Impact of heat-inactivated <Emphasis Type="Italic">Lactobacillus casei</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strains on cytokine responses in whole blood cell cultures of children with atopic dermatitis

Heat-inactivated Lactobacillus casei LOCK 0900, L. casei LOCK 0908 and Lactobacillus paracasei LOCK 0919 strains, applied to blood cell cultures obtained from children with atopic dermatitis induced production of anti-allergic T_H1 cytokines (interleukin-12, interleukin-18, interferon-γ, tumor necrosis factor-α) and regulatory transforming growth factor-β₁), but did not stimulate pro-allergic interleukin-5. The lactobacilli-mixture remarkably enhanced the T_H1 response compared to single strains. This synergistic effect was not observed for transforming growth factor-β₁. In contrast, the amount of interleukin-10 was found to be considerably lower when cells were stimulated with lactobacilli-mixture compared to single strains. The mixture of Lactobacillus strains represents a probiotic bacterial preparation modulating in vitro cytokine profile of allergic children towards anti-allergic T_H1 response.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

<Emphasis Type="Italic">Singulispaera mucilagenosa</Emphasis> sp. nov., a novel acid-tolerant representative of the order <Emphasis Type="Italic">Planctomycetales</Emphasis>

Two novel strains of budding bacteria, Z-0071^T and Z-0072, were isolated from dystrophic humified waters formed by xylotrophic fungi in the course of spruce wood degradation. The cells of both strains are coccoid (0.95–1.80 μm), nonmotile, single or arranged in pairs. The cells have a complex system of intracellular membranes and are covered with fimbriae and surrounded by a mucous capsule up to 0.3 μm thick. Both strains are aerobic organoheterotrophic, mesophilic, and acid-tolerant microorganisms that are able to grow under microaerobic conditions. They utilize N-acetyl-glucosamine, carbohydrates, and lactate as growth substrates. The strains grow in a pH range of 4.0–7.5 with an optimum at 6.0–6.5. The temperature range for growth is 4–30°C, with an optimum at 25–28°C. Strains Z-0071^T and Z-0072, inhabitants of dystrophic low-mineral waters, are NaCl-sensitive: the NaCl content in the media above 0.5 g/l inhibited growth. The main fatty acids of strains Z-0071^T and Z-0072 are C_16:0, C_18:1ω9c, and C_{18:2ω9c, 12c}. The DNA G + C base content is 51.2–51.7 mol %. The sequences of the 16S rRNA gene fragments (1310 bp) of strains Z-0071^T and Z-0072 were found to be identical. The obtained sequences showed a 94.3% similarity with the sequences of the type strain of the most closely related species Singulisphaera acidiphila MOB10≅T. The phenotypic and phylogenetic properties of strains Z-0071^T and Z-0072 support classification of these strains within the genus Singulisphaera as a new species Singulisphaera mucilagenosa sp. nov., with the type strain Z-0071^T (VKM B-2626).     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pisum sativum in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm⁻³ kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T₀ putative transformants and their seed progenies (T₁ to T₃ generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T₀ – T₃ plants were morphologically normal and fertile.This research was supported by the National Agency for Agricultural Research (grants No. QE 0046 and QF 3072) and Ministry of Education of the Czech Republic (grant No. ME 433).     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

The <Emphasis Type="Italic">dnaK</Emphasis> gene as a molecular marker for the classification and discrimination of the <Emphasis Type="Italic">Lactobacillus casei</Emphasis> group

It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.     

<Emphasis Type="Italic">Flavobacterium koreense</Emphasis> sp. nov., <Emphasis Type="Italic">Flavobacterium chungnamense</Emphasis> sp. nov., and <Emphasis Type="Italic">Flavobacterium cheonanense</Emphasis> sp. nov., isolated from a freshwater reservoir

Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6–99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42^T, ARSA-103^T, and ARSA-108^T. The isolates contained menaqui-none-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:0 3-OH, iso-Ci_15:1 G, and iso-C_16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4–33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42^T =KCTC 23182^T =JCM 17066^T =KACC 14969^T), Flavobacterium chungnamense sp. nov. (strain ARSA-103^T =KCTC 23183^T =JCM 17068^T =KACC 14971^T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108^T =KCTC 23184^T =JCM 17069^T =KACC 14972) are proposed.     

