Defining the healthy "core microbiome" of oral microbial communities

Background  

Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing).     

Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.     

The microtubules dance and the spindle poles swing

Using live imaging and computer simulation, Kozlowski et al. (2007) show that an interplay between spindle pole movements, microtubule dynamics, and microtubule bending contribute to asymmetric spindle placement in the C. elegans embryo.     

Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism

This study on human cytochrome P450 reductase (CPR) presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH*,FADH*; FMN,FADH2; FMNH2,FAD) is independent of pH. No shift from the blue, neutral di-semiquinone (FMNH*,FADH*) towards the red, anionic species is observed upon increasing the pH from 6.5 to 8.5. Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH (1 to 1) in a stopped-flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH, indicative of a pH-gating mechanism. The final level of blue di-semiquinone formation is found to be pH independent. Stopped-flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di-semiquinone intermediate, yet the observed rate constants are essentially pH independent. Thus, the kinetic pH-gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four-electron reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH-dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH-dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein.     

The proteolysis of immunoglobulin G with long-acting thyroid-stimulating activity

1. Proteolysis of immunoglobulin G with long-acting thyroid-stimulating activity with papain and pepsin shortened the duration of thyroid stimulation in mice prepared for the McKenzie (1958) bioassay. The time-course appeared to be related to molecular size. 2. The activity after proteolysis was recovered solely in the electrophoretically slow F(ab)-fragment. 3. There was no immunological cross-reaction between pituitary thyroid-stimulating hormone and the active fragments of the long-acting thyroid stimulator despite their similar behaviour in the bioassay. 4. The implications of these findings are discussed.     

Simultaneous total correction of temporomandibular ankylosis and facial asymmetry

Ankylosis of the temporomandibular joint not only prevents mouth opening and chewing, but also affects the growth and position of the mandible. This produces progressive facial distortion with devastating psychosocial effects compounding the already difficult problem of not being able to open the mouth. Over the past 6 years, 18 patients in Canada and Taiwan were treated by excision of a large block of bone at the ankylosis and repositioning of the jaw, with the addition of osteotomies as necessary to produce a symmetrical face with good occlusion. Bilateral cases were treated at one operation in a similar way. The temporomandibular joint and absent ramus were constructed with a costochondral graft taken from the opposite chest. Some patients were treated with intermaxillary fixation for 8 weeks, while others had no fixation, but there was no difference in the effectiveness of correction of the ankylosis.