Interactions between aromatase (estrogen synthase) and dopamine in the control of male sexual behavior in quail

In male quail, like in other vertebrates including rodents, testosterone acting especially through its estrogenic metabolites is necessary for the activation of male sexual behavior. Also, the administration of dopamine agonists and antagonists profoundly influences male sexual behavior. How the steroid-sensitive neural network and dopamine interact physiologically, remains largely unknown. It is often implicitly assumed that testosterone or its metabolite estradiol, stimulates male sexual behavior via the modification of dopaminergic transmission. We have now identified in quail two possible ways in which dopamine could potentially affect sexual behavior by modulating the aromatization of testosterone into an estrogen. One is a long-acting mechanism that presumably involves the modification of dopaminergic transmission followed by the alteration of the genomic expression of aromatase. The other is a more rapid mechanism that does not appear to be dopamine receptor-mediated and may involve a direct interaction of dopamine with aromatase (possibly via substrate competition). We review here the experimental data supporting the existence of these controls of aromatase activity by dopamine and discuss the possible contribution of these controls to the activation of male sexual behavior.     

Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.     

Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils

Degradation of agricultural land and the resulting loss of soil biodiversity and productivity are of great concern. Land-use management practices can be used to ameliorate such degradation. The soil bacterial communities at three separate arable farms in eastern England, with different farm management practices, were investigated by using a polyphasic approach combining traditional soil analyses, physiological analysis, and nucleic acid profiling. Organic farming did not necessarily result in elevated organic matter levels; instead, a strong association with increased nitrate availability was apparent. Ordination of the physiological (BIOLOG) data separated the soil bacterial communities into two clusters, determined by soil type. Denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism analyses of 16S ribosomal DNA identified three bacterial communities largely on the basis of soil type but with discrimination for pea cropping. Five fields from geographically distinct soils, with different cropping regimens, produced highly similar profiles. The active communities (16S rRNA) were further discriminated by farm location and, to some degree, by land-use practices. The results of this investigation indicated that soil type was the key factor determining bacterial community composition in these arable soils. Leguminous crops on particular soil types had a positive effect upon organic matter levels and resulted in small changes in the active bacterial population. The active population was therefore more indicative of short-term management changes.     

New donor vector for generation of histidine-tagged fusion proteins using the Gateway Cloning System

An optimized donor/shuttle vector, pENTR-His-ccdB, was generated that readily produces a histidine-tagged recombinant protein in multiple expression systems using Gateway Technology. In the current Gateway System, six histidines and the tobacco etch virus protease cleavage site are encoded upstream of the attR1 recombination site such that the histidine-tagged destination/expression vector adds 15 residues to the amino-terminus of recombinant proteins. Our new vector introduces the histidine tag at the donor level and places the multiple cloning sites within the attL recombination sites producing cleavable histidine-tagged proteins with a short, neutral linker of five residues. Two histidine-tagged clones were produced and fusion proteins expressed using the newly engineered vector.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Incorporation of alkaline phosphatase into layer-by-layer polyelectrolyte films on the surface of affi-gel heparin beads: physicochemical characterization and evaluation of the enzyme stability

The preparation of functionalized beads in the micrometer size range that can be used to probe the action of immobilized biomolecules on cell cultures during controlled periods of time is of fundamental importance in cell biology. However, the preparation and characterization of such particles is tedious because of their fast sedimentation. It is hence difficult to prepare such beads in a reproducible manner. This highlights the need to prepare an important batch of functionnalized particles and to store them under conditions where the loss of biological activity is minimized. The aim of this paper was to immobilize alkaline phosphatase (AP) as a model enzyme on the surface of Affi-gel heparin beads functionnalized by means of a layer-by-layer (LBL) film made of poly-l-glutamic (PGA) acid and poly-l-lysine (PLL). The enzyme has been adsorbed either on the top of the LBL film or embedded under five polyelectrolyte layers. When embedded, the enzyme was not released in buffer and retained more than 30% of its initial activity after 3 months of storage at 4 degrees C. However, when the enzyme was adsorbed on top of the LBL film, about 80% of the adsorbed enzyme was released in the buffer after a few days of storage. Longer storage did not lead to any further desorption and the remaining enzyme displayed the same evolution of its activity with time as the embedded enzyme. The time evolution of the enzyme activity on the beads is compared with that in solution alone and in the presence of PGA and PLL separately.     

Bactericidal effect of chlorine on Mycobacterium paratuberculosis in drinking water

AIMS: One possible route of transmission of Mycobacterium paratuberculosis from cattle to humans is via contaminated water supplies. The aim of this work was to determine whether this organism can survive standard water treatment processes. METHODS AND RESULTS: Two strains of M. paratuberculosis (bovine strain, NCTC 8578 and human strain Linda, ATCC 43015) were subjected to various chlorine concentrations (0.5, 1.0 and 2.0 microg ml(-1)) for 15 and 30 min. Chlorine test solutions were made up in two types of water, sterile water that had been deionized and subjected to reverse osmosis (DRO) and DRO water containing MgCl(2), CaCl(2), NaHCO(3) and bovine serum albumin (0.3% w/v), the latter to mimic conditions the organism would experience in commercial water treatment operations. CONCLUSION: The data showed that when initial inoculum levels were high (10(6) cfu ml(-1)) neither M. paratuberculosis strain was completely killed at the free chlorine concentrations and contact times applied. Log10 reductions in the range 1.32-2.82 were observed. The greatest log(10) reduction in cell numbers (2.82 and 2.35 for the bovine and human strains, respectively) was observed at the highest chlorine concentration (2 microg ml(-1)) and longest contact time (30 min). SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the need for further research into the survival of M. paratuberculosis during water treatment.     

Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.