Type="Italic">Candida rugosa</Emphasis>

Whole cells of Candida rugosa catalyzed the conversion of (R)-2-octanol from the corresponding racemate with the optical purity of 97% e.e. and yield of 92% in 10 h. The product was formed through a stereoinversion involving enantioselective oxidation and asymmetric reduction with 2-octanone as the intermediate.Revisions requested 22 September 2004; Revisions received 2 November 2004     

Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of <Emphasis Type="Italic">Saussurea medusa</Emphasis>

Three previously established cell lines (yellow, red and white) of Saussurea medusa were investigated for jaceosidin and hispidulin production. Maximum yields of the jaceosidin and hispidulin were obtained in the red cell line at 75 ± 0.41 and 6.4 ± 0.31 mg l^-1. Production of jaceosidin and hispidulin correlated with the sizes of compact callus aggregates (CCA) and cellular viability. In the red cell line, the sizes of CCA were predominantly of 2–4mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.Revisions requested 3 September 2004/27 October 2004; Revisions received 22 October 2004/18 November 2004     

Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein

A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.Revisions requested/16 November 2004; Revisions received 12 November 2004/14 December 2004     

An improved method for the enzymatic transformation of nucleosides into 5′-monophosphates

An improved method to transform nucleosides into 5-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn²⁺. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5-monophosphates were 41–91% (mol).Revisions requested 22 September 2004; Revisions received 11 October 2004     

Acid hydrolysis and quantitative determination of total hexosamines of an exopolysaccharide produced by <Emphasis Type="Italic">Citrobacter</Emphasis> sp.

During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 m HCl at 115 °C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol^–1 and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson–Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l^–1 (29% of crude EPS) after 96 h of fed-batch culture.Revisions requested 18 August 2004; Revisions received 2 November 2004     

Cloning and expression of human lectin-like oxidized low density lipoprotein receptor-1 in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

Lectin-like oxidatively-modified LDL receptor-1 (LOX-1) is a major receptor for oxidized low-density lipoprotein (oxLDL) in aortic endothelial cells. Human LOX-1 (hLOX-1) gene (cDNA) was cloned from the monocytic leukemic cell line THP-1 and expressed in Pichia pastoris. The recombinant protein (rhLOX-1) was purified by his-tag affinity chromatography. Preliminary identification was performed by Western blot analysis and a ligand-receptor binding assay showed that the protein had specific oxLDL-binding activity.Revisions requested 21 September 2004; Revisions received 10 November 2004     

Production of extracellular polymeric substances in anoxic/oxic process with zeolite carriers for nitrogen removal

During the hydraulic retention time (HRT) from 12 to 6 h, the production of extracellular polymeric substance (EPS) decreased from 82 to 70 EPSmg volatile suspended solids (VSS) g^-1 in an anoxic/oxic (A/O) reactor with zeolite carriers. Added zeolite could successfully control the EPS production by the formation of stable biofilm and the maintenance the lower chemical oxygen demand/ammonium (C/N) ratio on its surface.Revisions requested 20 July 2004; Revisions received 7 september 2004     

Production of an anti-fungal substance for biological control of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> causing phytophthora blight in red-peppers by <Emphasis Type="Italic">Streptomyces halstedii</Emphasis>

The culture broth of Streptomyces halstedii AJ-7 suppressed the growth of Phytophthora capsici, which causes phytophthora blight in red-peppers, with less than 1% survival of the pathogen after 12 h of treatment. The low molecular fraction ( 10 kDa) of the culture broth retained anti-fungal activity against P. capsici after being held at 100 °C for 6 h.Revisions requested/26 August 2004; Revisions received 16 August 2004/10 December 2004     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Perinuclear translocation of hsp 27 in shear stress exposed human endothelial cells

Expression of hsp 27 in human umbilical vein endothelial cells exposed to a shear stress was investigated. Using immunostaining, it was concluded that shear stress results in perinuclear translocation of hsp 27. Polymerization of actin microfilaments plays an important role in this process.Revisions requested 11 October 2004; Revisions received 2 February 2005     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004     

Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 g g^-1 dry mycelia and the A₂₆₀/A₂₈₀ ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A₂₆₀/A₂₈₀ ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.Revisions requested 8 September 2004; Revisions received 1 November 2004     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005     

Enzymatic Synthesis and Antioxidant Properties of <Emphasis Type="SmallCaps">L</Emphasis>-Ascorbyl Oleate and <Emphasis Type="SmallCaps">L</Emphasis>-Ascorbyl Linoleate

L-Ascorbyl oleate and L-ascorbyl linoleate were synthesized by an immobilized lipase from Candida antarctica with yields of 38% and 44%, respectively. L-Ascorbyl oleate was stable in sterile culture medium over 12 h at 37 °C but L-ascorbyl linoleate degraded by 17%. Ascorbyl oleate had a better protective effect on human umbilical cord vein endothelial cells treated with H₂O₂ than of L-ascorbic acid-2-phosphate-6-palmitate (Asc2P6P).Revisions requested 21 July 2004/26 August 2004; Revisions received 20 August 2004/27 September 2004