Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).     

Conjuring Medical Science: The 1986 Referendum on AIDS/HIV Policy in California

Medical science occupies a peculiar status in American life. On the one hand, people often view medical science as a privileged and authoritative body of knowledge that transcends other kinds of knowledge. On the other handy medical–scientific authority can be easily conjured from the popular symbols of science, e.g., credentials, technical terms, and white lab coats. This problem can be converted into an anthropological question of meanings and symbols, based on Geertz's interpretive anthropology and Baudrillard' s sociology of hyperreality. This article uses these frameworks to explore the cultural construction of medical-scientific authority in the case of a 1986 referendum on AIDS/HIV policy in California. The interpretation of that construction raises some difficult problems concerning anthropology's treatment of medical science. [AIDS, HIV, cultural construction of science, anthropology of science, hermeneutics of science]     

The design and discovery of novel amide CCR5 antagonists

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.     

Copper induced oxidation of serotonin: analysis of products and toxicity

Serotonin is a major neurotransmitter that controls many functions, ranging from mood and behaviour through to sleep and motor functions. The non-enzymatic oxidation of serotonin is of significant importance as some oxidation products are considered to be neurotoxic. An interaction between copper and serotonin has been suggested by symptoms observed in a number of neurodegenerative diseases such as Wilson's and Prion diseases. Using PC12 cells as a model of neuronal cells, we show that the interaction between copper and serotonin is toxic to undifferentiated cells. The toxicity is largely due to reactive oxygen species as cell death is significantly reduced in the presence of the antioxidant mannitol. Differentiation of the PC12 cells also confers resistance to the oxidative process. In vitro oxidation of serotonin by copper results in the eventual formation of a coloured pigment, thought to be a melanin-like polymeric species. Using spectroscopic methods we provide evidence for the formation of a single intermediate product. This dimeric intermediate was identified and characterized as 5,5'-dihydroxy-4,4'-bitryptamine. These results indicate that copper structurally alters serotonin and this process may play a role in copper related neurodegenerative diseases.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Structural and spectroscopic studies of a model for catechol oxidase

A binuclear copper complex, [Cu₂(BPMP)(OAc)₂][ClO₄]·H₂O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled (J = −0.6 cm⁻¹). Simulation of the dipole–dipole-coupled EPR spectrum showed that in solution the Cu–O–Cu angle was increased from 126° to 160° and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity (k _cat = 15 ± 1.5 min⁻¹, K _M = 6.4 ± 1.8 mM), which is compared with other dicopper catechol oxidase models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Fatigue in Multiple Sclerosis: Assessing Pontine Involvement Using Proton MR Spectroscopic Imaging

Background/Objective

The underlying mechanism of fatigue in multiple sclerosis (MS) remains poorly understood. Our study investigates the involvement of the ascending reticular activating system (ARAS), originating in the pontine brainstem, in MS patients with symptoms of fatigue.

Methods

Female relapsing-remitting MS patients (n = 17) and controls (n = 15) underwent a magnetic resonance spectroscopic imaging protocol at 1.5T. Fatigue was assessed in every subject using the Fatigue Severity Scale (FSS). Using an FSS cut-off of 36, patients were categorized into a low (n = 9, 22 ± 10) or high (n = 10, 52 ± 6) fatigue group. The brain metabolites N-acetylaspartate (NAA) and total creatine (tCr) were measured from sixteen 5x5x10 mm³ spectroscopic imaging voxels in the rostral pons.

Results

MS patients with high fatigue had lower NAA/tCr concentration in the tegmental pons compared to control subjects. By using NAA and Cr values in the cerebellum for comparison, these NAA/tCr changes in the pons were driven by higher tCr concentration, and that these changes were focused in the WM regions.

Discussion/Conclusion

Since there were no changes in NAA concentration, the increase in tCr may be suggestive of gliosis, or an imbalanced equilibrium of the creatine and phosphocreatine ratio in the pons of relapsing-remitting MS patients with fatigue.