Differential expression of <Emphasis Type="Italic">Gnrh2</Emphasis>, <Emphasis Type="Italic">Gthβ</Emphasis>, and <Emphasis Type="Italic">Gthr</Emphasis> genes in sterile triploids and fertile tetraploids

Gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GTH), and gonadotropin hormone receptor (GTHR) are the pivotal signal molecules of the hypothalamic-pituitary-gonad (HPG) axis, which plays a crucial role in regulating gonadal development in vertebrate. In this study, we comparatively analyze the expression characteristics of Gnrh2, Gthβ, and Gthr in red crucian carp diploids, triploids, and allotetraploids. The expression patterns of these genes are similar in the three fish ploidy types: the Gnrh2 gene is expressed in midbrains, pituitaries, and gonads; the Gthβ gene is expressed in pituitaries; the Gthr gene is mainly expressed in gonads. These results indicate that the three genes participate in the regulation of gonadal development. By real-time polymerase chain reaction and in situ hybridization, we find that, among these three fish ploidy types, the expression level of Gthr in the gonads of triploids is lower than those of diploids and tetraploids; this weakens the combination of GTHR with GTH released from the pituitary and leads to the sterility of triploids, since the gonad cannot produce enough sex steroids. In addition, the low expression of Gthr in triploids may affect the down-regulation of Gthβ, which then affects the down-regulation of Gnrh2; hence, the expression levels of Gnrh2 and Gthβ genes in triploids are the highest after the breeding season. In conclusion, the differential expression of Gnrh2, Gthβ, and Gthr in triploids and tetraploids is related to their sterility and bisexual fertility, respectively.     

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     

Gene–environment interactions between <Emphasis Type="Italic">ERCC2</Emphasis>, <Emphasis Type="Italic">ERCC3</Emphasis>, <Emphasis Type="Italic">XRCC1</Emphasis> and cadmium exposure in nasal polyposis disease

Gene–environment interactions have long been known to play an important role in complex disease aetiology, such as nasal polyposis (NP). The present study supports the concept that DNA repair gene polymorphisms play critical roles in modifying individual susceptibility to environmental diseases. In fact, we investigated the role of polymorphisms in DNA repair genes and cadmium as risk factors for Tunisian patients with NP. To the best of our knowledge, this is the first report on the impact of combined effects of cadmium and ERCC3 7122 A>G (rs4150407), ERCC2 Lys751Gln (rs13181) and XRCC1 Arg399Gln (rs25487) genes in the susceptibility to NP disease. Significant associations between the risk of developing NP disease and ERCC2 [odds ratio (OR)?=?2.0, 95 % confidence interval (CI)?=?1.1–3.7, p?=?0.023] and ERCC3 (OR?=?2.2, 95 % CI?=?1.2–4.1, p?=?0.013) genotypes polymorphisms were observed. Blood concentrations of Cd in NP patients (2.2 μg/L) were significantly higher than those of controls (0.5 μg/L). A significant interaction between ERCC3 (7122 A>G) polymorphism and blood-Cd levels (for the median of blood-Cd levels: OR?=?3.8, 95 % CI?=?1.3–10.8, p?=?0.014 and for the 75th percentiles of blood-Cd levels: OR?=?2.7, 95 % CI?=?1.1–7.2, p?=?0.041) was found in association with the risk of NP disease. In addition, when we stratified ERCC2, ERCC3 and XRCC1 polymorphism genotypes by the median and 75th percentiles of blood-Cd levels, we found also significant interactions between ERCC2 (Lys751Gln) and ERCC3 (7122 A>G) genotypes polymorphism and this metal in association with NP disease. However, no interaction was found between XRCC1 (Arg399Gln) polymorphism genotypes and Cd in association with NP disease.     

