The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.     

Involvement of S6 kinase and p38 mitogen activated protein kinase pathways in strain-induced alignment and proliferation of bovine aortic smooth muscle cells

Bovine aortic smooth muscle cell (SMC) phenotype can be altered by physical forces. This has been demonstrated by cyclic strain-induced changes in proliferation and alignment. However, the intracellular coupling pathways remain ill defined. In the present study, we examined whether the p38 and S6 kinase pathway were involved in the mitogenic and morphological changes seen in SMCs exposed to cyclic strain. We seeded bovine aortic SMCs on silastic membranes that were deformed with 150-mmHg vacuum. Cyclic strain induced both alignment and proliferation of SMCs. SB202190, a specific inhibitor of p38, hindered SMC alignment, but not proliferation. Rapamycin, a specific inhibitor of the mTOR-S6 kinase pathway, attenuated strain-induced proliferation, but not alignment. Peak activation of p38 and S6 kinase was 351 +/- 76.9% at 5 min and 363 +/- 56.2% at 60 min compared with static control, respectively (P < 0.05). The results suggest that strain-induced SMC alignment is dependent on activation of p38, but not S6 kinase. Strain induced SMC proliferation is S6 kinase, but not p38 activation, dependent.     

Age and anesthetic effects on murine electrocardiography

Murine models offer potential insights regarding human cardiac disease, but efficient and reliable methods for phenotype evaluation are necessary. We employed non-invasive electrocardiography (ECG) in mice, investigating statistical reliability of these parameters with respect to anesthetic and animal age. Mice (C57BL/6, 8 or 48 weeks) were anesthetized by ketamine/xylazine (K/X, 80/10 mg/kg ip) or by inhalation anesthetic (halothane, HAL; sevoflurane, SEV) and 6 lead ECGs were recorded. P wave duration and QT interval was significantly prolonged with K/X compared to HAL and SEV, indicating slowed atrial and ventricular conduction. P-R interval (atrio-ventricular conduction) was significantly increased in aged mice under all anesthetics. Heart rate was inversely correlated to QT interval and P wave duration. We also detected significant age effects with respect to optimal approaches for QT interval corrections. Power analysis showed 4-fold higher number of mice/group, were required for K/X, to achieve identical statistical sensitivity. These data demonstrate the importance of anesthetic selection for relevant and reliable ECG analysis in mice and illustrate the selective influences of anesthetics and age on cardiac conductance in this species.     

Leaf biomechanics, morphology, and anatomy of the deciduous mesophyte Prunus serrulata (Rosaceae) and the evergreen sclerophyllous shrub Heteromeles arbutifolia (Rosaceae)

Leaf tensile properties were compared between the mesic deciduous tree Prunus serrulata (var. "Kwanzan") and the xeric and sclerophyllous chaparral evergreen shrub Heteromeles arbutifolia (M. Roem). All values for biomechanical parameters for H. arbutifolia were significantly greater than those of P. serrulata. The fracture planes also differed between the two species with P. serrulata fracturing along the secondary veins, while H. arbutifolia most often fractured across the leaf irrespective of the vein or mesophyll position, thus yielding qualitative differences in the stress-strain curves of the two species. Anatomically, P. serrulata exhibits features typical for a deciduous mesophytic leaf such as a thin cuticle, a single layer of palisade mesophyll, isodiametric spongy mesophyll, and extensive reticulation of the laminar veins. Heteromeles arbutifolia leaves, however, are typically two- to three-fold thicker with a 35% higher dry mass/fresh mass ratio. The vascular tissue is restricted to the interface of the palisade and spongy mesophyll near the center of the leaf. Both epidermal layers have a thick cuticle. The palisade mesophyll is tightly packed and two to three layers thick. The spongy mesophyll cells are ameboid in shape and tightly interlinked both to other spongy cells as well as to the overlying palisade layer. We conclude that the qualitative and quantitative biomechanical differences between the leaves of these two species are likely due to a complex interaction of internal architectural arrangement and the physical/chemical differences in the properties of their respective cell walls. These studies illustrate the importance that morphological and anatomical correlates play with mechanical behavior in plant material and ultimately reflect adaptations present in the leaves of chaparral shrubs that are conducive to surviving in arid environments.     

Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme

Heterodisulfide reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe-4S](3+) cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe-4S] cluster using CoM-(33)SH as substrate and purified Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-(33)SH in the presence of duroquinone as an oxidant resulted in a significant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyperfine coupling between the (33)S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe-4S] cluster in the active site of the enzyme.     

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.     

The role of tissue expansion in the management of large congenital pigmented nevi of the forehead in the pediatric patient

The authors present a cohort of 21 consecutive patients who had congenital pigmented nevi covering 15 to 65 percent of the forehead and adjacent scalp and who were treated at their institution within the last 12 years. All patients were treated with an expansion of the adjacent texture- and color-matched skin as the primary modality of treatment. The median age at presentation was approximately 1 year; mean postoperative follow-up was 4 years. Nevi were classified according to the predominant anatomic areas they occupied (temporal, hemiforehead, and midforehead/central); some of the lesions involved more than one aesthetic subunit.The authors propose the following guidelines: (1) Midforehead nevi are best treated using an expansion of bilateral normal forehead segments and advancement of the flaps medially, with scars placed along the brow and at or posterior to the hairline. (2) Hemiforehead nevi often require serial expansion of the uninvolved half of the forehead to minimize the need for a back-cut to release the advancing flap. (3) Nevi of the supraorbital and temporal forehead are preferentially treated with a transposition of a portion of the expanded normal skin medial to the nevus. (4) When the temporal scalp is minimally involved with nevus, the parietal scalp can be expanded and advanced to create the new hairline. When the temporoparietal scalp is also involved with nevus, a transposition flap (actually a combined advancement and transposition flap because the base of the pedicle moves forward as well) provides the optimal hair direction for the temporal hairline and allows significantly greater movement of the expanded flap, thereby minimizing the need for serial expansion. (5) Once the brow is significantly elevated on either the ipsilateral or contralateral side from the reconstruction, it can only be returned to the preoperative position with the interposition of additional, non-hair-bearing forehead skin. Expansion of the deficient area alone will not reliably lower the brow once a skin deficiency exists. (6) In general, one should always use the largest expander possible beneath the uninvolved forehead skin, occasionally even carrying the expander under the lesion. Expanders are often overexpanded.     

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50°C) were such as enabling >94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (R_g) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.     

Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1

AIMS: Optimization of the growth of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1 by traditional fermentation methods. METHODS AND RESULTS: Fermentation studies were conducted in De Man Rogosa and Sharpe (MRS) broth (Oxoid), preadjusted to specific pH values, and in MRS broth supplemented with various nitrogen sources, MnSO4, MgSO4 and Tween 80. The production of pediocin PD-1 closely followed the growth curve of Ped. damnosus NCFB 1832. Maximum levels of bacteriocin activity (3249 AU ml(-1)/O.D.max) were recorded in MRS broth with an initial pH of 6.7. In media with an initial pH of 4.5 bacteriocin activity as low as 222 AU ml(-1)/O.D.max was recorded. The highest bacteriocin activity was recorded in growth conditions allowing the greatest pH variation (highest DeltapH). The addition of bacteriological peptone (1.7%, w/v), MnSO4 (0.014%, w/v) and Tween 80 (3%, v/v) to MRS and adjustment of the medium pH to 6.7 resulted in a further increase in activity (from 3249 to 5078 AU ml(-1)/O.D.max). The same medium, but with an initial pH of 6.2, resulted in an 82.5% decrease in bacteriocin activity. CONCLUSIONS: Pediocin PD-1 production is not only stimulated by the presence of specific growth factors (e.g., bacteriological peptone, MnSO4 or Tween 80), but may also be stimulated by the lowering in pH during growth (highest DeltapH), and thus also the amount of organic acids produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of pediocin PD-1 by the wild-type producer strain was significantly improved by using a defined medium and traditional fermentation methods.