X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures

The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods. Phases have been obtained by the multiple isomorphous replacement method. The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence. The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter. This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes. Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis. Each subunit is folded into eight helices and irregular loops. Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A. The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem. All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation. alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop. The shortest centre-to-centre distance between chromophores in trimer is 22 A.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Laboratory confirmation of Buruli ulcer disease in Togo, 2007-2010

Background

Since the early 1990s more than 1,800 patients with lesions suspicious for Buruli ulcer disease (BUD) have been reported from Togo. However, less than five percent of these were laboratory confirmed. Since 2007, the Togolese National Buruli Ulcer Control Program has been supported by the German Leprosy and Tuberculosis Relief Association (DAHW). Collaboration with the Department for Infectious Diseases and Tropical Medicine (DITM), University Hospital, Munich, Germany, allowed IS2404 PCR analysis of diagnostic samples from patients with suspected BUD during a study period of three years.

Methodology/Principal Findings

The DAHW integrated active BUD case finding in the existing network of TB/Leprosy Controllers and organized regular training and outreach activities to identify BUD cases at community level. Clinically suspected cases were referred to health facilities for diagnosis and treatment. Microscopy was carried out locally, external quality assurance (EQA) at DITM. Diagnostic samples from 202 patients with suspected BUD were shipped to DITM, 109 BUD patients (54%) were confirmed by PCR, 43 (29.9%) by microscopy. All patients originated from Maritime Region. EQA for microscopy resulted in 62% concordant results.

Conclusions/Significance

This study presents a retrospective analysis of the first cohort of clinically suspected BUD cases from Togo subjected to systematic laboratory analysis over a period of three years and confirms the prevalence of BUD in Maritime Region. Intensified training in the field of case finding and sample collection increased the PCR case confirmation rate from initially less than 50% to 70%. With a PCR case confirmation rate of 54% for the entire study period the WHO standards (case confirmation rate ≥50%) have been met. EQA for microscopy suggests the need for intensified supervision and training. In January 2011 the National Hygiene Institute, Lomé, has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.     

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from <Emphasis Type="Italic">Mycoplasma suis</Emphasis>

Background  

Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.     

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K(+) channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na(+) channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNa(V)1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state ("ultra-slow inactivation," I(US)) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of I(US). The fact that mutations at site 1575 modulate I(US) produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.     

Do Mirror Glasses Have the Same Effect on Brain Activity as a Mirror Box? Evidence from a Functional Magnetic Resonance Imaging Study with Healthy Subjects

Since its original proposal, mirror therapy has been established as a successful neurorehabilitative intervention in several neurological disorders to recover motor function or to relieve pain. Mirror therapy seems to operate by reactivating the contralesional representation of the non-mirrored limb in primary motor- and somatosensory cortex. However, mirror boxes have some limitations which prompted the use of additional mirror visual feedback devices. The present study evaluated the utility of mirror glasses compared to a mirror box. We also tested the hypothesis that increased interhemispheric communication between the motor hand areas is the mechanism by which mirror visual feedback recruits the representation of the non-mirrored limb. Therefore, mirror illusion capacity and brain activations were measured in a within-subject design during both mirror visual feedback conditions in counterbalanced order with 20 healthy subjects inside a magnetic resonance imaging scanner. Furthermore, we analyzed task-dependent functional connectivity between motor hand representations using psychophysiological interaction analysis during both mirror tasks. Neither the subjective quality of mirror illusions nor the patterns of functional brain activation differed between the mirror tasks. The sensorimotor representation of the non-mirrored hand was recruited in both mirror tasks. However, a significant increase in interhemispheric connectivity between the hand areas was only observed in the mirror glasses condition, suggesting different mechanisms for the recruitment of the representation of the non-mirrored hand in the two mirror tasks. We conclude that the mirror glasses might be a promising alternative to the mirror box, as they induce similar patterns of brain activation. Moreover, the mirror glasses can be easy applied in therapy and research. We want to emphasize that the neuronal mechanisms for the recruitment of the affected limb representation might differ depending on conceptual differences between MVF devices. However, our findings need to be validated within specific patient groups.     

The twin-arginine translocation (Tat) system is essential for Rhizobium-legume symbiosis

The Tat (twin-arginine translocation) system mediates export of periplasmic proteins in folded conformation. Proteins transported via Tat contain a characteristic twin-arginine motif in their signal peptide. Genetic determinants (tatABC genes) of the Tat system from Rhizobium leguminosarum bv. viciae were cloned and characterized, and a tatBC deletion mutant was constructed. The mutant lacked the ability for membrane targeting of hydrogenase, a known Tat substrate, and was impaired in hydrogenase activity. Interestingly, in the absence of a functional Tat system, only small, white nodules unable to fix nitrogen were induced in symbiosis with pea plants. Analysis of nodule structure and location of green fluorescent protein (GFP)-tagged bacteria within nodules indicated that the symbiotic process was blocked in the tat mutant at a stage previous to bacteria release into cortical cells. The R. leguminosarum Tat-deficient mutant lacked a functional cytochrome bc1 complex. This was consistent with the fact that R. leguminosarum Rieske protein, a key component of the symbiosis-essential cytochrome bc1 complex, contained a typical twin-arginine signal peptide. However, comparative analyses of nodule structure indicated that nodule development in the tat mutant was arrested at an earlier step than in a cytochrome bc1 mutant. These data indicate that the Tat pathway is also critical for proteins relevant to the initial stages of the symbiotic process.     

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

Background

As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.

Methodology/Principal Findings

Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.

Conclusions/Significance

Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.