Chemical warfare agents: verification of compounds containing the phosphorus-methyl linkage in waste water.

The chemical analysis of the waste water from plants that manufacture chemicals is a nonintrusive method for confirming a suspected violation of the prohibition against the production of chemical warfare agents. The chemical structure of most nerve gases is related to that of methylphosphonic acid, whereas most organo-phosphorus pesticides have the phosphoric acid structure. On the basis of this characteristic difference, a procedure has been developed in which the presence of a nerve gas, its decomposition products, or its starting materials in waste water (Rhine River and Meuse River water) is reflected by the appearance of methylphosphonic acid as a breakdown product after hydrolysis. This acid is concentrated and converted into a volatile compound by methylation. After cleanup, the ester may be separated from related compounds by gas chromatography and is detected by means of a specific detector for phosphorus. The detection limit of nerve gases by this procedure is approximately 1 nanomole per liter of water. The scope and limitations of the method are discussed.

Selective and sensitive trace analysis of sulfur mustard with thermal desorption and two-dimensional gas chromatography-mass spectrometry.

An improved method is presented for the trace analysis of sulfur mustard (HD) in biological samples, such as blood and tissue from laboratory animals. Using the internal standard method and liquid-liquid extraction with ethyl acetate, up to 400 microL of the extract was injected by thermal desorption from Tenax and analyzed by two-dimensional GC-MS/EI in SIM mode. The analysis was compared with a direct GC injection. Reversed thermal desorption was used as a tool for handling heavily contaminated (fat) samples, thus preventing contamination of the injection system and pre-column. A successful analytical configuration has been set up for the bioanalysis of HD at the low, toxicologically relevant pM level. A detection limit of 10 pg mL(-1) blood or pg g(-1) tissue of sulfur mustard (S/N=3) was established by using this configuration.

Expression and function of endothelial Ca(2+)-activated K(+) channels in human mesenteric artery: A single-cell reverse transcriptase-polymerase chain reaction and electrophysiological study in situ.

Ca(2+)-activated K(+) (K(Ca)) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase-polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K(Ca) channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K(Ca) channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non-cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K(Ca) channel (hIK1) was detected in non-cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K(Ca) currents, whereas respective K(Ca) currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non-cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P:<0.05). However, K(Ca) current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K(Ca) channel (hSlo) was detected in single ECs from CA patients. The increased K(Ca) channel expression in CA patients resulted in a 2. 7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P:<0.05). This increased expression and function of K(Ca) channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Impaired hyperpolarization in regenerated endothelium after balloon catheter injury.

Ca(2+)-activated K(+) (K(Ca)) channels control endothelial Ca(2+) homeostasis and the formation of vasodilators. After angioplasty, dysfunction of the regenerated endothelium leads to abnormal vasoregulation. In this study, we tested the expression and function of K(Ca) channels in regenerated endothelium at 6 weeks after balloon catheter injury of rat carotid arteries (CAs) by using single-cell reverse transcription-polymerase chain reaction, patch-clamp techniques, and analysis of vasoreactivity. In single regenerated endothelial cells (ECs), the percentage of ECs expressing the K(Ca) genes, rSK3 (12+/-8%) and rIK1 (22+/-9%), was significantly lower compared with the percentage of native ECs expressing these genes (rSK3 58+/-8%, rIK1 64+/-10%). In patch-clamp experiments, K(Ca) currents and acetylcholine-induced hyperpolarization were markedly reduced in regenerated ECs (shift of membrane potential -6+/-3 mV) compared with those in native ECs (shift of membrane potential -21+/-5 mV). In pressure myograph experiments, acetylcholine-induced dilation was impaired in reendothelialized CAs compared with normal CAs. Intraluminal application of the K(Ca) blocker apamin and charybdotoxin inhibited dilation by 30% in normal CAs but was without effect in reendothelialized CAs. Intraluminal application of 1-ethyl-2-benzimidazolinone (100 micromol/L), an opener of K(Ca) channels, evoked dilation by 29% in normal CAs but had no effect in reendothelialized CAs. In conclusion, the impaired expression of K(Ca) channels in regenerated endothelium results in defective hyperpolarization and impaired dilation. Thus, the impaired K(Ca) channel function contributes to functional alterations of regenerated endothelium after angioplasty.

Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells.

OBJECTIVE: Ca(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells. METHODS: By combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial cell line EA.hy926. RESULTS: At the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry. CONCLUSIONS: Our findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.

Effects of parathyroidectomy on tissue calcium, phosphorus, magnesium, and copper concentrations in aluminum-loaded uremic rats.

Rats were subjected to a two-stage 5/6 nephrectomy and treated with Al for 2 and 4 wk with a cumulative dose of 4.2 and 8.4 mg of Al, respectively. Other animals were parathyrectomized (PTx) and loaded with 8.4 mg of Al for 4 wk. Total Al, Ca, P, Mg, and Cu contents were analyzed in the liver, kidney, and bone by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that Al given to growing uremic rats significantly increased the content of Al in the liver, kidney, and bone. Moreover, Al treatment increased the liver and kidney Ca levels and decreased the Ca and P values in bone. Previous parathyroidectomy significantly reduced Al accumulation within organs and changes in the Ca and P levels in the bone, liver, and kidney. The result was not influenced by different degrees of renal failure.

