One-step purification of a functional, constitutively activated form of visual arrestin

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact? fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho?). The results show that both prodomain/Profinity eXact? fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale? Mini Profinity eXact? cartridge on the Profinia? purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho?.     

Existence of memory in membrane channels: analysis of ion current through a voltage‐dependent potassium single channel

Ion current fluctuation of voltage‐dependent potassium channel in LβT2 cells has been investigated by autocorrelation function and DFA (detrended fluctuation analysis) methods. The calculation of the autocorrelation function exponent and DFA exponent of the sample was based on the digital signals or the 0–1 series corresponding to closing and opening of channels after routine evolution, rather than the sequence of sojourn times. The persistent character of the correlation of the time series was evident from the slow decay of the autocorrelation function. DFA exponent α was significantly greater than 0.5. The main outcome has been the demonstration of the existence of memory in this ion channel. Thus, the ion channel current fluctuation provided information about the kinetics of the channel protein. The result suggests the correlation character of the ion channel protein non‐linear kinetics indicates whether the channel is open or not.     

Mechanisms of Prostate Permeability Triggered by Microbubble-Mediated Acoustic Cavitation

The objective of this research was to study the mechanisms of opening of the blood?Cprostate barrier and increased permeability of prostate tissue induced by microbubble cavitation. Thirty-five rabbits were randomly divided into four study groups: (1) control group and groups exposed to (2) microbubble alone, (3) ultrasound alone, or (4) combined intervention (ultrasound?+?microbubble group). Evans blue (EB) tracer was used to gauge the changes of permeability of prostate tissue. Furthermore, light and electron microscopy analyses were conducted, as well as the western blot analysis of expression of gap junction (Cx43) protein. We observed that EB concentration in prostate tissue was significantly greater in the ultrasound?+?microbubble group compared with either intervention alone (p?<?0.05, both comparisons). Furthermore, light microscopy of tissue samples from animals exposed to ultrasound?+?microbubble showed epithelial cell disarrangement, loss of interstitial structure, and thickness of fibrous stroma. In line with these findings, electron microscopy analysis demonstrated widening of cell gaps and broken cell connections, as well as more dense lysosomes and secretary granules, and mitochondrial swelling. These changes were absent in the animals exposed to microbubble or ultrasound alone. Finally, only combined treatment with microbubble or ultrasound significantly elevated expression of Cx43 (p?<?0.05 vs. control group). In conclusion, increases of permeability of prostate tissue by acoustic cavitation appear to involve opening of tight junctions, widening of intracellular spaces, changes in the structure of acinar cell membrane, enhancement of vesicular transport, and loosening of fibrous stroma. Increased expression of cell gap junction protein will help to restore normal connections between cells and the blood?Cprostate barrier after the treatment.     

Usefulness of Speckle Tracking Imaging to Assess Myocardial Contractility in Intra-Abdominal Hypertension: Study in a Mini-Pig Model

This study evaluated the usefulness of speckle tracking imaging (STI) in assessment of myocardial contractility in intra-abdominal hypertension experimentally induced in mini-pigs. To this effect, 12 mini-pigs were anesthetized with intravenous injection of 3?% sodium pentobarbital, hemorrhaged to reach the shock status, and resuscitated with excessive volume of lactated Ringer??s solution. The animals were either sham-operated (study group 1) or underwent treatment with intra-abdominal volume increment (study group 2). Observations were made prior to induction of shock, 1?h after shock, 2?h after induction of intra-abdominal hypertension, and 8 and 12?h after treatment. The heart rate and mean artery pressure were conventionally measured. STI was used to assess radial and circumferential strains of segmental ventricular wall. The results obtained demonstrated that myocardial contractility, as manifested by radial and circumferential strains of different ventricular wall segments, was decreased after induction of intra-abdominal hypertension. Treatment with intra-abdominal volume increment was able to decrease heart rate and intra-bladder pressure (indicator of effectiveness of treatment) and significantly improved myocardial contractility of involved ventricular wall segments. In conclusion, STI is a useful method to assess myocardial regional functions.     

Polymorphisms in the ERCC5 Gene and Risk of Esophageal Squamous Cell Carcinoma (ESCC) in Eastern Chinese Populations

Background

Excision repair cross complementing group 5 (ERCC5 or XPG) plays an important role in regulating DNA excision repair; its functional single nucleotide polymorphisms (SNPs) may alter DNA repair capacity and thus contribute to cancer risk.

Methodology/Principal Findings

In a hospital-based case-control study of 1115 esophageal squamous cell carcinoma (ESCC) cases and 1117 cancer-free controls, we genotyped three potentially functional SNPs of ERCC5 (SNPs, rs2296147T>C, rs2094258C>T and rs873601G>A) and estimated crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for their associations with risk of ESCC using unconditional logistic regression models. We also calculated false-positive report probabilities (FPRPs) for significant findings. We found that compared with the TT genotype, ERCC5 rs2296147 C variant genotypes were associated with a significantly lower ESCC risk (CT: adjusted OR = 0.76, 95% CI = 0.63–0.93, CT/CC: adjusted OR = 0.80, 95% CI = 0.67–0.96); however, this risk was not observed for the other two SNPs (rs2094258C>T and rs873601 G>A), nor in further stratification and haplotype analysis.

Conclusions/Significances

These findings suggested that ERCC5 polymorphisms may contribute to risk of ESCC in Eastern Chinese populations, but the effect was weak and needs further validation by larger population-based case-control studies.     

Dynamics of Yersinia pestis and Its Antibody Response in Great Gerbils (Rhombomys opimus) by Subcutaneous Infection

Background

Rhombomys opimus (great gerbil) is a reservoir of Yersinia pestis in the natural plague foci of Central Asia. Great gerbils are highly resistant to Y. pestis infection. The coevolution of great gerbils and Y. pestis is believed to play an important role in the plague epidemics in Central Asia plague foci. However, the dynamics of Y. pestis infection and the corresponding antibody response in great gerbils have not been evaluated. In this report, animal experiments were employed to investigate the bacterial load in both the liver and spleen of infected great gerbils. The dynamics of the antibody response to the F1 capsule antigen of Y. pestis was also determined.

Methodology

Captured great gerbils that tested negative for both anti-F1 antibodies and bacterial isolation were infected subcutaneously with different doses (10⁵ to 10¹¹ CFU) of a Y. pestis strain isolated from a live great gerbil during routine plague surveillance in the Junggar Basin, Xinjiang, China. The clinical manifestations, changes in body weight, anal temperature, and gross anatomy of the infected animals were observed. The blood cell count, bacterial load, and anti-F1 antibody titers were determined at different time points after infection using a blood analyzer, plate counts, and an indirect hemagglutination assay, respectively.

Conclusions/Significance

The dynamics of bacterial load and the anti-F1 antibody concentration in great gerbils are highly variable among individuals. The Y. pestis infection in great gerbils could persist as long as 15 days. They act as an appropriate reservoir for plague in the Junggar Basin, which is part of the natural plague foci in Central Asia. The dynamics of the Y. pestis susceptibility of great gerbil will improve the understanding of its variable resistance, which would facilitate the development of more effective countermeasures for controlling plague epidemics in this focus.