Isolation and characterization of heparan sulfate from various murine tissues

Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes, has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin was purified from 6–8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and ¹H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental understanding of certain aspects of protein-carbohydrate interactions.     

Forward genetic analysis of the circadian clock separates the multiple functions of ZEITLUPE

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.     

Stereoselective synthesis of some 17beta-dihydrooxazinyl steroids, as novel presumed inhibitors of 17alpha-hydroxylase-C17,20-lyase

17beta-Dihydrooxazinyl steroids 5a-l and 6a-l were synthetized. The acid-catalyzed reactions of 21-azidomethyl-20-hydroxy- and 21-hydroxymethyl-20-azidosteroids with substituted aromatic aldehydes led to the formation of androst-5-en-3beta-ols substituted in position 17beta with dihydrooxazine residues. The inhibitory effects of these compounds on rat testicular C(17,20)-lyase were investigated with an in vitro radioincubation technique.     

Expression of cyclooxygenase 1 and 2 in the canine corpus luteum during diestrus

In the dog, unlike most other domestic animal species, corpus luteum (CL) life span is not affected by hysterectomy. Only in pregnant dogs, during the immediate prepartum decline of progesterone, does PGF2alpha clearly seem to act as an endogenous luteolytic agent. Whether endogenous PGF2alpha plays a role in the slow regression of the corpora lutea of the nonpregnant cycle is not known. To test for possible paracrine/autocrine effects of locally produced PGF2alpha, luteal expression of the key rate-limiting enzymes in prostaglandin biosynthesis, i.e. cyclooxygenase 1 and 2 (Cox1 and Cox2), was examined in dogs during diestrus, including the periods of CL formation, as well as early and late CL regression. Corpora lutea were collected by ovariohysterectomy from nonpregnant bitches 5, 15, 25, 35, 45 and 65 days after ovulation. On the mRNA-level, expression of Cox1 and Cox2 was tested by qualitative and quantitative, Real Time (Taq Man) RT-PCR; on the protein level, expression of Cox2 was studied by immunohistochemistry. The mRNA for Cox1 and Cox2 were detected at all stages of diestrus. Expression of Cox1 was lowest on Day 5 (ovulation = Day 0) and higher and nearly constant thereafter. Expression of Cox2-mRNA was distinctly cycle related and highest on Day 5; it decreased by Day 15 and remained constantly low until Day 65. Immunohistochemistry localized expression of Cox2 in the cytoplasm of luteal cells. Staining was restricted to Days 5 and 15, with stronger signals on Day 5. These data suggested that increased expression of Cox2 is associated with luteal growth and development and not luteal regression. Furthermore, the expression of Cox1 more likely reflected activity of a housekeeping gene.     

Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9alpha-hydroxylase in Mycobacterium smegmatis mc(2)155

Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc(2)155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9alpha-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9alpha-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9alpha-hydroxylase in M. smegmatis mc(2)155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9alpha position.     

Use of Pleurotus dryinus for lignocellulolytic enzymes production in submerged fermentation of mandarin peels and tree leaves

It has been shown that the wood-rotting mushroom Pleurotus dryinus IBB 903 is able to effectively produce cellulases, xylanase, laccase, and manganese peroxidase in submerged fermentation of mandarin peels and tree leaves. Gradual increasing of lignocellulosic substrates concentration from 1 to 4–6% enhanced enzyme accumulation in culture liquid. A simple and inexpensive medium containing mandarin peels and yeast extract as sole carbon and nitrogen sources allowed simultaneous production of high levels of both hydrolases and oxidases by P. dryinus IBB 903. Supplementation of this medium by copper and manganese caused earlier and faster accumulation of laccase and manganese peroxidase increasing their yield by 1.5 and 7.5 times, respectively. In addition, by adding manganese to the medium it is possible to regulate the ratio of laccase and MnP in enzyme preparation. The presence of lignocellulosic substrate is the requisite for MnP production by P. dryinus IBB 903 since there was no production of MnP when mushroom has been cultivated in the synthetic medium with different carbon source. Among carbon source tested only utilization of glucose resulted to 21-fold increase of fungus laccase specific activity compared to control medium without carbon source. Carboxymethyl cellulase and xylanase appeared to be inducible enzymes.     

Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation

Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.     

Channel activity of OmpF monitored in nano-BLMs

Free-standing lipid bilayer membranes can be formed on small apertures (60 nm diameter) on highly ordered porous alumina substrates. The formation process of the membranes on a 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol submonolayer was followed by impedance spectroscopy. After lipid bilayers had thinned, the reconstitution and ionic conducting properties of the outer membrane protein OmpF of E. coli were monitored using single-channel recordings. The characteristic conductance states of the three monomers, fast kinetics, and subconductance states were observed. Blockade of the ion flow as a result of interaction of the antibiotic ampicillin with the protein was verified, indicating the full functionality of the protein channel in nanometer-scale bilayer membranes.     

First records of soil ciliates (Protozoa, Ciliophora) from classes Prostomea, Nassophorea, Spirotrichea, and Colpodea in Slovakia

In total, seven ciliate species were recorded in leaf-litter, moss and soil from a variety of sites in Slovakia for the first time: Chilophrya terricola Foissner, 1984; Holostichides typicus (Song et Wilbert, 1988) Eigner, 1994; Keronella gracilis Wiackowski, 1985; Notoxoma parabryophryides Foissner, 1993; Parafurgasonia sorex (Penard, 1922) Foissner et Adam, 1981; Paragonostomum multinucleatum Foissner, Agatha et Berger, 2002, and Territricha stramenticola Berger et Foissner, 1988. The paper deals with their distribution, ecology, and comparison with similar species. The shape and nuclear variants of Paragonostomum multinucleatum are presented and populations of P. multinucleatum and T. stramenticola are morphometrically characterized.