Growth optimization of a probiotic candidate,<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4, in milk medium using response surface methodology

Bifidobacterium pseudocatenulatum G4, a wild strain isolated from infant stools that has previously exhibited probiotic characteristics, was used in this study. The aim of this research was to improve the growth potential of this strain in milk-based medium. An initial screening study using a 2³ full factorial design was carried out to identify the impact on biomass production of the various components of the medium which were skim milk, yeast extract, and glucose. Statistical analysis suggested that yeast extract had a significant positive effect on viable cell count whereas glucose had a negative effect. Response surface methodology (RSM) was then applied to optimize the use of skim milk and yeast extract. A quadratic model was derived using a 3² face-centered central composite design to represent cell mass as a function of the two variables. The optimized medium composition was found to be 2.8% skim milk and 2.2% yeast extract, w/v. The optimized medium allowed a maximum biomass of 9.129 log₁₀ cfu/mL, 3.329 log units higher than that achieved with 10% skim milk, which is the amount commonly used. The application of RSM resulted in an improvement in the biomass production of this strain in a more cost-effective milk medium, in which skim milk use was reduced by 71.8%.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

Ligand binding to six recombinant pheromone-binding proteins of<Emphasis Type="Italic"> Antheraea polyphemus</Emphasis> and<Emphasis Type="Italic"> Antheraea pernyi</Emphasis>

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, (E,Z)-6,11-hexadecadienyl acetate (³H-Ac1) and (E,Z)-6,11-hexadecadienal (³H-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone (³H-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of ³H-Ac1. In competition experiments with ³H-Ac1 and the third unlabelled pheromone component, (E,Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound ³H-Ald only. All of the six PBPs strongly bound ³H-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for ³H-Ac1 and ³H-Ald bound to PBPs. No pH influence was found for ³H-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1–3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation.Abbreviations Ac1 (E,Z)-6,11-hexadecadienyl acetate - Ac2 (E,Z)-4,9-tetradecadienyl acetate - Ald (E,Z)-6,11-hexadecadienal - AMA 1-amino-anthracene - cpm counts per min - DTFP decyl-thio-1,1,1-trifluoropropanone - ES-MS electrospray mass spectrometry - OH (E,Z)-6,11-hexadecadienol - PAGE polyacrylamide gel electrophoresis - PCR polymerase chain reaction - PBP pheromone-binding protein - SDS sodium dodecyl sulphate - Z-11 OH Z-11 hexadecenolCommunicated by G. Heldmaier     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Effects of pH on NaCl tolerance of American elm (<Emphasis Type="Italic">Ulmus americana</Emphasis>) seedlings inoculated with <Emphasis Type="Italic">Hebeloma crustuliniforme</Emphasis> and <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

In the present study, we investigated the effects of pH treatments on NaCl tolerance in mycorrhizal and non-mycorrhizal American elm. American elm (Ulmus americana) seedlings were inoculated with Hebeloma crustuliniforme, Laccaria bicolor or with both mycorrhizal fungi and subsequently subjected to different pH solutions (pH 3, 6 and 9) containing 0 mM (control) and 60 mM NaCl for 4 weeks. Inoculation with the mycorrhizal fungi did not have a large effect on seedling dry weights when the pH and NaCl treatments were considered independently. However, when the inoculated seedlings were treated with 60 mM NaCl at pH 3 or 6, shoot to root ratios and root hydraulic conductivity were higher compared with non-inoculated plants, likely reflecting changes in seedling water flow properties. At pH 6, transpiration rates were about twofold lower in non-inoculated plants treated with NaCl compared with non-treated controls. For NaCl-treated H. crustuliniforme- and L. bicolor-inoculated plants, the greatest reduction of transpiration rates was at pH 9. Treatment with 60 mM NaCl reduced leaf chlorophyll concentrations more in non-inoculated compared with inoculated plants, with the greatest, twofold, decrease occurring at pH 6. At pH 3, root Na concentrations were higher in inoculated than non-inoculated seedlings; however, there was no effect of inoculation on root Na concentrations at pH 6 and 9. Contrary to the roots, the leaves of inoculated plants had lower Na concentrations at pH 6 and 9, but not at pH 3. The results point to an interaction between ECM fungi and root zone pH for salt tolerance of American elm.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Mutation of<Emphasis Type="Italic">gyrA</Emphasis> and<Emphasis Type="Italic">parC</Emphasis> in clinical isolates of<Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> and its relationship with antimicrobial drugs resistance in Taiwan

Acinetobacter baumannii is a species of non fermentative Gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Herein we have determined the mutation frequency of two hot spot residue 83 in gyrA of gyrase and residue 80 in parC of topoisomerase IV and performed a comparative screen the drug resistance ability in 128 clinical isolates ofAcinetobacter baumannii in Taiwan region. Low frequency of mutation was found (11.7%, 11.7%, and 10.2% ingyrA, parC, or both, respectively). Mutation of these sites was not correlated with drug resistance. Our study suggested that mutation ofgyrA andparC may play a minor role in quinolone resistance and other mechanisms may contribute to the drug resistance ofA. Baumannii.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel     

Horticultural characterization of<Emphasis Type="Italic"> Angelonia salicariifolia</Emphasis> plants transformed with wild-type strains of<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis>

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Sexual identification of<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>

The absence of sexual dimorphism in Pyrenean desmanGalemys pyrenaicus (E. Geoffroy, 1811) makes it difficult to sex live animals. This paper presents a method of sexing desmans observing their external genitals. Based in a sampling of 49 animals from the north of Spain, it was possible to identify the penis of males, independently of the age or season.     

pH stability of penicillin acylase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The inactivation kinetics of penicillin acylase from Escherichia coli have been investigated over a wide pH range at 25 and 50 degrees C. The enzyme was very stable in neutral solutions and quickly lost its catalytic activity in acidic and alkaline solutions. In all cases, the inactivation proceeded according to first order reaction kinetics. Analysis of the pH dependence of enzyme stability provides evidence that stable penicillin acylase conformation is maintained by salt bridges. Destruction of the salt bridges due to protonation/deprotonation of the amino acid residues forming these ion pairs causes inactivation by formation of the unstable "acidic" EH(4)(3+), EH(3)(2+), EH(2)(+) and "alkaline" E(-) enzyme forms. At temperatures above 35 degrees C penicillin acylase apparently undergoes a conformational change that is accompanied by destruction of one of these salt bridges and change in the catalytic properties.