Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the two-component signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors. Chemotaxis receptors require dimerization for activity and are in vivo-organized in large clusters. In this study we investigated the oligomerization and aggregation states of pHtrII-cyt by using chemical cross-linking, analytical gel-filtration chromatography, and small-angle neutron scattering. We show that pHtrII-cyt is monomeric in dilute buffers, but forms dimers in 4 M KCl, the physiological salt concentration for halophilic archaea. At high ammonium sulfate concentration, the protein forms higher-order aggregates. The monomeric protein has a rod-like shape, 202 A in length and 14.4 A in diameter; upon dimerization the length increases to 248 A and the diameter to 18.2 A. These results suggest that under high salt concentration the shape and oligomerization state of pHtrII-cyt are comparable to those of chemotaxis receptors.     

The gene expression and activity of calpains and the muscle wasting-associated ubiquitin ligases, atrogin-1 and MuRF1, are not altered in patients with primary hyperparathyroidism

Hyperparathyroidism (HPT) can be associated with muscle atrophy and weakness. Muscle atrophy is typically caused by increased muscle protein breakdown. The influence of HPT on calpains and the ubiquitin-proteasome pathway, which are important regulators of muscle proteolysis, is not yet known. We examined the expression in skeletal muscle of mu- and m-calpain and the ubiquitin ligases, atrogin-1 and MuRF1, in patients with primary HPT. A biopsy was obtained from the sternohyoid muscle in patients undergoing surgery for primary HPT (n=8) and in normocalcemic control patients undergoing thyroid surgery (n=11). mRNA levels for atrogin-1, MuRF1 and the calcium-regulated proteases, mu- and m-calpain, were determined by real-time PCR. Calpain activity was measured using the calpain-specific substrate, BODIPY-FL-casein, and by zymography. Serum calcium was 11.4+/-0.46 and 9.5+/-0.10 mg/dl in HPT and control patients, respectively (p<0.01). The corresponding phosphate levels were 2.7+/-0.2 and 3.6+/-0.1 mg/dl (p<0.05). Parathyroid hormone serum concentration was 286+/-103 pg/ml (range, 77-946 pg/ml) in patients with HPT and was not measured in control patients. There were no significant differences in mRNA levels for atrogin-1, MuRF1, mu- or m-calpain and in calpain activity between HPT and control patients. The results suggest that the ubiquitin-proteasome and calpain systems are not activated in skeletal muscle in patients with primary HPT, at least not in patients with moderate hypercalcemia.     

Beta-amyloid-dependent expression of NOS2 in neurons: prevention by an alpha2-adrenergic antagonist

The neurotransmitter noradrenaline (NA) exerts important antiinflammatory effects on glial cells including suppression of the inducible form of nitric oxide synthase (NOS2). The authors examined the consequences of manipulating NA in vivo by treating adult rats with the neurotoxin DSP4, which selectively lesions noradrenergic neurons of the locus ceruleus (LC), and reduces cortical NA levels. Following LC lesion, intracortical injection of aggregated amyloid beta 1-42 (Abeta1-42) caused appearance of NOS2 within neurons, and increased neuronal damage assessed by staining for nonphosphorylated neurofilament proteins with antibody SMI-32. Co-treatment with a selective alpha2-adrenergic antagonist reduced neuronal NOS2 staining as well as SMI-32 staining. Neuronal damage was dependent on NOS2 expression since injection of Abeta1-42 into DSP4-treated NOS2-deficient mice did not result in neuronal damage. These results demonstrate that decrease of NA levels in vivo can exacerbate inflammatory responses and neuronal damage due to inflammatory stimuli such as Abeta. These findings suggest that alpha2-adrenergic antagonists could provide therapeutic benefit in neurological diseases such as AD or PD where LC loss is known to occur.     

Random phase detection in multidimensional NMR

Despite advances in resolution accompanying the development of high-field superconducting magnets, biomolecular applications of NMR require multiple dimensions in order to resolve individual resonances, and the achievable resolution is typically limited by practical constraints on measuring time. In addition to the need for measuring long evolution times to obtain high resolution, the need to distinguish the sign of the frequency constrains the ability to shorten measuring times. Sign discrimination is typically accomplished by sampling the signal with two different receiver phases or by selecting a reference frequency outside the range of frequencies spanned by the signal and then sampling at a higher rate. In the parametrically sampled (indirect) time dimensions of multidimensional NMR experiments, either method imposes an additional factor of 2 sampling burden for each dimension. We demonstrate that by using a single detector phase at each time sample point, but randomly altering the phase for different points, the sign ambiguity that attends fixed single-phase detection is resolved. Random phase detection enables a reduction in experiment time by a factor of 2 for each indirect dimension, amounting to a factor of 8 for a four-dimensional experiment, albeit at the cost of introducing sampling artifacts. Alternatively, for fixed measuring time, random phase detection can be used to double resolution in each indirect dimension. Random phase detection is complementary to nonuniform sampling methods, and their combination offers the potential for additional benefits. In addition to applications in biomolecular NMR, random phase detection could be useful in magnetic resonance imaging and other signal processing contexts.     

