New simple molecular fluorescent probes for rapid and highly selective sensing of hydrazine by aggregate-induced emission.

With an aim towards the design of efficient and straightforward fluorescent probes for hydrazine, the synthesis of (2-acetoxyaryl) methylene diacetate derivatives (1-4) was carried out by reacting substituted aromatic α-hydroxy aldehydes with acetyl chloride and sodium acetate in excellent yields. As a preliminary investigation, the ability of probe 1 was examined for the detection of substituted aliphatic and aromatic amines, amino acids, and other ions in Britton-Robinson buffer solution (50 mM, water/ethanol v/v of 99/1 at pH 7.4). Probe 1 selectively exhibited an intense blue fluorescence with hydrazine in less than 2 minutes, whereas light green or no fluorescence was noticed with substituted amines and amino acids. Among all the probes employed (1-4) in the present study, probes 1 and 2 were found efficient towards the rapid detection of hydrazine. Furthermore, the fluorescence sensing ability of probes 1 and 2 was tested not only under varying pH conditions but also by varying water-fraction from 0-99%. Moreover, the detection limits of hydrazine using 1 and 2 were found as 8.4 and 8.7 ppb, respectively, which is less than the acceptable limit as per the standards of the US Environment Protection Agency. In this contribution, the probes 1 and 2 demonstrate rapid, selective, sensitive, and ratiometric detection of highly toxic hydrazine by OFF-ON fluorescence switch in water samples as well as living cells.

Radiation damage reveals promising interaction position.

High-resolution structural data of protein inhibitor complexes are the key to rational drug design. Synchrotron radiation allows for atomic resolutions but is frequently accompanied by radiation damage to protein complexes. In this study a human aldose reductase mutant complexed with a bromine-substituted inhibitor was determined to atomic resolution [Protein Data Bank (PDB) code 3onc]. Though the radiation dose was moderate, a selective disruption of a bromine-inhibitor bond during the experiment was observed while the protein appears unaffected. A covalent bond to bromine is cleaved and the displaced atom is not scattered throughout the crystal but can most likely be assigned as a bromide to an additional difference electron density peak observed in the structure. The bromide relocates to an adjacent unoccupied site where promising interactions to protein residues stabilize its position. These findings were verified by a second similar structure determined with considerably higher radiation dose (PDB code 3onb).

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle.

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.

Absorption of 193- and 213-nm laser wavelengths in sodium chloride solution and balanced salt solution.

OBJECTIVE: To determine absorption coefficients for sodium chloride solution (saline) and balanced salt solution at the 193- and 213-nm laser wavelengths. METHODS: Absorption coefficients were obtained for each of the component species found in balanced salt solution. This was achieved by measuring laser pulse transmission through solutions of varying concentration. The experiments were repeated using the 193-nm excimer and 213-nm solid-state laser wavelengths. Results for each species were then used to obtain an overall absorption coefficient and penetration depth for balanced salt solution and 0.9% sodium chloride solution. RESULTS: Absorption coefficients in balanced salt solution for the 193- and 213-nm wavelengths were found to be 140 and 6.9 cm(-1), respectively. In 0.9% sodium chloride solution, the absorption coefficient was 81 cm(-1) at 193 nm and 0.05 cm(-1) at 213 nm. At 193 nm, absorption in balanced salt solution was dominated by sodium chloride. Sodium citrate emerged as the dominant species of absorption at 213 nm. CONCLUSIONS: For the species investigated, we found reduced absorption for the longer wavelength of 213 nm. While the difference in wavelength between 193 and 213 nm is within about 10%, the respective molar absorption coefficients varied by 1 to 4 orders of magnitude. This indicates that predictions for the wavelength-dependent changes of absorption coefficients of other solutions are unreliable. CLINICAL RELEVANCE: Fluid placed on the surface of the cornea during keratorefractive surgery has proved to be a barrier to ablation for the 193-nm wavelength. The increased penetration depth through sodium chloride solution and balanced salt solution for the longer 213-nm laser wavelength may mean that these solutions cannot be used as a masking agent for keratorefractive procedures performed with this wavelength.

Determination of montelukast sodium in human plasma by column-switching high-performance liquid chromatography with fluorescence detection.

MK-0476 (montelukast sodium) is a potent and selective cysteinyl leukotriene receptor antagonist that is being investigated in the treatment of asthma. A simple and sensitive method for the determination of MK-0476 in human plasma was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. A plasma sample was injected directly onto the HPLC system consisting of a pre-column (Capcell pak MF) and an analytical column (Capcell pak C18) which were connected with a six-port switching valve. The column eluate was monitored with a fluorescence detector (excitation at 350 nm; emission at 400 nm). The calibration curve was linear in a concentration range of 1-500 ng ml(-1) for MK-0476 in human plasma. The intra-day coefficients of variation of all concentrations within the range was less than 9.2%, and the intra-day accuracy values were between 97.2 and 114.6%. This method was used to measure the plasma concentration of MK-0476 following oral administration of the drug in humans.

Capacitative Ca2+ entry into Xenopus oocytes is sensitive to omega-conotoxins GVIA, MVIIA and MVIIC.

We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca(2+)-activated Cl- current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1-10 mM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists omega-conotoxin GVIA, MVIIA and MVIIC (0.3-10 microM) and the inhibition of Ca2+ entry in individual oocytes by omega-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 microM) and the imidazoles SK&F 96365 (10 microM), miconazole (40 microM) and econazole (40 microM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or alpha-bungarotoxin (1 microM). The possibility that these compounds might inhibit the Ca(2+)-activated Cl- current rather than capacitative Ca2+ entry itself was examined by recording the Cl- current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2-10 microM) markedly inhibited, and La3+ (1 mM but not 100 microM) potentiated the increase in Ca(2+)-activated Cl- current. In contrast, omega-conotoxins and Mg2+ had no effect on the Ca(2+)-activated Cl- current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

Absorption of 308-nm excimer laser radiation by balanced salt solution, sodium hyaluronate, and human cadaver eyes.

Absorption of the excimer laser radiations of 193-nm argon fluorine and 308-nm xenon chloride in balanced salt solution, sodium hyaluronate, and human cadaver eyes was measured. The absorption of these materials as considerably different for the two wavelengths; we found that 308-nm light experienced much less absorption than the 193-nm light. The extinction coefficient (k) for 308 nm was k = 0.19/cm for balanced salt solution and k = 0.22/cm for sodium hyaluronate. In contrast to this, the extinction coefficient for 193 nm was k = 140/cm for balanced salt solution and k = 540/cm for sodium hyaluronate. Two 1-day-old human phakic cadaver eyes showed complete absorption with both wavelengths. Using aphakic eyes, incomplete absorption was noted at the posterior pole with 308 nm and complete absorption was noted with 193 nm. The extinction in the anterior part of aphakic eyes (the first 6 mm) was 4.2/cm for 308 nm, meaning that the intensity of the light is reduced by a factor of 10 after traveling the first 5.5 mm. However, we observed that the material in the eye fluoresces, meaning the 308 nm is transformed into other (longer) wavelengths that travel through the total eye with minimal absorption. Conclusions drawn from this experiment are that the use of the 308-nm wavelength may have undesirable side effects, while the use of the 193-nm wavelength should be consistent with ophthalmic use on both the cornea and the lens.