Proton exclusion by an aquaglyceroprotein: a voltage clamp study

BACKGROUND INFORMATION: In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. RESULTS: To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7x10(-14) cm(3).subunit(-1).s(-1). Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H(+) (OH(-)) to H(2)O molecules transport stoichiometry of 2x10(-9). A significant GlpF-mediated ion conductivity was also not detectable. CONCLUSIONS: The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.     

An absorbable catheter system for use in microvascular and peripheral vascular surgery: an experimental study in the canine.

An absorbable catheter for use in regional anticoagulation in microvascular and peripheral vascular surgery was studied in 20 sites in 10 adult beagle dogs to answer three questions: (1) Could the polyglycolic acid and trimethylene carbonate catheter withstand intraarterial pressures of infusion and completely absorb over a predictable time interval? (2) Could the catheter be filled with heparin and maintain patency for reuse after a 24-hour interval? (3) Could the catheter be placed in a side branch of a major artery and, after catheter dissolution, maintain long-term patency of the primary feeding artery? The catheters were completely absorbed from 24 to 34 weeks following implantation. The catheters were able to withstand intraarterial pressures, and no evidence of significant thrombosis of the primary feeding artery was seen in any animal studied. No complications of catheter leak, hematoma formation within the catheter placement sites, or sepsis were noted in any of the 20 catheter sites studied.     

Beta clamp directs localization of mismatch repair in Bacillus subtilis

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an interaction with beta clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of the beta clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with beta clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and beta is the major molecular interaction facilitating focus formation and that beta clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with beta clamp.     

Surgical repair of an esophageal perforation after radiofrequency catheter ablation for atrial fibrillation

Recent reports have described the incidence of atrioesophageal fistulas (AEF), often resulting in death, from radiofrequency (RF) catheter ablation of atrial fibrillation (AF).¹ Cases of esophageal perforation without concomitant AEF have not been described as extensively.¹ The precise mechanisms leading to esophageal injury after catheter ablation without involvement of the left atrium are not fully understood. The surgical approach to treat esophageal perforation is strongly recommended.² However, a unified surgical treatment approach has not yet been established. We describe a case of successful surgical repair of an esophageal perforation after ablation using surgical repair in combination with an omental wrap.     

Determination of the hydraulic conductivity of Lamprothamnium by use of the pressure clamp

By use of the pressure-clamp technique, the hydraulic conductivity of the brackish-water alga Lamprothamnium was found to be 5·10^-6 cm s^-1 bar^-1. The dimensions of the internodes are so small that it is possible, for the first time, to measure a complete volume relaxation upon clamping the turgor pressure to a preset value by a feedback control of the pressure probe. As theoretically predicted, the values of the hydraulic conductivity obtained from the initial slope of the volume relaxation induced by the pressure clamp are in agreement (within experimental error) with those obtained from the half-time of the relaxation process. The cell volume also obtained from the analysis of the volume relaxation is the osmotically effective cell volume and is therefore slightly smaller than the value obtained by taking the dimensions of the cell including the cell wall.Abbreviations and symbols Lp hydraulic conductivity - P turgor pressure - S_v initial slope of volume relaxion - T_1/2 half-time of volume relaxation Dedicated to Professor Dr. H. Ziegler on the occasion of his 60th birthday     

Studying mechanosensitive ion channels with an automated patch clamp

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.     

Parotid secretory protein is an HDL-associated protein with anticandidal activity

High-density lipoprotein (HDL) is part of innate immunity, protecting against infection and inflammation. Using a proteomic approach, we identified an amino acid sequence in a hamster HDL protein that showed homology to rat and mouse parotid secretory protein (PSP), a salivary protein secreted from the parotid glands. We cloned the cDNA encoding a putative hamster homolog of rat and mouse PSP. Searches for conserved domains of the protein showed that the COOH terminus of hamster PSP contains a region homologous to the NH2 termini of a family of HDL-associated proteins, including LPS-binding protein, cholesteryl ester transfer protein, and phospholipid transfer protein. In mice, PSP was also associated with HDL but was not detected in very-low-density lipoprotein, low-density lipoprotein, or lipoprotein-deficient sera. In addition to salivary glands, we found that PSP mRNA was expressed in lung, testis, and ovary. The level of PSP in HDL was increased after endotoxin injection in hamsters, but not in mice. Recombinant PSP inhibits growth of Candida albicans in culture. In summary, our results showed that PSP is a novel anticandidal protein associated with HDL.     

Reduction of gene repair by selenomethionine with the use of single-stranded oligonucleotides

Background  

The repair of single base mutations in mammalian genes can be directed by single-stranded oligonucleotides in a process known as targeted gene repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which gene repair takes place.     

The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair

The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.     

Escherichia coli mismatch repair protein MutL interacts with the clamp loader subunits of DNA polymerase III

It has been hypothesized that DNA mismatch repair (MMR) is coupled with DNA replication; however, the involvement of DNA polymerase III subunits in bacterial DNA MMR has not been clearly elucidated. In an effort to better understand the relationship between these 2 systems, the potential interactions between the Escherichia coli MMR protein and the clamp loader subunits of E. coli DNA polymerase III were analyzed by far western blotting and then confirmed and characterized by surface plasmon resonance (SPR) imaging. The results showed that the MMR key protein MutL could directly interact with both the individual subunits delta, delta', and gamma and the complex of these subunits (clamp loader). Kinetic parameters revealed that the interactions are strong and stable, suggesting that MutL might be involved in the recruitment of the clamp loader during the resynthesis step in MMR. The interactions between MutL, the delta and gamma subunits, and the clamp loader were observed to be modulated by ATP. Deletion analysis demonstrated that both the N-terminal residues (1-293) and C-terminal residues (556-613) of MutL are required for interacting with the subunits delta and delta'. Based on these findings and the available information, the network of interactions between the MMR components and the DNA polymerase III subunits was established; this network provides strong evidence to support the notion that DNA replication and MMR are highly associated with each other.     

Successful replantation of an amputated nasal tip by microvascular anastomosis

Successful microsurgical replantation of a completely avulsed nasal tip is presented. The result is excellent. The technical challenge involved no sizable veins for drainage. This problem was solved by an artery-to-vein shunting. For facial avulsions, there is nothing superior to the original tissue, and replantation should always be attempted first.     

Completely successful replantation of an amputated ear by microvascular anastomosis

A completely successful microvascular replantation of an avulsed ear using the superficial temporal bundle is presented, and the advantages of this procedure are discussed.     

Effective use of a joystick by an infant monkey

The acquisition of the ability to use a joystick to maintain contact between a cursor and moving target is described for an infant bonnet macaque (Macaca radiata). The infant was first exposed to a very elementary joystick task while living in a social group at the age of 3.5 months. With task difficulty increased in small increments over a total of only 9 weeks of access to the tasks, the infant was able to maintain contact with a small moving target for over 2.5 sec by the age of 7.5 months. © 1994 Wiley-Liss, Inc.