Structure dependent prototropy in 4-hydroxy-3-formylideneamino-1-methyl/phenylquinolin-2-ones.

The electronic absorption spectra of 4-hydroxy-3-formyl quinolin-2-ones and their Schiff bases were investigated in various solvents of varying polarity. The three aromatic transitions of napthalene in quinolin-2-one are shifted to longer wavelength on their transformation to anils. Electron-donating group in the anils lead to enolimine form, while electron-withdrawing group leads to an equilibrium mixture of enolimine and ketoamine forms and the Schiff base derived from alkyl amine exist in ketoamine form. The prototropic interconversion of enolimine and ketoimine forms in the anils with the electron-withdrawing substituted anils is further supported by proton NMR studies. The spectral shifts are solvent dependent. Dipolar aprotic solvents bring bathochromic shift while polar protic solvents cause blue shift in the longer wavelength absorption maxima. In the case of Schiff bases substituted by electron-donating group the bathochromic shift is directly related to the polarity of the solvents.

Wavelength dependant quenching of 2,5-diphenyloxazole fluorescence by nucleotides.

The quenching of 2,5-diphenyloxazole (PPO) fluorescence by nucleotides has been investigated by electronic absorption and steady state fluorescence spectra. Five purine nucleotides AMP, ADP, ATP, GMP and dGMP, one pyrimidine nucleotide UMP and one dinucleotide NAD have been employed in the present study. Electronic absorption studies indicate that there is no ground state complexation between the nucleotides and PPO. The quenching of PPO fluorescence was investigated at two different wavelengths. When excited at 304 nm, the lambda(max) of PPO, the fluorescence spectra of PPO is quenched following Stern-Volmer kinetics. The quenching ability of nucleotides are in the order NAD>AMP>ADP>GMP>dGMP>UMP. The K(SV) and k(q) values obtained indicate that AMP is a better quencher of PPO fluorescence than GMP, which is contrary to commonly observed pattern. The quenching is found to be dynamic in nature. However, when excited at 260 nm, the absorption maximum of the nucleotides, the fluorescence intensity of PPO is reduced with increase in the concentration of the nucleotide. This is attributed to the primary inner filter effect arising due to the absorption of the incident radiation by the nucleotides. Thus the inner filter effect phenomenon can be employed to assay the non-fluorescent molecules by fluorimetry.

Molecular adducts between a few phenolic donors and pi-acceptors in solid-state.

Electron donor-acceptor molecular complexes of a few phenolic donors with some quinonoid and tetracyanoethylene acceptors have been prepared by two different methods, i.e., by simple grinding of the respective component pair in the solid-state and in solution. Both the methods yielded identical dark colored 1:1 stoichiometric complexes. Spectral studies revealed that the complexes are ionic in nature. The g values obtained in ESR spectral studies for all these molecular adducts vary between 2.000 and 2.022, confirming the free radical nature of the adducts. The electronic absorption spectral studies proved that the donor-acceptor complexes formed initially, exhibit new electronic transitions at longer wavelengths, are less stable and disassociate readily into ionic type of adducts. The absorption maximum at longer wavelengths, i.e. >or=550 nm, are assigned to the charge transfer complexes, while the new transition at around 410 +/- 5 nm is attributed to the anion radical of the adducts. The ease of complexation not only depends on the ionization potential and electron affinities of the phenolic donors and the acceptors but is also structure sensitive. Complexation is confirmed by the shift in IR absorption of the phenolic hydroxyl group and the carbonyl group of quinone acceptors and the cyano group of tetracyanoethylene. Proton magnetic resonance studies indicate an interaction between the phenolic donors and acceptors on basis of the altered chemical shifts. Further, IR and NMR studies show that the stability of the adducts is governed not only by the charge transfer interaction but also by hydrogen bonding.

Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus.

A single tube fluorogenic RT-PCR-based 'TaqMan' assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan-PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5' untranslated region (5' UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT-PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT-PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values (C(T)) versus copy number. Single tube TaqMan-PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.

Single-tube single-enzyme reverse transcriptase PCR assay for detection of bovine viral diarrhea virus in pooled bovine serum.

A reverse transcriptase PCR (RT-PCR) was developed for use as a diagnostic screening test for the detection of bovine viral diarrhea virus (BVDV) in pooled bovine serum samples. Individual serum samples from 60 dairy cattle herds located in Pennsylvania were evaluated by the microplate virus isolation method, and pooled sera were analyzed by RT-PCR. RT-PCR was sensitive and specific and detected a single viremic serum sample in up to 100 pooled serum samples. RT-PCR analysis of pooled sera provides a rapid and cost-effective method for the screening of cattle herds for the presence of animals persistently infected with BVDV.