Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector

A new method based on fluorescence derivatization with 5‐(dimethylamino) naphthalene‐1‐sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high‐performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C₁₈ column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o‐phosphoric acid, 1.2 ml/min. The injection volume was 20 μl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emission 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27–70.99 and 18.81–212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24–0.59 and 0.35–0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13–0.51 and 0.04–0.15, respectively.     

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.     

Negligible Senescence during Reproductive Dormancy in Drosophila melanogaster

Some endemic Drosophila overwinter in a state of adult reproductive diapause where egg maturation is arrested in previtellogenic stages. When maintained at cool temperatures, adult Drosophila melanogaster enter reproductive dormancy, that is, diapause or diapause-like quiescence. The ability to survive for extended periods is a typical feature of diapause syndromes. In adults this somatic persistence may involve reduced or slowed senescence. Here we assess whether reproductively dormant D. melanogaster age at slow rates. Adults were exposed to dormancy-inducing conditions for 3, 6, or 9 wk. After this period, demographic parameters were measured under normal conditions and compared to the demography of newly eclosed cohorts. The age-specific mortality rates of postdormancy adults were essentially identical to the mortality rates of newly eclosed, young flies. Postdormancy reproduction, in contrast, declined with the duration of the treatment; somatic survival during dormancy may tradeoff with later reproduction. Adults in reproductive dormancy were highly resistant to heat and to oxidative stress. Suppressed synthesis of juvenile hormone is known to regulate reproductive diapause of many insects. Treatment of dormant D. melanogaster with a juvenile hormone analog restored vitellogenesis, suppressed stress resistance, and increased demographic senescence. We conclude that D. melanogaster age at slow rates as part of their reproductive dormancy syndrome; the data do not agree with an alternative hypothesis based on heat-dependent "rate of living." We suggest that low temperature reduces neuroendocrine function, which in turn slows senescence as a function of altered stress response, nutrient reallocation, and metabolism.     

The molecular mechanisms of vulpinic acid induced programmed cell death in melanoma

Molecular Biology Reports - Malignant melanoma is an aggressive skin tumor with a rapidly increasing incidence and there is not yet a successful treatment strategy. Vulpinic acid (VA) is derived...     

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.     

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.     

Effects of some metals on paraoxonase activity from shark Scyliorhinus canicula

Paraoxonase (PON) is an organophosphate hydrolyser enzyme which also has antioxidant properties in metabolism. Due to its crucial functions, the inhibition of the enzyme is undesirable and very dangerous. PON enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Ni(2+), Cd(2+), Cu(2+) and Hg(2+), on the activity of shark serum PON (SPON). For this purpose, we first purified the enzyme from shark Scyliorhinus canicula (LINNAEUS, 1758) serum and analysed the alterations in the enzyme activity in the presence of metal ions. The K(M) and V(max) is 0.227 mM and 454.545 U/mL, respectively. The results show that metal ions exhibit inhibitory effects on SPON1 at low concentrations with IC(50) values ranging from 0.29 to 2.00 mM. Copper was determined to be the most effective inhibitor with IC(50) of 0.29 mM.     

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.