Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Colonization patterns of mycorrhizal fungi associated with two rare orchids, <Emphasis Type="Italic">Cephalanthera</Emphasis> <Emphasis Type="Italic">falcata</Emphasis> and <Emphasis Type="Italic">C. erecta</Emphasis>

We investigated the spatial distribution and taxonomic identity of mycorrhizal fungi colonizing the root systems of two threatened Cephalanthera species, C. falcata and C. erecta, in naturally regenerated forests. Peloton formation was observed in both plant species, confirming the existence of orchid mycorrhizas. For C. falcata, mycorrhization was significantly different among individuals, ranging from 14 to 63%, and no significant difference among C. erecta individuals was detected (57–68%). Mycorrhization among three growth directions of roots and between orchid species was not significantly different. The spatial distribution of mycorrhizas in both orchids showed significant differences, being most frequent at an apical position. Based on DNA sequencing and phylogenetic analyses, we inferred that the families Thelephoraceae and Sebacinaceae were mycobionts for C. falcata and Thelephoraceae for C. erecta. Our findings indicated that mycorrhizal colonization occurs at a distal position from the base of these orchid root systems and that mycorrhizal fungi are restricted to few ectomycorrhizal fungal families.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

In vitro growth response of <Emphasis Type="Italic">Phytophthora cactorum</Emphasis>, <Emphasis Type="Italic">P. nicotianae</Emphasis> and <Emphasis Type="Italic">P.</Emphasis> × <Emphasis Type="Italic">pelgrandis</Emphasis> to antibiotics and fungicides

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

Peripheral Assessment of the Genes <Emphasis Type="Italic">AQP4</Emphasis>, <Emphasis Type="Italic">PBP</Emphasis> and <Emphasis Type="Italic">TH</Emphasis> in Patients with Parkinson’s Disease

Parkinson’s disease (PD) typically appears in late middle-aged and in elderly persons progressing over a period of several years. The characteristic pathological features of PD patients include defective motor function and cognitive function affecting the quality of life of PD patients. Oxidative stress is considered to a play a central role along with various other factors in the pathogenesis of PD and the incidence and prevalence of the disease is incessantly increasing worldwide. The objective of the current study was to assess mRNA expressional changes of AQP4, TH and PBP in blood samples of control and patients with PD. The study included 30 healthy controls and 90 PD patients subjected to treatment through the entire period of the study. RNA isolation was carried out using blood samples of the subjects recruited in the study and used for RT-PCR analysis of AQP4, TH as well as PBP. The mRNA expressions of AQP4 and TH were found to be reduced whereas that of PBP was found to be elevated when compared with those of healthy control samples. The statistically analysed data were presented which could be helpful for appreciation of PD pathology reflecting in the blood samples of PD population.     

Effect of <Emphasis Type="Italic">POX3</Emphasis> gene disruption using self-cloning <Emphasis Type="Italic">CRF1</Emphasis> cassette in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> on the γ-decalactone production

γ-Decalactone, an important flavor compound, can be produced by Yarrowia lipolytica, but the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded). A yeast strain Yarrowia lipolytica TA1 of high γ-decalactone yield was constructed by integrating copper-resistance gene CRF1 from Yarrowia lipolytica into the locus of POX3 genes. After being cultured in shake-flask at 28°C for 90 h, TA1 reached a γ-decalactone yield of 0.531 g/l (highest at 63 h), being 2.9 times higher than that of Yarrowia lipolytica As2.1045 (0.194 g/l, highest at 57 h). It was free of heterologous DNA sequences and drug-resistance genes and could be safely used in γ-decalactone production. And this work may throw a new light on addressing the problems in commercial fragrance manufacture.     

A Single and Robust Critical Nitrogen Dilution Curve for <Emphasis Type="Italic">Miscanthus</Emphasis> × <Emphasis Type="Italic">giganteus</Emphasis> and <Emphasis Type="Italic">Miscanthus sinensis</Emphasis>

The sustainable development of miscanthus as a bioenergy feedstock requires optimizing its fertilizer inputs and, therefore, determining its nitrogen (N) requirements. The ‘critical nitrogen dilution curve’ is a powerful tool to characterize such N requirements; it relates the N concentration ([N]) in aboveground organs to their biomass, defining two domains depending on whether the N factor limits biomass growth or not. We aimed to develop such a tool in miscanthus. Using a rhizome N depletion strategy with green cutting pre-treatment over several years before the start of the experiment, we grew, in 2014, two cultivated species, Miscanthus × giganteus (M×g) and Miscanthus sinensis (Msin), at four fertilizer levels (0, 80, 160 and 240 kg N ha^?1). We found a strong nitrogen fertilization effect. The shoot [N] decreased as the aboveground biomass increased in both species and in all of the treatments. [N] was strongly correlated with leaf/stem biomass ratio. The N treatments enabled the identification of the observed critical points, i.e. points with the maximum biomass (W) and the lowest [N], on each measurement date. These points could be fitted to the following critical dilution curve that was common between M×g and Msin: N concentration (Nc) (critical [N], g N kg^?1) = 27.0 W ^?0.48 when W > 1 t ha^?1 and Nc = 27.0 when W ≤ 1. This curve was validated by literature data, separated into N-limited or not-limited conditions. The similarity of the curves between the two species was due to compensation between leaf/stem biomass ratio and [N] in the stems. This curve is helpful to diagnose the crop N status and define the optimal fertilizer requirements of miscanthus crops.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

<Emphasis Type="Italic">Astyanax</Emphasis> <Emphasis Type="Italic">bockmanni</Emphasis> Vari and Castro, 2007: an ambiguous karyotype in the <Emphasis Type="Italic">Astyanax</Emphasis> genus

Despite the widespread distribution of Astyanax bockmanni in streams from Upper Paraná River system in central, southeastern, and southern Brazil, just recently, it has been identified as a distinct Astyanax species. Cytogenetic studies were performed in two populations of this species, revealing conservative features. A. bockmanni shows 2n = 50 chromosomes, a karyotypic formula composed of 10 M + 12SM + 12ST + 16A and multiple Ag-NORs. Eight positive signals in subtelocentric/acrocentric chromosomes were identified by fluorescent in situ hybridization (FISH) with 18S rDNA probes. After FISH with 5S rDNA probes, four sites were detected, comprising the interstitial region of a metacentric pair and the terminal region on long arms of another metracentric pair. Little amounts of constitutive heterochromatin were observed, mainly distributed at distal region in two chromosomal pairs. Additionally, heterochromatin was also located close to the centromeres in some chromosomes. No positive signals were detected in the chromosomes of A. bockmanni by FISH with the As-51 satellite DNA probe. The studied species combines a set of characteristics previously identified in two different Astyanax groups. The chromosomal evolution in the genus Astyanax is discussed.