+<Emphasis Type="Italic">276G</Emphasis>><Emphasis Type="Italic">T</Emphasis> single nucleotide polymorphism in the adiponectine (<Emphasis Type="Italic">APM1</Emphasis>) gene in donors from the Kharkiv population

+276G > T polymorphism in the adiponectine gene (APM1) was studied in 103 samples obtained from donors in the Kharkiv population (men/women: 64/38; 70 Ukrainian, 33 Russian). The T- and G-allele frequencies did not significantly differ between either men and women or between Russians and Ukrainians and comprised p _T = 0.55 and p _G = 0.45 in the general group. The genotype distribution deviated from the Hardy-Weinberg equilibrium: the heterozygote frequency was 1.55 times higher compared to the selectively neutral value, while the TT- and GG-homozygote frequencies were 0.55 and 0.33 of the equilibrium value, respectively.     

Functional Characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum</Emphasis> F1

In this study, Lactobacillus fermentum (L. fermentum) F1 reduced cholesterol 48.87%. The strain was screened from cattle feces using an API 50 CHL system and the 16S rRNA sequence contrasting method. L. fermentum F1 showed acid and bile tolerance, and antimicrobial activity against pathogenic Escherichia coli and Staphylococcus aureus. L. fermentum F1 deconjugated 0.186 mM of free cholalic acid after it was incubated at 37°C in 0.20% sodium taurocholate (TCA) broth for 24 h. Heat-killed and resting cells of L. fermentum F1 showed small amounts of cholesterol removal (6.85 and 25.19 mg/g, respectively, of dry weight) compared with growing cells (33.21 mg/g of dry weight). The supernatant fluid of the broth contained 50.85% of the total cholesterol, while the washing buffer and cell extracts had 13.53 and 35.39%, respectively. These findings suggest that L. fermentum F1 may remove cholesterol by co-precipitating with deconjugated bile salt, assimilating with cells and by incorporation into cellular membranes. Cholesterol assimilated by cells held 72.0% of the reduced cholesterol, while 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells.     

Proposal of three novel species of soil bacteria, <Emphasis Type="Italic">Variovorax ureilyticus,Variovorax rhizosphaerae</Emphasis>, and <Emphasis Type="Italic">Variovorax robiniae</Emphasis>, in the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>

Three novel bacterial strains (UCM-2^T, UCM-G28^T, and UCM-G35^T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C_16:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), and C_17:0 cyclo as the major fatty acids, and C_10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2^T, UCM-G28^T, and UCMG35^T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2^T was most closely related to Variovorax soli NBRC 106424^T, whereas strains UCM-G28^T and UCM-G35^T were most similar to Variovorax ginsengisoli Gsoil 3165^T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2^T (= KACC 18899^T = NBRC 112306^T), UCMG28^T (= KACC 18900^T = NBRC 112307^T), and UCM-G35^T (= KACC 18901^T = NBRC 112308^T), respectively.     

Acclimation of photosynthesis to temperature in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Plants differ in how much the response of net photosynthetic rate (P _N) to temperature (T) changes with the T during leaf development, and also in the biochemical basis of such changes in response. The amount of photosynthetic acclimation to T and the components of the photosynthetic system involved were compared in Arabidopsis thaliana and Brassica oleracea to determine how well A. thaliana might serve as a model organism to study the process of photosynthetic acclimation to T. Responses of single-leaf gas exchange and chlorophyll fluorescence to CO₂ concentration measured over the range of 10–35 °C for both species grown at 15, 21, and 27 °C were used to determine the T dependencies of maximum rates of carboxylation (V_Cmax), photosynthetic electron transport (J_max), triose phosphate utilization rate (TPU), and mesophyll conductance to carbon dioxide (g’_m). In A. thaliana, the optimum T of P _N at air concentrations of CO₂ was unaffected by this range of growth T, and the T dependencies of V_Cmax, J_max, and g’_m were also unaffected by growth T. There was no evidence of TPU limitation of P _N in this species over the range of measurement conditions. In contrast, the optimum T of P _N increased with growth T in B. oleracea, and the T dependencies of V_Cmax, J_max, and g’_m, as well as the T at which TPU limited P _N all varied significantly with growth T. Thus B. oleracea had much a larger capacity to acclimate photosynthetically to moderate T than did A. thaliana.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Ability of <Emphasis Type="Italic">Lactobacillus</Emphasis> to inhibit enteric pathogenic bacteria adhesion on Caco-2 cells

