Solute Crystallization in Frozen Systems–Use of Synchrotron Radiation to Improve Sensitivity

Purpose

To demonstrate the sensitivity of low temperature synchrotron X-ray diffractometry (SXRD) for detecting solute crystallization in frozen sodium phosphate buffer solutions. To determine the effect of annealing on solute crystallization in frozen solutions.

Materials and Methods

Sodium phosphate buffer solutions, at initial buffer concentrations ranging from 1 to 100 mM (pH 7.4) were cooled to ?50°C. The crystallization of disodium hydrogen phosphate dodecahydrate (Na₂HPO₄ ?12H₂O) was monitored using a laboratory as well as a synchrotron source. At selected concentrations, the effect of annealing (at ?20°C) was investigated.

Results

With the laboratory source, solute crystallization, based on the appearance of one diagnostic peak with a d-spacing of 5.4 Å, was evident only when the initial buffer concentration was at least 50 mM. In contrast, using SXRD, crystallization was detected at initial buffer concentrations down to 1 mM. In addition, the use of a high-resolution 2D detector enabled the visualization of numerous diffraction rings of the crystalline solute. At both 10 and 100 mM buffer concentration, there was no increase in solute crystallization due to annealing.

Conclusion

By using synchrotron radiation, solute crystallization was detected with substantially increased sensitivity, making the technique useful for freeze-drying cycles of practical and commercial importance. Since numerous peaks of the crystalline solute appeared, the technique has potential utility in complex, multi-component systems.     

Evaluation of rk39 immunochromatographic test with urine for diagnosis of visceral leishmaniasis

This study evaluates commercially available rK39 immunochromatographic strips using urine for diagnosis of visceral leishmaniasis (VL). Freshly collected urine and serum samples of 280 parasitologically confirmed VL patients and 66 endemic healthy controls (EHC), 48 nonendemic healthy controls (NEHC) and 45 different diseases were tested with rK39 strips. The sensitivity of rK39 in urine was 96.4% while the specificity was low varying from 66.7% in EHC, 77.08% in NEHC to 62.2% in different diseases. With serum, sensitivity was 100% whereas the specificity was 100%, 92.4% and 95.55% for the respective control groups. In the present format, the immunochromatographic strips cannot be used for the diagnosis of VL using urine samples.     

3'-Intercalation of a N2-dG 1R-trans-anti-benzo[c]phenanthrene DNA adduct in an iterated (CG)3 repeat

The conformation of the 1 R,2 S,3 R,4 S-benzo[ c]phenanthrene- N (2)-dG adduct, arising from trans opening of the (+)-1 S,2 R,3 R,4 S- anti-benzo[ c]phenanthrene diol epoxide, was examined in 5'- d(ATCGC XCGGCATG)-3'.5'-d(CATGCCG CGCGAT)-3', where X = 1 R,2 S,3 R,4 S-B[ c]P- N (2)-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG) 3 iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X (4) and C (5), and in the complementary strand, they showed another disconnection between G (18) and C (19). In the imino region of the (1)H NMR spectrum, a resonance was observed at the adducted base pair X (4) x C (19). The X (4) N1H and G (18) N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X (4) N1H and C (19) N (4)H and between C (5) N (4)H and G (18) N1H, indicating that base pairs X (4) x C (19) and C (5) x G (18) maintained Watson-Crick hydrogen bonding. No NOE connectivity was observed between X (4) and G (18) in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X (4) and C (5) in the modified strand and G (18) and C (19) in the complementary strand. A total of 13 NOEs between the protons of the 1 R-B[ c]Ph moiety and the DNA were observed between B[ c]Ph and major groove aromatic or amine protons at base pairs X (4) x C (19) and 3'-neighbor C (5) x G (18). Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[ c]Ph moiety intercalated on the 3'-face of the X (4) x C (19) base pair such that the terminal ring of 1 R-B[ c]Ph threaded the duplex and faced into the major groove. The torsion angle alpha' [X (4)]-N3-C2-N2-B[ c]Ph]-C1 was calculated to be -177 degrees, maintaining an orientation in which the X (4) exocyclic amine remained in plane with the purine. The torsion angle beta' [X (4)]-C2-N2-[B[ c]Ph]-C1-C2 was calculated to be 75 degrees. This value governed the 3'-orientation of the B[ c]Ph moiety with respect to X (4). The helical rise between base pairs X (4) x C (19) and C (5) x G (18) increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[ c]Ph moiety were below the Watson-Crick hydrogen-bonding face of the modified base pair X (4) x C (19). The B[c]Ph moiety was stacked above nucleotide G (18), in the complementary strand.     

Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens

Rubia cordifolia L. (Rubiaceae) is an important medicinal plant used in the Ayurvedic medicinal system. Its use as a traditional therapeutic has been related to the treatment of skin disorders and cancer. Besides its medicinal value, anthraquinones from this plant are used as natural food colourants and as natural hair dyes. Dyes derived from natural sources have emerged as important alternatives to synthetic dyes. Alizarin (1,2-dihydroxyanthraquinone) was isolated and characterized from R. cordifolia L. and evaluated for its antigenotoxic potential against a battery of mutagens viz. 4-nitro-o-phenylenediamine (NPD) and 2-aminofluorene (2-AF) in Ames assay using TA98 tester strain of Salmonella typhimurium; hydrogen peroxide (H₂O₂) and 4-nitroquinoline-1-oxide (4NQO) in SOS chromotest using PQ37 strain of Escherichia coli and in Comet assay using human blood lymphocytes. Our results showed that alizarin possessed significant modulatory role against the genotoxicity of mutagens.     

Synthesis, characterization, and ion exchange behavior of antimony(V) phosphate: selective adsorption of cadmium and mercury on its column

Antimony(V) phosphate has been prepared as a new inorganic ion exchanger suitable for the selective adsorption of cadmium and mercury, the two chief polluting metals. The materials possesses good ion exchange capacity and stability, both thermal and chemical. It has also been characterized and an empirical formula has been proposed on the basis of its chemical analysis and certain instrumental studies like infrared spectroscopy, X-rays, thermogravimetry, derivative thermogravimetry, differential thermal analysis, and pH titrations.     

Effects of calmodulin antagonists on radiation-induced lipid peroxidation in microsomes

Rat liver microsomes were irradiated with gamma-rays at a dose rate of 1.31 Gys-1. The extent of lipid peroxidation, measured in terms of malondialdehyde (MDA) formed, increased with radiation dose. The presence of calmodulin antagonists during irradiation decreased lipid peroxidation. The order of their protective efficiency was: chlorpromazine (CPZ) greater than promethazine (PMZ) greater than trimeprazine (TMZ). Their protective effect was diminished in the presence of ferrous (Fe2+) ions and was restored on addition of EDTA. However, calmodulin antagonists considerably inhibited radiation-induced lipid peroxidation in the presence of ferric (Fe3+) ions. Calmodulin antagonists also decreased the cytochrome P-450 content of microsomes. These results are discussed with respect to their applicability to radiotherapy. A possible mechanism for the inhibition of radiation-induced lipid peroxidation is suggested.     

Atrial paroxysmal tachycardia in dogs and its management with homeopathic Digitalis--two case reports

Homeopathic Digitalis 6c was evaluated in two clinical cases of atrial paroxysmal tachycardia in dogs. Tachycardias are common cardiac problems in dogs, and atrial paroxysmal tachycardia is a serious cardiac arrhythmia that may lead to syncope. Both adult dogs (Labrador and German Shepherd) were treated with Digitalis 6c, 4 drops orally four times daily for 7 days. Following treatment with Digitalis 6c heart rate stabilised and synchronized atrial and ventricular electrical activity was restored in 7 days.