Purification of extracellular laccase from Cerrena unicolor

Cerrena unicolor was found to produce large amounts of extracellular laccase when grown aerobically on the optimized Lindenberg and Holm medium in fermenter culture with an automatic pH control. The laccase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, affinity chromatography, and chromatofocusing. The enzymes isoforms were recovered with a 65- to 92-fold increase in specific activity and a yield for Ia1?=?6.7%; Ia2?=?27.5%; Ib?=?9.7%; and IIa1?=?21%. The molecular mass of the purified enzymes proved to be 45, 47, 54, and 62?kD, respectively, as determined by size-exclusion high-performance liquid chromatography (HPLC). The isoelectric points were in the range of 4.7 to 4.2, and the carbohydrate content in the purified enzymes was between 1.6 and 3.5%.     

A novel pattern of follicular epithelium morphogenesis in higher dipterans

In fly ovaries, the follicular epithelium surrounding germline cells diversifies into several morphologically distinct cell subpopulations. This complex process is crucial for the formation of a regionally complex eggshell and establishment of polarity of the future embryo. Morphogenetic changes accompanying patterning of the follicular epithelium have been best characterized in the model fly, Drosophila melanogaster. Here, we analyze follicular epithelium diversification in the ovaries of Tachypeza nubila, a brachyceran fly closely related to the group Cyclorrhapha, which also includes Drosophila. We provide morphological evidence that in Tachypeza, the diversification process differs from that described in the Drosophila model system in several important respects: (i) follicle cells differentiate into five subpopulations (versus eight in Drosophila); (ii) only one of these subpopulations (i.e. border cells) is migratory (versus four in Drosophila); (iii) the main body follicle cells form a uniform epithelium with no distinct border between follicle cells covering the nurse cell compartment and the oocyte; (iv) chorionic material is deposited not only on the surface of the oocyte but also on the nurse cells; (v) there is no centripetal migration of the follicle cells; (vi) the resulting eggshell is morphologically simple with no regional specializations except for the micropylar apparatus at the anterior pole of the oocyte. Our findings provide novel insights into the evolution of the follicle cell patterning and functioning in dipterans. A critical analysis of these processes in different dipteran groups strongly indicates that in Tachypeza, follicular epithelium diversification follows a distinct pattern, novel for higher dipterans.     

Asymmetric addition of allyltrimethylsilane to N-tosylimine of (1R)-8-phenylmenthyl glyoxylate.

The addition of allyltrimethylsilane to chiral N-tosylimine derived from (1R)-8-phenylmenthyl glyoxylate is discussed. Various Lewis acids were applied in order to postulate the stereochemical model for this type of nucleophilic addition.     

Asymmetric induction in the [4+2]cycloaddition of cyclopentadiene and furan to chiral derivatives of fumaric acid.

[4+2]Cycloaddition reactions of cyclopentadiene (1a) and furan (1b) to N,N'-fumaroyldi[(2R)-bornane-10,2-sultam] (2) and to N,N'-fumaroyldi[(2R)-bornane-10,2-(2'-phenyl-pyrazol-3'-one)] (3) are presented. A correlation between the solvent polarity and the logarithm of the diastereoisomer ratio (dr) was found for the uncatalyzed [4+2]cycloaddition of 1a to 3.     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.     

Hoc protein regulates the biological effects of T4 phage in mammals

We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours. Maria Zembala and Janusz Boratynski contributed equally to this work.     

Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.     

The effects of acute corticosterone administration on anxiety, endogenous corticosterone, and c-Fos expression in the rat brain

The effects of acute pretreatment of rats with corticosterone (5 and 20 mg/kg, s.c.) on emotional behavior, expression of c-Fos protein in brain structures, and serum concentration of corticosterone were studied to model the short-term glucocorticoid-dependent changes in brain functions. Corticosterone was administered 90 min before training of a conditioned fear reaction (a freezing response), and behavioral, hormonal and immunocytochemical effects were examined 1 day later, on the test day. Pretreatment of rats with corticosterone significantly attenuated the freezing reaction in the conditioned fear test. The effect of the corticosterone was accompanied by a selective enhancement of the aversive context-induced c-Fos expression in some brain structures: the parvocellular and magnocellular neurons of the paraventricular hypothalamic nucleus (pPVN and mPVN), the medial amygdala nucleus (MeA), and the cingulate cortex, area 1 (Cg1), as well as an increase in the concentration of aversive context-induced endogenous serum glucocorticoid, 1.5 h and 10 min after the test session, respectively. It is suggested that the behavioral effects of acute pretreatment of rats with corticosterone could be due to changes in the mnemonic processes in the brain, inhibition of brain corticotropin releasing factor (CRF) synthesis, or stimulation of GABA-A receptor modulating neurosteroids synthesis. It is hypothesized that the enhanced activity of Cg1, MeA, pPVN, and mPVN, and the hypothalamic-pituitary-adrenal axis with concomitant increased serum glucocorticoid concentration, might serve to facilitate active coping behavior in a threatening situation.