Radiation Synthesis of Linear and Crosslinked Poly[2‐(methacryloyloxy)ethyl]trimethylammonium Chloride and Complex Formation with Potassium Hexacyanoferrates (II,III) in Aqueous Solutions

Linear and crosslinked poly[2‐(methacryloyloxy)ethyl]trimethylammonium chloride was synthesized by γ‐irradiation polymerization in aqueous solutions. Interactions between linear poly[2‐(methacryloyloxy) ethyl]trimethylammonium chloride and low molecular coordination compounds K₃[Fe(CN)₆] and K₄[Fe(CN)₆] have been studied in aqueous solutions by viscometric and turbidimetric measurements. The formation of complexes with certain stoichiometry depending on the nature of the low molecular ion was detected. Hydrogels of poly[2‐(methacryloyloxy)ethyl]trimethylammonium chloride absorb these salts and undergo contraction. The stability of the complexes formed in different ionic strength media has been evaluated.     

Anemia in patient with primary hyperoxaluria and bone marrow involvement by oxalate crystals

We present a rare case of anaemia secondary to bone marrow infiltration by oxalate crystals and renal failure in a patient diagnosed with primary hyperoxaluria. In our case, the anaemia was recovered after the double liver and kidney transplantation, the latter was performed on two occasions after the failure of the first graft.     

HbiF regulates type 1 fimbriation independently of FimB and FimE

Type 1 fimbriae have been suggested to play a role in the pathogenesis of extraintestinal Escherichia coli infection. Type 1 fimbriation in E. coli is phase variable and known to be dependent upon FimB and FimE, the two recombinases that invert the molecular switch fimS and control the expression of the downstream fim operon. Here we showed that HbiF, a novel site-specific recombinase, inverted fimS independently of FimB and FimE. HbiF-mediated fimS inversion appeared to be predominantly switching from "off" (termed OFF) to "on" (termed ON) orientation. This is different from the fimS inversion mediated by either FimB (bidirectional ON to OFF and OFF to ON) or FimE (unidirectional ON to OFF). Constitutive expression of the hbiF gene in E. coli resulted in a fimS-locked-ON phenotype, which resulted in the pathogenic E. coli K1 strain being incapable of inducing a high degree of bacteremia in neonatal rats. Discovery of HbiF-mediated OFF-to-ON fimS switching provides a new opportunity to develop a strategy for the prevention and therapy of extraintestinal E. coli infection including bacteremia and meningitis.     

Phase‐contrast magnetic resonance imaging measurements in intracranial aneurysms in vivo of flow patterns,velocity fields,and wall shear stress: Comparison with computational fluid dynamics

Evolution of intracranial aneurysms is known to be related to hemodynamic forces such as wall shear stress (WSS) and maximum shear stress (MSS). Estimation of these parameters can be performed using numerical simulations with computational fluid dynamics (CFD), but can also be directly measured with magnetic resonance imaging (MRI) using a time‐dependent 3D phase‐contrast sequence with encoding of each of the three components of the velocity vectors (7D‐MRV). To study the accuracy of 7D‐MRV in estimating these parameters in vivo, in comparison with CFD, 7D‐MRV and patient‐specific CFD modeling was performed for 3 patients who had intracranial aneurysms. Visual and quantitative analyses of the flow pattern and distribution of velocities, MSS, and WSS were performed using the two techniques. Spearman's coefficients of correlation between the two techniques were 0.56 for the velocity field, 0.48 for MSS, and 0.59 for WSS. Visual analysis and Bland–Altman plots showed good agreement for flow pattern and velocities but large discrepancies for MSS and WSS. These results indicate that 7D‐MRV can be used in vivo to measure velocity flow fields and for estimating MSS and WSS. Currently, however, this method cannot accurately quantify the latter two parameters. Magn Reson Med 61:409–417, 2009. © 2009 Wiley‐Liss, Inc.