Facile detection of protein-protein interactions by one-dimensional NMR spectroscopy

Two methods for detecting protein-protein interactions in solution using one-dimensional (1D) NMR spectroscopy are described. Both methods rely on measurement of the intensity of the strongest methyl resonance (SMR), which for most proteins is observed at 0.8-0.9 ppm. The severe resonance overlap in this region facilitates detection of the SMR at low micromolar and even sub-micromolar protein concentrations. A decreased SMR intensity in the 1H NMR spectrum of a protein mixture compared to the added SMR intensities of the isolated proteins reports that the proteins interact (SMR method). Decreased SMR intensities in 1D 13C-edited 1H NMR spectra of 13C-labeled proteins upon addition of unlabeled proteins or macromolecules also demonstrate binding (SMRC method). Analysis of the interaction between XIAP and Smac, two proteins involved in apoptosis, illustrates both methods. A study showing that phospholipids compete with the neuronal core complex for Ca2+-dependent binding to the presynaptic Ca2+-sensor synaptotagmin 1 illustrates the usefulness of the SMRC method in studying multicomponent systems.     

Sporotrichosis Successfully Treated with Terbinafine and Potassium Iodide: Case Report and Review of the Literature

Sporotrichosis is rare in Turkey. We report a 40-year-old woman who had subcutaneous sporotrichosis caused by sporothrix schenckii that was successfully treated with terbinafine (250 mg, twice a day) for a period of 6 months. She received a saturated solution of potassium iodide orally for two months. Terbinafine and potassium iodide are suggested to be the agents of choice for treatment of subcutaneous sporotrichosis.     

Enzymic activity of the K5-type yeast killer toxin and its characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).     

Association between circulating irisin levels and epicardial fat in patients with treatment-naïve overt hyperthyroidism

Abstract

Background: Hyperthyroidism is associated with increased metabolic activity and thermogenesis. Irisin is a key molecule in thermogenesis and energy expenditure via adipose tissue browning. Epicardial fat was previously defined as brown-like fat. Thus, here we aimed to evaluate the association between serum irisin level and epicardial fat thickness (EFT) in patients with hyperthyroidism.

Methods: A total of 25 hyperthyroid patients and 24 age-, sex- and BMI-matched healthy controls were enrolled. Serum irisin levels, thyroid hormone levels, and body compositions were compared. EFT was measured via transthoracic echocardiography.

Results: Serum irisin level and EFT were significantly higher in the hyperthyroid group (p?<?0.001 and p?=?0.001, respectively). The distributions of fat-free mass, muscle mass and fat mass were similar between the study groups. Serum irisin level was negatively correlated with TSH (p?<?0.001) and positively correlated with fT3 (p?<?0.001), fT4 (p?<?0.001) and TSH receptor antibody (p?=?0.002) levels and EFT (p?=?0.001). In multivariate linear regression analysis, TSH (β?=??0.475, p?<?0.001) and EFT (β?=?0.290, p?=?0.023) levels were significantly associated with serum irisin levels.

Conclusions: An increased serum irisin level associated with EFT might contribute to metabolic derangement in hyperthyroidism. Further studies are needed to elucidate whether irisin levels and EFT are affected by hyperthyroidism or vice versa.     