Retiolitid graptolites from the collection of Hermann Jaeger II: <Emphasis Type="Italic">Cometograptus</Emphasis>, <Emphasis Type="Italic">Spinograptus</Emphasis> and <Emphasis Type="Italic">Plectograptus</Emphasis>

Graptolites from the Jaeger collection at the Museum für Naturkunde (Berlin, Germany) provide important information on structural details of Silurian (Wenlock–Ludlow) retiolitids as well as for the biostratigraphic and biogeographic distribution of these magnificent graptolites. Species of the genera Cometograptus, Spinograptus and Plectograptus are described from isolated glacial boulder material, collected in northern Germany and from shale specimens found in the Lower Graptolite Shale of Thuringia. The biostratigraphic placement of material derived from glacial erratic boulders, however, is far from being precise. The fauna associated with the neotype of Plectograptus macilentus in the ‘Unterer Graptolithenschiefer’ of Thuringia is discussed and illustrated. Cometograptus alfeisenacki from the Cyrtograptus lundgreni Biozone is recognized as a new species. The genus is discovered for the first time in North German glacial erratic boulders.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

<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.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

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.     

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.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Optimization of ethanol,citric acid,and α-amylase production from date wastes by strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, <Emphasis Type="Italic">Aspergillus niger</Emphasis>, and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The present study deals with submerged ethanol, citric acid, and α-amylase fermentation by Saccharomyces cerevisiae SDB, Aspergillus niger ANSS-B5, and Candida guilliermondii CGL-A10, using date wastes as the basal fermentation medium. The physical and chemical parameters influencing the production of these metabolites were optimized. As for the ethanol production, the optimum yield obtained was 136.00 ± 0.66 g/l under optimum conditions of an incubation period of 72 h, inoculum content of 4% (w/v), sugars concentration of 180.0 g/l, and ammonium phosphate concentration of 1.0 g/l. Concerning citric acid production, the cumulative effect of temperature (30°C), sugars concentration of 150.0 g/l, methanol concentration of 3.0%, initial pH of 3.5, ammonium nitrate concentration of 2.5 g/l, and potassium phosphate concentration of 2.5 g/l during the fermentation process of date wastes syrup did increase the citric acid production to 98.42 ± 1.41 g/l. For the production of α-amylase, the obtained result shows that the presence of starch strongly induces the production of α-amylase with a maximum at 5.0 g/l. Among the various nitrogen sources tested, urea at 5.0 g/l gave the maximum biomass and α-amylase estimated at 5.76 ± 0.56 g/l and 2,304.19 ± 31.08 μmol/l/min, respectively after 72 h incubation at 30°C, with an initial pH of 6.0 and potassium phosphate concentration of 6.0 g/l.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">Z</Emphasis>, <Emphasis Type="Italic">N</Emphasis>′-Formyl α-Amino Acid Derived Gem-Diamines

A variety of N-carbobenzoxy, N′-formyl gem-diaminoalkyl derivatives have been obtained through Goldsmith-Wick reaction of Z-α-amino acid/peptide acid derived isocyanates with 96% HCOOH in presence of 4-dimethylaminopyridine (DMAP) as catalyst. The reaction proceeds to completion within 2–4 h and results in good yields of the products isolated as stable solids.     

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.     

<Emphasis Type="Italic">Irpex lacteus</Emphasis>, a white-rot fungus with biotechnological potential — <Emphasis Type="Italic">review</Emphasis>

White-rot fungi that are efficient lignin degraders responsible for its turnover in nature have appeared twice in the center of biotechnological research — first, when the lignin degradation process started being systematically investigated and major enzyme activities and mechanisms involved were described, and second, when the huge remediation potential of these organisms was established. Originally, Phanerochaete chrysosporium became a model organism, characterized by a secondary metabolism regulatory pattern triggered by nutrient (mostly nitrogen) limitation. Last decade brought evidence of more varied regulatory patterns in white-rot fungi when ligninolytic enzymes were also abundantly synthesized under conditions of nitrogen sufficiency. Gradually, research was focused on other species, among them Irpex lacteus showing a remarkable pollutant toxicity resistance and biodegradation efficiency. Systematic research has built up knowledge of biochemistry and biotechnological applicability of this fungus, stressing the need to critically summarize and estimate these scattered data. The review attempts to evaluate the information on I. lacteus focusing on various enzyme activities and bioremediation of organopollutants in water and soil environments, with the aim of mediating this knowledge to a broader microbiological audience.