Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2.     

Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.     

Desflurane consumption during automated closed-circuit delivery is higher than when a conventional anesthesia machine is used with a simple vaporizer-O2-N2O fresh gas flow sequence

Background

Current analgesics have drawbacks such as delays in acquisition, lag-times for effect, and side effects. We recently presented a preliminary report of a new analgesic method involving a two-minute sciatic nerve press, which resulted in immediate short-term relief of pain associated with dental and renal diseases. The present study investigated whether this technique was effective for pain associated with other disease types, and whether the relief was effective for up to one hour.

Methods

This randomized, placebo-controlled, parallel-group trial was conducted in four hospitals in Anhui Province, China. Patients with pain were sequentially recruited by participating physicians during clinic visits, and 135 patients aged 15 – 80 years were enrolled. Dental disease patients included those with acute pulpitis and periapical abscesses. Renal disease patients included those with kidney infections and/or stones. Tumor patients included those with nose, breast, stomach and liver cancers, while Emergency Room patients had various pathologies. Patients were randomly assigned to receive a "sciatic nerve press" in which pressure was applied simultaneously to the sciatic nerves at the back of both thighs, or a "placebo press" in which pressure was applied to a parallel region on the front of the thighs. Each fist applied a pressure of 11 – 20 kg for 2 minutes. Patients rated their level of pain before and after the procedure.

Results

The "sciatic nerve press" produced immediate relief of pain in all patient groups. Emergency patients reported a 43.5% reduction in pain (p < 0.001). Significant pain relief for dental, renal and tumor patients lasted for 60 minutes (p < 0.001). The peak pain relief occurred at the 10 – 20^th minutes, and the relief decreased 47% by the 60^th minutes.

Conclusion

Two minutes of pressure on both sciatic nerves produced immediate significant short-term conduction analgesia. This technique is a convenient, safe and powerful method for the short-term treatment of clinical pain associated with a diverse range of pathologies.

Trial registration

Current Controlled Trials ACTRN012606000439549     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

Diffusion Tensor Imaging and Decision Making in Cocaine Dependence

Background

Chronic stimulant abuse is associated with both impairment in decision making and structural abnormalities in brain gray and white matter. Recent data suggest these structural abnormalities may be related to functional impairment in important behavioral processes.

Methodology/Principal Findings

In 15 cocaine-dependent and 18 control subjects, we examined relationships between decision-making performance on the Iowa Gambling Task (IGT) and white matter integrity as measured by diffusion tensor imaging (DTI). Whole brain voxelwise analyses showed that, relative to controls, the cocaine group had lower fractional anisotropy (FA) and higher mean of the second and third eigenvalues (λ⊥) in frontal and parietal white matter regions and the corpus callosum. Cocaine subjects showed worse performance on the IGT, notably over the last 40 trials. Importantly, FA and λ⊥ values in these regions showed a significant relationship with IGT performance on the last 40 trials.

Conclusions

Compromised white matter integrity in cocaine dependence may be related to functional impairments in decision making.