Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Optimization of pulsed electromagnetic field therapy for management of arthritis in rats

Studies were undertaken to find out the effects of low frequency pulsed electromagnetic field (PEMF) in adjuvant induced arthritis (AIA) in rats, a widely used model for screening potential therapies for rheumatoid arthritis (RA). AIA was induced by an intradermal injection of a suspension of heat killed Mycobacterium tuberculosis (500 mug/0.1 ml) into the right hind paw of male Wistar rats. This resulted in swelling, loss of body weight, increase in paw volume as well as the activity of lysosomal enzymes viz., acid phosphatase, cathepsin D, and beta-glucuronidase and significant radiological and histological changes. PEMF therapy for arthritis involved optimization of three significant factors, viz., frequency, intensity, and duration; and the waveform used is sinusoidal. The use of factorial design in lieu of conventional method resulted in the development of an ideal combination of these factors. PEMF was applied using a Fransleau-Braunbeck coil system. A magnetic field of 5 Hz x 4 muT x 90 min was found to be optimal in lowering the paw edema volume and decreasing the activity of lysosomal enzymes. Soft tissue swelling was shown to be reduced as evidenced by radiology. Histological studies confirmed reduction in inflammatory cells infiltration, hyperplasia, and hypertrophy of cells lining synovial membrane. PEMF was also shown to have a membrane stabilizing action by significantly inhibiting the rate of release of beta-glucuronidase from lysosomal rich and sub-cellular fractions. The results indicated that PEMF could be developed as a potential therapy in the treatment of arthritis in humans.     

Efficient expression of transgenes in adult zebrafish by electroporation

Background  

Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish.     

Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas. We have characterized a genetic locus (pig) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Evidence-based de-implementation for contradicted, unproven, and aspiring healthcare practices

Abandoning ineffective medical practices and mitigating the risks of untested practices are important for improving patient health and containing healthcare costs. Historically, this process has relied on the evidence base, societal values, cultural tensions, and political sway, but not necessarily in that order. We propose a conceptual framework to guide and prioritize this process, shifting emphasis toward the principles of evidence-based medicine, acknowledging that evidence may still be misinterpreted or distorted by recalcitrant proponents of entrenched practices and other biases.     

Associations between COPD related manifestations: a cross-sectional study

Background

Cardiovascular disease, osteoporosis and emphysema are associated with COPD. Associations between these factors and whether they predict all-cause mortality in COPD patients are not well understood. Therefore, we examined associations between markers of cardiovascular disease (coronary artery calcification [CAC], thoracic aortic calcification [TAC] and arterial stiffness), bone density (bone attenuation of the thoracic vertebrae), emphysema (PI-950 and 15th percentile) and all-cause mortality in a COPD cohort.

Methods

We assessed CAC, TAC, bone attenuation of the thoracic vertebrae, PI-950 and 15th percentile on low-dose chest computed tomography in COPD subjects. We measured arterial stiffness as carotid-radial pulse wave velocity (PWV), and identified deaths from the national register.

Results

We studied 119 COPD subjects; aged 67.8 ±7.3, 66% were males and mean FEV₁% predicted was 46.0 ±17.5. Subjects were classified into three pre-specificed groups: CAC = 0 (n = 14), 0 < CAC ≤ 400 (n = 41) and CAC > 400 (n = 64). Subjects with higher CAC were more likely to be older (p < 0.001) and male (p = 0.03), and more likely to have higher systolic blood pressure (p = 0.001) and a history of hypertension (p = 0.002) or ischemic heart disease (p = 0.003). Higher CAC was associated with higher PWV (OR 1.62, p = 0.04) and lower bone attenuation (OR 0.32, p = 0.02), but not with 15th percentile, after adjustment for age, sex and pack-years of smoking. In a Cox proportional hazards model, CAC, TAC and 15th percentile predicted all-cause mortality (HR 2.01, 2.09 and 0.66, respectively).

Conclusions

Increased CAC was associated with increased arterial stiffness and lower bone density in a COPD cohort. In addition, CAC, TAC and extent of emphysema predicted all-cause mortality.

Trial registration

Lothian NHS Board, Lothian Research Ethics Committee, LREC/2003/8/28.