Assignment of all four disulfide bridges in echistatin.

Echistatin is a 49-amino-acid protein from Echis carinatus venom. It contains four disulfide bonds. Since the disulfide bonding is critical for biological activity; it is very important to assign the disulfide linkage in this protein. Echistatin was incubated in 250 mM oxalic acid at 100 degrees C for 4 hr under nitrogen. Under these conditions, many overlapping disulfide-containing peptides were identified by ionspray mass spectrometry. Ionspray MS/MS data indicate that the four disulfide bonds are Cys 2-Cys 11, Cys 7-Cys 32, Cys 8-Cys 37, and Cys 20-Cys 39. To our knowledge, this is the first time all four disulfide bonds in echistatin have been assigned in one experiment without disulfide bond exchange. This approach, which combines oxalic acid hydrolysis and ionspray MS/MS, may be very useful for assigning disulfide bridges in other proteins from the disintegrin family.

Interaction of frustrated magnetic sublattices in ErMnO3.

A spontaneous or field induced "hidden" phase transition with antiferromagnetic-to-ferromagnetic reordering is disclosed in multiply frustrated hexagonal ErMnO3. It is revealed by Faraday rotation and second harmonic generation as sublattice-sensitive probes to the Er and Mn systems. The acquired phase diagram in the magnetic-field-temperature plane is shown to be a consequence of a broken geometric frustration between the Er and Mn lattices, giving way to anisotropic superexchange between the 3d and 4f ions.

The influence of early parathyroidectomy on aluminum-induced rickets in growing uremic rats.

Rats were subjected to a two-stage 5/6 nephrectomy and treated with aluminum for 2 and 4 weeks with a cumulative dose of 4.2 and 8.4 mg of aluminum, respectively. Other animals were parathyroidectomized and loaded with 8.4 mg of aluminum for 4 weeks. Histomorphometry and electron microscopy (tibiae), aluminum tissue (bone, kidney, liver) determination, serum (Ca, Mg, Zn, P, urea, creatinine, alkaline phosphatase, 1,25(OH)2D3, PTH) and urine (creatinine, A1) revealed that: (a) a dose of 8.4 mg aluminum was sufficient to induce rickets within 4 weeks of treatment and was associated with decreased serum calcitriol values and high aluminum accumulation within organs (electron-dense material was found in osteoblasts only); (b) previous parathyroidectomy prevented the occurrence of any aluminum-induced alteration of bone. It was associated with higher calcitriol and phosphorus values than in corresponding non-parathyroidectomized rats and significantly reduced aluminum accumulation within organs. The results was influenced neither by a drop in serum calcium values nor by different degrees of renal failure. We suggest that aluminum-induced rickets in growing uremic rats is prevented or delayed when previous parathyroidectomy has been performed.

Optical clock with ultracold neutral atoms.

We demonstrate how to realize an optical clock with neutral atoms that is competitive to the currently best single ion optical clocks in accuracy and superior in stability. Using ultracold atoms in a Ca optical frequency standard, we show how to reduce the relative uncertainty to below 10(-15). We observed atom interferences for stabilization of the laser to the clock transition with a visibility of 0.36, which is 70% of the ultimate limit achievable with atoms at rest. A novel scheme was applied to detect these atom interferences with the prospect to reach the quantum projection noise limit at an exceptional low instability of 4 x 10(-17) in 1 s.

Stabilization and gas chromatographic analysis of the four stereoisomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) in rat blood.

A method for the stabilization and gas chromatographic analysis of the four stereoisomers of C(+/-)P(+/-)-1,2,2-trimethylpropyl methylphosphonofluoridate (C(+/-)P(+/-)-soman) in rat blood samples is described. Satisfactory stabilization of all four stereoisomers is obtained by (i) acidification of the blood sample to pH 4.2 at 0 degrees C, to stabilize the C(+/-)P(+) isomers, (ii) addition of aluminum ions (2.5 mM) for complexation of fluoride ions, which prevents regeneration of C(+/-)P(-)-soman by free fluoride ions from soman-inhibited aliesterase, and (iii) addition of 2,2-dimethylpropyl methylphosphonofluoridate in order to occupy covalent binding sites for C(+/-)P(-)-soman. The stereoisomers of soman and internal standard are extracted from the blood-stabilizing buffer mixture with a Sep-Pak C18 cartridge and are subsequently eluted with ethyl acetate with overall extraction recoveries of 52 +/- 8%. The four soman stereoisomers are resolved and analyzed on a wide-bore capillary Chirasil Val column, synthesized, and coated in house, which also resolves the internal standard C(+/-)P(+/-)-1,2,2-[U-2H]trimethylpropyl methylphosphonofluoridate from C(+/-)P(+/-)-soman. Alternatively, the gas chromatographic analysis can be performed on a wide-bore capillary Chirasil Val column, identical with the commercially available Chirasil Val column, when combined in series with a Carbowax 20M column. This system resolves the four stereoisomers of soman and the internal standard C(-)P(+)-1,2,2-trimethylpropyl [U-2H]methylphosphonofluoridate. Using an alkali flame ionization detector, the detection limit of our procedure is ca. 250 pg soman isomer/blood sample.