Purging of inbreeding depression within the Irish Holstein-Friesian population

The objective of this study was to investigate whether inbreeding depression in milk production or fertility performance has been partially purged due to selection within the Irish Holstein-Friesian population. Classical, ancestral (i.e., the inbreeding of an individual's ancestors according to two different formulae) and new inbreeding coefficients (i.e., part of the classical inbreeding coefficient that is not accounted for by ancestral inbreeding) were computed for all animals. The effect of each coefficient on 305-day milk, fat and protein yield as well as calving interval, age at first calving and survival to second lactation was investigated. Ancestral inbreeding accounting for all common ancestors in the pedigree had a positive effect on 305-day milk and protein yield, increasing yields by 4.85 kg and 0.12 kg, respectively. However, ancestral inbreeding accounting only for those common ancestors, which contribute to the classical inbreeding coefficient had a negative effect on all milk production traits decreasing 305-day milk, fat and protein yields by -8.85 kg, -0.53 kg and -0.33 kg, respectively. Classical, ancestral and new inbreeding generally had a detrimental effect on fertility and survival traits. From this study, it appears that Irish Holstein-Friesians have purged some of their genetic load for milk production through many years of selection based on production alone, while fertility, which has been less intensely selected for in the population demonstrates no evidence of purging

Chronic Obstructive Pulmonary Disease, inflammation and co-morbidity – a common inflammatory phenotype?

Chronic Obstructive Pulmonary Disease (COPD) is and will remain a major cause of morbidity and mortality worldwide. The severity of airflow obstruction is known to relate to overall health status and mortality. However, even allowing for common aetiological factors, a link has been identified between COPD and other systemic diseases such as cardiovascular disease, diabetes and osteoporosis. COPD is known to be an inflammatory condition and neutrophil elastase has long been considered a significant mediator of the disease. Pro-inflammatory cytokines, in particular TNF-α (Tumour Necrosis Factor alpha), may be the driving force behind the disease process. However, the roles of inflammation and these pro-inflammatory cytokines may extend beyond the lungs and play a part in the systemic effects of the disease and associated co-morbidities. This article describes the mechanisms involved and proposes a common inflammatory TNF-α phenotype that may, in part, account for the associations

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

Fungal G-protein-coupled receptors::mediators of pathogenesis and targets for disease control

G-protein signalling pathways are involved in sensing the environment, enabling fungi to coordinate cell function, metabolism and development with their surroundings, thereby promoting their survival, propagation and virulence. G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in fungi. Despite the apparent importance of GPCR signalling to fungal biology and virulence, relatively few GPCR–G-protein interactions, and even fewer receptor-binding ligands, have been identified. Approximately 40% of current pharmaceuticals target human GPCRs, due to their cell surface location and central role in cell signalling. Fungal GPCRs do not belong to any of the mammalian receptor classes, making them druggable targets for antifungal development. This Review Article evaluates developments in our understanding of fungal GPCR-mediated signalling, while substantiating the rationale for considering these receptors as potential antifungal targets. The need for insights into the structure–function relationship of receptor–ligand interactions is highlighted, which could facilitate the development of receptor-interfering compounds that could be used in disease control

Properties of p-Hydroxybenzoate Hydroxylase When Stabilized in Its Open Conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts:  (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608−613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis

Luciferase from 'Vibrio campbellii' is more thermostable and binds reduced FMN better than its homologues

A new luciferase from 'V. campbellii' (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH-). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4°C and pH 8. The measured Kd at 4°C for the binding of FMNH- to Lux_Vc was 1.8 μM whereas to Lux_Vh, it was 11 μM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2 of 1020 min while Lux_Vh has t1/2 of 201 min at 37°C. The superior thermostability and tighter binding of FMNH- make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before

Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase

p-Hydroxyphenylacetate hydroxylase (HPAH) from 'Acinetobacter baumannii' catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins:  a flavin mononucleotide (FMN) reductase (C₁) and an oxygenase (C₂). C₁ catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C₂ in the hydroxylation reaction. C₁ is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C₁-bound FMN can be reduced and released to be used efficiently as the substrate for the C₂ reaction; additional FMN is not necessary. Reactions in which O₂ is rapidly mixed with solutions containing C₁-FMNH- and C₂ are very similar to those in which solutions containing O₂ are mixed with one containing the C₂-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C₂ and has already been completely transferred to C₂ before it reacts with oxygen. Rate constants for the transfer of FMNH- from C₁ to C₂ were found to be 0.35 and ≥74 s⁻¹ in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C₁. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s⁻¹, while with HPA present it dissociates at 80 s⁻¹; these are the same rates as those for the transfer from C₁ to C₂. Therefore, the dissociation of FMNH- from C₁ is rate-limiting in the intermolecular transfer of FMNH- from C₁ to C₂, and this process is regulated by the presence of HPA. This regulation avoids the production of H₂O₂ in the absence of HPA. Our findings indicate that no protein-protein interactions between C₁ and C₂ are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C₁

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

The 'luxG' gene is part of the 'lux' operon of marine luminous bacteria. 'luxG' has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, 'luxG' from 'Photobacterium leiognathi' TH1 was cloned and expressed in 'Escherichia coli' in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of 'LuxG' were purified to homogeneity, and their biochemical properties were characterized. Purified 'LuxG' is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the 'LuxG' reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s‾¹, respectively. Coupled assays between LuxG and luciferases from 'P. leiognathi' TH1 and Vibrio campbellii also showed that LuxG could supply FMNH‾ for light emission in vitro. A 'luxG' gene knockout mutant of 'P. leiognathi' TH1 exhibited a much dimmer luminescent phenotype compared to the native 'P. leiognathi' TH1, implying that 'LuxG' is the most significant source of FMNH‾ for the luminescence reaction in vivo

Probabilistic Consequences of Imperfect NDE

This paper presents results of Monte Carlo simulation of the Retirement-for-Cause (RFC) engine maintenance system as developed by Pratt and Whitney Aircraft and the U. S. Air Force. The Retirement-for-Cause concept is addressed, conventional Monte Carlo modeling techniques are explained, and an alternative approach developed at Pratt and Whitney is presented. Next, a simplified non-ideal Non-Destructive-Evaluation (NDE) model with fixed probabilities of Type I and Type II errors is described and simulation results obtained using this model are presented and discussed. An appendix presents a survey of various methods used to model NDE.</p