Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Congenital stationary night blindness (CSNB) is an inherited and non‐progressive retinal dysfunction. Here, we present the crystal structure of CSNB‐causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin‐II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light‐activated state of the CSNB‐causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43. To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28. We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.     

Phylogenetic studies of the genus <Emphasis Type="Italic">Cebus</Emphasis>(Cebidae-Primates) using chromosome painting and G-banding

Background  

Chromosomal painting, using whole chromosome probes from humans and Saguinus oedipus, was used to establish karyotypic divergence among species of the genus Cebus, including C. olivaceus, C. albifrons, C. apella robustus and C. apella paraguayanus. Cytogenetic studies suggested that the species of this genus have conservative karyotypes, with diploid numbers ranging from 2n = 52 to 2n = 54.     

The second coordination sphere of FIH controls hydroxylation

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the α-subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, αKG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects; autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn(205) → Ala and Asn(294) → Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238) → Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238) → Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239) → Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239) → Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro–stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.     

Relative shortening and functional tethering of spinal cord in adolescent scoliosis – Result of asynchronous neuro-osseous growth, summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The Statement for this debate was written by Dr WCW Chu and colleagues who examine the spinal cord to vertebral growth interaction during adolescence in scoliosis. Using the multi-planar reconstruction technique of magnetic resonance imaging they investigated the relative length of spinal cord to vertebral column including ratios in 28 girls with AIS (mainly thoracic or double major curves) and 14 age-matched normal girls. Also evaluated were cerebellar tonsillar position, somatosensory evoked potentials (SSEPs), and clinical neurological examination. In severe AIS compared with normal controls, the vertebral column is significantly longer without detectable spinal cord lengthening. They speculate that anterior spinal column overgrowth relative to a normal length spinal cord exerts a stretching tethering force between the two ends, cranially and caudally leading to the initiation and progression of thoracic AIS. They support and develop the Roth-Porter concept of uncoupled neuro-osseous growth in the pathogenesis of AIS which now they prefer to term ' asynchronous neuro-osseous growth'. Morphological evidence about the curve apex suggests that the spinal cord is also affected, and a 'double pathology' is suggested. AIS is viewed as a disorder with a wide spectrum and a common neuroanatomical abnormality namely, a spinal cord of normal length but short relative to an abnormally lengthened anterior vertebral column. Neuroanatomical changes and/or abnormal neural function may be expressed only in severe cases. This asynchronous neuro-osseous growth concept is regarded as one component of a larger concept. The other component relates to the brain and cranium of AIS subjects because abnormalities have been found in brain (infratentorial and supratentorial) and skull (vault and base). The possible relevance of systemic melatonin-signaling pathway dysfunction, platelet calmodulin levels and putative vertebral vascular biology to the asynchronous neuro-osseous growth concept is discussed. A biomechanical model to test the spinal component of the concept is in hand. There is no published research on the biomechanical properties of the spinal cord for scoliosis specimens. Such research on normal spinal cords includes movements (kinematics), stress-strain responses to uniaxial loading, and anterior forces created by the stretched cord in forward flexion that may alter sagittal spinal shape during adolescent growth. The asynchronous neuro-osseous growth concept for the spine evokes controversy. Dr Chu and colleagues respond to five other concepts of pathogenesis for AIS and suggest that relative anterior spinal overgrowth and biomechanical growth modulation may also contribute to AIS pathogenesis.     

Renal Carcinogenesis After Uninephrectomy

Nephrectomized rats have widely been used to study chronic renal failure. Interestingly, renal cell carcinoma occurred in the remnant kidney after uninephrectomy (UNX). In this study, we probed insulin-like growth factor (IGF)-1 signaling pathway in UNX-induced renal cancer. Adult male Sprague-Dawley rats were randomized into two groups: UNX rats (n = 22) and sham-operated rats (n = 12). Rats were killed at 3, 7, and 10 months. After 7 months after nephrectomy, the UNX rats developed renal cell carcinoma with increased expression of proliferating cell nuclear antigen, and 68.2% (15/22) of the animals exhibited invasive carcinoma. Western blot demonstrated significant down-regulation of IGF binding protein 3 contrasting with the up-regulation of protein kinase Cζ and Akt/protein kinase B in the renal cancer tissues. These findings indicate a unique rat model of UNX-induced renal cancer associated with enhanced IGF-1 signaling pathway.