The antagonistic effects of Lactobacillus against pathogenic bacteria were evaluated in vitro on cultured Caco-2 cells. Lactobacilli were added simultaneously with enteric pathogenic strains (enterotoxigenic Escherichia coli K88 or Salmonella typhimurium SARB21 and SL1344), before pathogenic strains and after pathogenic strains for competition, exclusion and displacement assays. The six lactobacilli significantly limited the adhesion and invasion of the pathogenic bacteria. In the simulating competition and exclusion assays, the adhesion of pathogenic strains was reduced by Lactobacillus strains significantly, whereas the inhibiting effect on pathogenic strains adhesion was a little weaker in the displacement assay. Furthermore, we found that the antagonistic effects of lactobacilli against K88, SARB21, and SL1344 were various. Strain R4 showed a strong inhibitory effect on the adhesion of K88 to Caco-2 cells. In the competition assay of R4, the number of viable cell-associated K88 (3.84 ± 0.10 log CFU/well) was much lower than the control group without Lactobacillus (5.98 ± 0.02 log CFU/well). Compared to the control group (6.07 ± 0.02 log CFU/well), the six Lactobacillus strains all performed strong antagonistic effects against SL1344, particularly D17 showed a higher inhibitory effect in the displacement assays (4.15 ± 0.04 log CFU/well). These results implied that several Lactobacillus strains might be useful for protecting against enteric pathogenic infection.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

<Emphasis Type="Italic">Sphingomonas jejuensis</Emphasis> sp. nov., isolated from marine sponge <Emphasis Type="Italic">Hymeniacidon flavia</Emphasis>

A Gram-negative, non-motile, rod shaped, and orange-pigmented chemoheterotrophic bacterium, strain MS-31^T was isolated from the marine sponge Hymeniacidon flavia, collected from near Jeju Island, Korea. The Strain MS-31^T was subjected to a polyphasic taxonomic study. The phylogenetic analysis based on the 16S rRNA gene sequences revealed that the novel isolate could be affiliated within the genus Sphingomonas. The strain MS-31^T showed 95.6% of 16S rRNA gene sequence similarity with the most closely related species Sphingomonas koreensis JSS26^T. The DNA G+C content of the strain MS-31^T was 69.4 mol%. The major isoprenoid quinone was ubiqunone 10 and predominant cellular fatty acids were summed feature 7 (comprising C_18:1 ω7c, C_18:1 Ω9t and/or C_18:1 ωl2t, 39.7%), C_16:0 (16.3%), C_14:0 2OH (15.9%) and summed feature 3 (comprising C_16:1 ω7c and/or C_15:0 iso 2OH, 11.7%). The polar lipids were sphingoglycolipid, phosphatidyletha-nolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified glycolipid. Based on the evidence from the polyphasic taxonomic study, the strain should be classified as a new species of the genus Sphingomonas. As a result, the name Sphingomonas jejuensis sp. nov. (type strain MS-31^T =KCTC 23321^T =NBRC 107775^T) is proposed.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

<Emphasis Type="Italic">In vitro</Emphasis> assessment of immunomodulating activity of the two <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from traditional fermented milk

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacillus coryniformis subsp torquens T3L (L. coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 10⁶ c.f.u. ml⁻¹, cell wall at 20 μg protein ml⁻¹ and DNA at 50 μg DNA ml⁻¹ of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.