Pretreatment Effect of Resveratrol on Streptozotocin-Induced Diabetes in Rats

This study was planned to investigate the pretreatment effect of resveratrol on streptozotocin-induced diabetic rats. The control group consisted of 10 male albino Sprague–Dawley rats, 10–12 weeks of age, weighing approximately 295 g. The first experimental group consisted of 15 albino Sprague–Dawley rats, 10–12 weeks of age, weighing approximately 305 g. This group was administered streptozotocin (55 mg/kg, intraperitoneally). The second experimental group (n = 15) was administered resveratrol (0.5 ml/day) 10 days before streptozotocin induction. A training period was performed for all groups before the experimental procedure, and systolic arterial blood pressures and heart rates were recorded daily. At the end of the 10th day, blood samples of control and experimental groups were drawn. Total nitrite, nitrite, nitrate, malondialdehyde (MDA), copper, and zinc concentrations in plasma were measured both in control and experimental groups. Additionally, superoxide dismutase, catalase activities, and copper and zinc concentrations in red cell were determined in each group. At the end of the study, increases in catalase activity, nitric oxide level, and zinc concentrations and decreases in lipid peroxidation product MDA and copper concentrations were found in the resveratrol-pretreated diabetic group when compared to the diabetic group. This study was presented at “The 5th International Congress of Pathophysiology (ISP2006)” June 28–July 1, 2006, Beijing, China.     

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

The tunnel region at triosephosphate isomerase (TIM)’s dimer interface, distant from its catalytic site, is a target site for certain benzothiazole derivatives that inhibit TIM’s catalytic activity in Trypanosoma cruzi, the parasite that causes Chagas disease. We performed multiple 100-ns molecular-dynamics (MD) simulations and elastic network modeling (ENM) on both apo and complex structures to shed light on the still unclear inhibitory mechanism of one such inhibitor, named bt10. Within the time frame of our MD simulations, we observed stabilization of aromatic clusters at the dimer interface and enhancement of intersubunit hydrogen bonds in the presence of bt10, which point to an allosteric effect rather than destabilization of the dimeric structure. The collective dynamics dictated by the topology of TIM is known to facilitate the closure of its catalytic loop over the active site that is critical for substrate entrance and product release. We incorporated the ligand’s effect on vibrational dynamics by applying mixed coarse-grained ENM to each one of 54,000 MD snapshots. Using this computationally efficient technique, we observed altered collective modes and positive shifts in eigenvalues due to the constraining effect of bt10 binding. Accordingly, we observed allosteric changes in the catalytic loop’s dynamics, flexibility, and correlations, as well as the solvent exposure of catalytic residues. A newly (to our knowledge) introduced technique that performs residue-based ENM scanning of TIM revealed the tunnel region as a key binding site that can alter global dynamics of the enzyme.     

The interrelations of radiologic findings and mechanical ventilation in community acquired pneumonia patients admitted to the intensive care unit: a multicentre retrospective study

Background

Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.

Findings

Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [³H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro.

Conclusions

These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.     

Nonionic, Water Self-Dispersible "Hairy-Rod" Poly(p-phenylene)-g-poly(ethylene glycol) Copolymer/Carbon Nanotube Conjugates for Targeted Cell Imaging

The generation and fabrication of nanoscopic structures are of critical technological importance for future implementations in areas such as nanodevices and nanotechnology, biosensing, bioimaging, cancer targeting, and drug delivery. Applications of carbon nanotubes (CNTs) in biological fields have been impeded by the incapability of their visualization using conventional methods. Therefore, fluorescence labeling of CNTs with various probes under physiological conditions has become a significant issue for their utilization in biological processes. Herein, we demonstrate a facile and additional fluorophore-free approach for cancer cell-imaging and diagnosis by combining multiwalled CNTs with a well-known conjugated polymer, namely, poly(p-phenylene) (PP). In this approach, PP decorated with poly(ethylene glycol) (PEG) was noncovalently (π-π stacking) linked to acid-treated CNTs. The obtained water self-dispersible, stable, and biocompatible f-CNT/PP-g-PEG conjugates were then bioconjugated to estrogen-specific antibody (anti-ER) via -COOH functionalities present on the side-walls of CNTs. The resulting conjugates were used as an efficient fluorescent probe for targeted imaging of estrogen receptor overexpressed cancer cells, such as MCF-7. In vitro studies and fluorescence microscopy data show that these conjugates can specifically bind to MCF-7 cells with high efficiency. The represented results imply that CNT-based materials could easily be fabricated by the described approach and used as an efficient "fluorescent probe" for targeting and imaging, thereby providing many new possibilities for various applications in biomedical sensing and diagnosis.