Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

Quartz crystal microbalance-based measurements of shear-induced senescence in human embryonic kidney cells

Fluid shear and other mechanical forces play an important role in the normal biophysical, biochemical, and gene regulatory responses of vertebrate tissue that are reflected in the expression of normal cell differentiation, growth, and function. Despite some promising work reported on the application of the quartz crystal microbalance (QCM) to both prokaryote and eukaryote cells over the last decade, QCM has yet to be successfully applied to cells in culture under conditions of flow-induced shear. In this study, high sensitivity QCM in conjunction with fluid modelling was used to monitor the onset of senescence in immortalised human embryonic kidney cells under laminar shear stresses of between 0.04 and 335 dyne/cm(2). The feasibility of this approach as a means of quantification and characterisation of cell physiological response and adhesion are explored and discussed.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

Chiral 2-pyridyl alcoholate copper complexes: crystal structures of [bis(2-pyridyl alcoholate)copper(II)] and the use of [(2-pyridyl alcoholate)copper(II)]and [(2-pyridyl alcoholate)copper(I)] in asymmetric cyclopropanation of styene and allylic oxidation of cyclohexene

Chiral N,O pyridine alcohols HL¹-HL⁶ were used to form complexes with copper(II) ions. Ligands HL¹ and HL² formed complexes with copper(II) ions when Cu(OAc)₂ and HL were refluxed in methanol/ethanol mixture. Ligand HL³ formed a complex with copper(II) when deprotonated with NaH and stirred in a Cu(II) acetate THF solution. Ligands HL⁴-HL⁶ did not form complexes with copper(II) under similar conditions. Two complexes, [Cu(L¹)₂] and [Cu(L²)₂], were isolated as single crystals and characterized by X-ray crystallography. These complexes showed low catalytic activities in asymmetric reactions. However, they became active when reacted with triflic acid. Copper complexes, [Cu(L)] or [Cu(L)]⁺, formed in situ by reacting ligands HL with copper(I) or (II) ions, respectively, were also found to be active copper catalysts for asymmetric cyclopropanation of styrene with ethyl diazoacetate and allylic oxidation of cyclohexene with t-butylperoxybenzoate. Enantioselectivities up to 56% and 38% were obtained in asymmetric cyclopropanation of styrene and asymmetric allylic oxidation of cyclohexene, respectively.     

Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement

Notch expression is frequently associated with progenitor cells, and its function is crucial for development. Our recent work showing that Notch1 is selectively expressed in basal epithelial cells of the prostate and higher Notch1 expression during development suggests that Notch1-expressing cells may define progenitor cells in the prostate. To test this hypothesis, we have generated a transgenic mouse line in which the Notch1-expressing cells can be ablated in a controlled manner. Specific targeting was achieved by expressing the bacterial nitroreductase, an enzyme that catalyzes its substrate into a cytotoxin capable of inducing apoptosis, under the Notch1 promoter. Cell death in transgenic prostate was confirmed by histological analyses including terminal dUTP nick-end labeling and caspase 3 immunocytochemical staining. We evaluated the consequences of ablation of Notch1-expressing cells in two systems, organ culture of early postnatal prostates and re-growth of prostate in castrated mice triggered by hormone replacement. Our data show that elimination of Notch1-expressing cells inhibited the branching morphogenesis, growth, and differentiation of early postnatal prostate in culture and impaired prostate re-growth triggered by hormone replacement in castrated mice. Furthermore, we found that Notch1 expression following castration and hormone replacement was concomitant with known basal cell markers p63 and cytokeratin 14 and was high in the proliferative human prostate epithelial cells. Taken together, these data suggest that Notch1-expressing cells define the progenitor cells in the prostatic epithelial cell lineage, which are indispensable for prostatic development and re-growth.     

PCTH: a novel orally active chelator of the aroylhydrazone class that induces iron excretion from mice

beta-Thalassaemia major is an inherited blood disorder which is complicated by repeated blood transfusion and excessive gastrointestinal iron (Fe) absorption, which leads to toxic Fe overload. Current treatment using the chelator, desferrioxamine (DFO), is expensive and cumbersome since the drug requires long subcutaneous infusions and it is not orally active. A novel chelator, 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), was recently designed and shown to have high Fe chelation efficacy in vitro. The aim of this investigation was to examine the Fe chelation efficacy of PCTH in vitro implementing primary cultures of cardiomyocytes and in vivo using mice. We showed that PCTH was significantly (P<0.005) more effective than DFO at mobilising (59)Fe from prelabelled cardiomyocytes. Moreover, PCTH prevented the incorporation of (59)Fe into ferritin during Fe uptake from (59)Fe-labelled transferrin. These effects were important to assess as cardiac complications caused by Fe deposition are a major cause of death in beta-thalassaemia major patients. Further studies showed that PCTH was orally active and well tolerated by mice at doses ranging from 50 to 200 mg/kg, twice daily (bd), for 2 days. A dose-dependent increase in faecal (59)Fe excretion was observed in the PCTH-treated group. This level of Fe excretion at 200 mg/kg was similar to the same dose of the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). Effective Fe chelation in the liver by PCTH was shown via its ability to reduce ferritin-(59)Fe accumulation. Mice treated for 3 weeks with PCTH at doses of 50 and 100 mg/kg/bd showed no overt signs of toxicity as determined by weight loss and a range of biochemical and haematological indices. In subchronic Fe excretion studies over 3 weeks, PIH and PCTH at 75 mg/kg/bd for 5 days/week increased faecal (59)Fe excretion to 140% and 145% of the vehicle control, respectively. This study showed that PCTH was well tolerated at 100 mg/kg/bd and induced considerable Fe excretion by the oral route, suggesting its potential as a candidate to replace DFO.     

Kinetics and comparative reactivity of human class I and class IIb histone deacetylases

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.     

Secretin controls anion secretion in the rat epididymis in an autocrine/paracrine fashion

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.     

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.