N-methyl-D-aspartate receptor mechanosensitivity is governed by C terminus of NR2B subunit

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.     

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.     

Role for the alpha-helix in aberrant protein aggregation

Is the alpha-helix structure capable of triggering the formation of aberrant protein aggregates? To answer this question, we investigate the in vitro aggregation of tau protein in the presence of the helix-inducing agent TFE. Tau is a natively unfolded protein that binds to microtubules and forms aggregates in Alzheimer's disease. We find that full-length tau has residual alpha-helix structure, which is further enhanced by three mutations involved in genetic neurological disorders. TFE concentrations matching an alpha-helical content of 40% in full-length tau and the triple mutant induce the formation of aggregates that are morphologically and structurally heterogeneous. A simple dilution experiment reveals that heterogeneity results from the competition between alpha-helical fibrillar aggregates and more classical amyloid-like aggregates. The alpha-helical aggregates are more resilient to dilution and have the spectroscopic features of alpha-helical coiled coils. We propose a general mechanism by which intrinsically stable alpha-helices can associate into aggregates with only coarse coiled-coil symmetry. In tau, high intrinsic alpha-helix stability and coarse coiled-coil symmetry could be byproducts of its biological function.     

A second serogroup of Legionella erythra serologically indistinguishable from Legionella rubrilucens.

Twenty-two red-autofluorescent Legionella strains were identified serologically as either Legionella rubrilucens or L. erythra. A rRNA probe was used for restriction fragment length polymorphism (RFLP) analysis of the strains and the patterns generated were used as an additional method of identifying the strains to species level. In two instances strains which were identified as L. rubrilucens by serology appeared to belong to the species L. erythra by RFLP analysis. This apparent contradiction was resolved by measurements of DNA/DNA homology which confirmed the existence of a second serogroup of L. erythra serologically indistinguishable from L. rubrilucens.     

Typing of Legionella pneumophila serogroups 2–14 strains by analysis of restriction fragment length polymorphisms

A typing method based on analysis of restriction fragment length polymorphisms has previously been developed for Legionella pneumophila serogroup 1. Here data are presented demonstrating the utility of this method for typing strains of all other L. pneumophila serogroups described to date. The method, which is highly discriminatory, should be of considerable value in epidemiological investigations of legionella infections.     

Tracheal aspiration cytology in neonates with respiratory distress: histopathologic correlation

This report evaluates the use of tracheal aspiration cytology in the differential diagnosis of respiratory distress in neonates by correlating cytologic features with histopathologic findings in 72 infants who died and were autopsied at Magee-Womens Hospital. It is concluded that the common causes of respiratory distress in neonates, including hyaline membrane disease (conventional and yellow), pneumonia, aspiration syndrome, pulmonary hemorrhage and bronchopulmonary dysplasia, may be diagnosed in adequate smears by this easily accessible, noninvasive, safe and repetitive method.     

Increased Inlet Blood Flow Velocity Predicts Low Wall Shear Stress in the Cephalic Arch of Patients with Brachiocephalic Fistula Access

BackgroundAn autogenous arteriovenous fistula is the optimal vascular access for hemodialysis. In the case of brachiocephalic fistula, cephalic arch stenosis commonly develops leading to access failure. We have hypothesized that a contribution to fistula failure is low wall shear stress resulting from post-fistula creation hemodynamic changes that occur in the cephalic arch.MethodsTwenty-two subjects with advanced renal failure had brachiocephalic fistulae placed. The following procedures were performed at mapping (pre-operative) and at fistula maturation (8–32 weeks post-operative): venogram, Doppler to measure venous blood flow velocity, and whole blood viscosity. Geometric and computational modeling was performed to determine wall shear stress and other geometric parameters. The relationship between hemodynamic parameters and clinical findings was examined using univariate analysis and linear regression.ResultsThe percent low wall shear stress was linearly related to the increase in blood flow velocity (p < 0.01). This relationship was more significant in non-diabetic patients (p < 0.01) than diabetic patients. The change in global measures of arch curvature and asymmetry also evolve with time to maturation (p < 0.05).ConclusionsThe curvature and hemodynamic changes during fistula maturation increase the percentage of low wall shear stress regions within the cephalic arch. Low wall shear stress may contribute to subsequent neointimal hyperplasia and resultant cephalic arch stenosis. If this hypothesis remains tenable with further studies, ways of protecting the arch through control of blood flow velocity may need to be developed.     

Heart rate measures in blind cave crayfish during environmental disturbances and social interactions

Most animals continually assess the environment in which they live and alter their behavior according to various stimuli. As an observer, one looks for changes in a behavior indicating that an animal responded to a particular event. When the animal does not make significant behavioral changes as measured by bodily movements, the animal may be characterized as unresponsive to a given stimulus. This study demonstrates that when behavioral body movements can not be observed an internal physiological measure of heart rate (HR) shows dramatic changes following presentation of defined stimuli. This study used the blind cave crayfish and examined their responsiveness to the following stimuli: light (infrared, dim red, and white), water-borne vibrations, removal of water, olfactory cues, and social interaction with partners. This study demonstrates that there is substantial individual variation of HR at basal levels and with the intensity of an social interaction. We find HR is a reasonable measure of the responsiveness of blind cave crayfish to given stimuli even in the absence of observable behavioral changes. This enables the observer to determine if an individual is responsive to and making an assessment of particular cues.