Synthesis and Application of D- and C-Labelled tert-Butyl Hoechst Dye.

Herein, the successful syntheses of D- and C-N-methyl and D-tert-butyl Hoechst dyes are presented. This includes the preparation of the labelled D- and C-N-methyl piperazines and D-tert-butylated hydroxytoluene precursors. The tert-butyl Hoechst dye is known to bind a specific RNA aptamer.

Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson.

Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution.

Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale.

Transforming planning and policy making processes at the intersections of climate, equity, and decolonization challenges.

Cities are facing increasing pressures to address complex challenges of climate change, equity, and reconciliation with Indigenous Peoples as intersecting issues, and innovation into planning and policy-making processes is urgently needed to achieve this. It is no longer good enough to work on these challenges discreetly, or solely within the dominant, western colonial paradigm and practices of governance. There are ongoing harms being caused by climate work that does not embed justice, and there are missed opportunities for synergies across these domains as they have the same systemic root causes.

How are decisions made to access a planned epidural in labour? Midwife-woman interactions in antenatal consultations.

Objective: The purpose of this study was to examine the ways in the decision to access a planned epidural in labour was topicalised and negotiated between pregnant women and midwives.

Design: This article uses conversation analysis to examine how decision-making unfolds in antenatal consultations in a large metropolitan hospital in South Australia. Data were sampled from naturally-occurring interactions between women and midwives in routine antenatal consultations.

"Cos You're Quite Normal, Aren't You?": Epistemic and Deontic Orientations in the Presentation of Model of Care Talk in Antenatal Consultations.

Women's involvement in decision-making around antenatal care is an issue of ongoing debate and discussion. Most research on the topic has used interview and focus group methods to examine women's perspectives. The present study uses a different kind of evidence.

"Trying to give birth naturally was out of the question": Accounting for intervention in childbirth.

Problem: Studies of women's childbirth preferences repeatedly show that natural birth remains highly valued, yet the majority of births involve some form of medical intervention. Reasons for this lack of correspondence have typically been investigated through interviews and focus-groups with women. Relatively little research explores the ways in which women describe their experiences of childbirth outside of such research settings.

The Sequential Intercept Model and Juvenile Justice: Review and Prospectus.

Behavioral health needs in justice-involved adolescents are an increasing concern, as it has been estimated that two-thirds of youths in the juvenile justice system now meet the criteria for one or more psychological disorders. This article describes the application of the Sequential Intercept Model (SIM), developed to describe five "points of interception" from standard prosecution into rehabilitation-oriented alternatives for adults (Munetz & Griffin, 2006), to juvenile justice. The five SIM intercepts are: (1) first contact with law enforcement or emergency services; (2) initial hearings and detention following arrest; (3) jails and courts (including problem-solving courts); (4) re-entry from jails, prisons and forensic hospitals; and (5) community corrections and community support, including probation and parole.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood.

Automated circular dichroism spectroscopy for medium-throughput analysis of protein conformation.

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g.

Ethical issues in new drug prescribing.

We use the format of a hypothetical case study to review issues related to pharmaceutical product approval and physician prescribing practices. In this case, a new FDA-approved drug is recommended for a patient who subsequently experiences an adverse event that may or may not be related to the prescription. This case raises a number of ethical and legal considerations physicians routinely face when deciding whether to recommend such drugs for their patients.

Binding to DNA protects Neisseria meningitidis fumarate and nitrate reductase regulator (FNR) from oxygen.

Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2-3x slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms.

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice.

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes.

Mutations in prokineticin 2 and prokineticin receptor 2 genes in human gonadotrophin-releasing hormone deficiency: molecular genetics and clinical spectrum.

Context: Mice deficient in prokineticin 2(PROK2) and prokineticin receptor2 (PROKR2) exhibit variable olfactory bulb dysgenesis and GnRH neuronal migration defects reminiscent of human GnRH deficiency.

Objectives: We aimed to screen a large cohort of patients with Kallmann syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (IHH) for mutations in PROK2/PROKR2, evaluate their prevalence, define the genotype/phenotype relationship, and assess the functionality of these mutant alleles in vitro.

Design: Sequencing of the PROK2 and PROKR2 genes was performed in 170 KS patients and 154 nIHH.

Delivery of nitric oxide for analysis of the function of cytochrome c'.

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas.

Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone (GnRH) deficiency in the human presents either as normosmic idiopathic hypogonadotropic hypogonadism (nIHH) or with anosmia [Kallmann syndrome (KS)]. To date, several loci have been identified to cause these disorders, but only 30% of cases exhibit mutations in known genes. Recently, murine studies have demonstrated a critical role of the prokineticin pathway in olfactory bulb morphogenesis and GnRH secretion.

Reversal of idiopathic hypogonadotropic hypogonadism.

Background: Idiopathic hypogonadotropic hypogonadism, which may be associated with anosmia (the Kallmann syndrome) or with a normal sense of smell, is a treatable form of male infertility caused by a congenital defect in the secretion or action of gonadotropin-releasing hormone (GnRH). Patients have absent or incomplete sexual maturation by the age of 18. Idiopathic hypogonadotropic hypogonadism was previously thought to require lifelong therapy.

Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH.

The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH(2) (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH(2) and O(2) in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions.

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.

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases.

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent.

Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al.

Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates p-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur.

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover.

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln.

A flavoprotein encoded in Selenomonas ruminantium is characterized after expression in Escherichia coli.

Selenomonas ruminantium is an obligate anaerobe that is very important for the provision of vitamin B12 to ruminants, which are particularly dependent upon this cofactor. One important use for vitamin B12 in anaerobic bacteria is for the utilization of glycerol as carbon source. A new flavoprotein has been found expressed by Escherichia coli from a plasmid created as part of a gene library of S.