Decreased S-nitrosation of peptide thiols in the membrane interior

It has been proposed that autoxidation of nitric oxide (NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of NO/O₂. The transmembrane cysteinyl peptides C₄ (AcNH-KKACALA(LA)₆KK-CONH₂) and C₈ (AcNH-KKALALACALA(LA)₃KK-CONH₂) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C₈ and C₄, and glutathione (GSH; 300 μM) were treated with a NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O₂ levels. Surprisingly, the more hydrophobic cysteinyl peptide, C₈, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C₄ peptide in the liposomes in the presence of NO/O₂. These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of NO/O₂.     

Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

Conformational Change in MSH2-MSH6 upon Binding DNA Coupled to ATPase Activity

Postreplication DNA mismatch repair is initiated by the eukaryotic protein MSH2-MSH6 or the prokaryotic protein MutS, both showing overall conserved structure and functionality. Crystal structures of MSH2-MSH6 and MutS bound to the mismatch DNA reveal a closed architecture of the clamp and the lever domains exhibiting strong contacts with the bent DNA backbone. Long molecular dynamics simulations of the human MSH2-MSH6 protein in the absence of a DNA show an altered conformation of the protein that reflects the protein's state before binding to DNA. The clamp and the lever domains of both MSH6 and MSH2 open in an asymmetric and dramatic fashion. The opening of the clamp and the lever domains in the absence of DNA is coupled to changes in the ATPase domains, which explains the experimentally observed diminished ATPase activity in DNA-free MSH2-MSH6 and illustrates the allosteric coupling between DNA binding and ATPase activity.     

Deciphering the Mismatch Recognition Cycle in MutS and MSH2-MSH6 Using Normal-Mode Analysis

Postreplication DNA mismatch repair is essential for maintaining the integrity of genomic information in prokaryotes and eukaryotes. The first step in mismatch repair is the recognition of base-base mismatches and insertions/deletions by bacterial MutS or eukaryotic MSH2-MSH6. Crystal structures of both proteins bound to mismatch DNA reveal a similar molecular architecture but provide limited insight into the detailed molecular mechanism of long-range allostery involved in mismatch recognition and repair initiation. This study describes normal-mode calculations of MutS and MSH2-MSH6 with and without DNA. The results reveal similar protein flexibilities and suggest common dynamic and functional characteristics. A strongly correlated motion is present between the lever domain and ATPase domains, which suggests a pathway for long-range allostery from the N-terminal DNA binding domain to the C-terminal ATPase domains, as indicated by experimental studies. A detailed analysis of individual low-frequency modes of both MutS and MSH2-MSH6 shows changes in the DNA-binding domains coupled to the ATPase sites, which are interpreted in the context of experimental data to arrive at a complete molecular-level mismatch recognition cycle. Distinct conformational states are proposed for DNA scanning, mismatch recognition, repair initiation, and sliding along DNA after mismatch recognition. Hypotheses based on the results presented here form the basis for further experimental and computational studies.     

Improving clinical research and cancer care delivery in community settings: evaluating the NCI community cancer centers program

Background

While the evidence suggests that the way physicians provide information to patients is crucial in helping patients decide upon a course of action, the field of knowledge translation and exchange (KTE) is silent about how the physician and the patient influence each other during clinical interactions and decision-making. Consequently, based on a novel relationship-centered model, EXACKTE² (EXploiting the clinicAl Consultation as a Knowledge Transfer and Exchange Environment), this study proposes to assess how patients and physicians influence each other in consultations.

Methods

We will employ a cross-sectional study design involving 300 pairs of patients and family physicians from two primary care practice-based research networks. The consultation between patient and physician will be audio-taped and transcribed. Following the consultation, patients and physicians will complete a set of questionnaires based on the EXACKTE² model. All questionnaires will be similar for patients and physicians. These questionnaires will assess the key concepts of our proposed model based on the essential elements of shared decision-making (SDM): definition and explanation of problem; presentation of options; discussion of pros and cons; clarification of patient values and preferences; discussion of patient ability and self-efficacy; presentation of doctor knowledge and recommendation; and checking and clarifying understanding. Patients will be contacted by phone two weeks later and asked to complete questionnaires on decisional regret and quality of life. The analysis will be conducted to compare the key concepts in the EXACKTE² model between patients and physicians. It will also allow the assessment of how patients and physicians influence each other in consultations.

Discussion

Our proposed model, EXACKTE², is aimed at advancing the science of KTE based on a relationship process when decision-making has to take place. It fosters a new KTE paradigm by putting forward a relationship-centered perspective and has the potential to reveal unknown mechanisms that underline effective KTE in clinical contexts. This will result in better understanding of the mechanisms that may promote a new generation of knowledge transfer strategies.     

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.Hui Zhang and Niko G. Gubernator contributed equally to this work.Download video file.^{(112M, mp4)}     

Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles

Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring β-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound β-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026. The inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.