DNA torsional dynamics by multifrequency phase fluorometry.

The time decay of the fluorescence polarization anisotropy of calf thymus DNA-ethidium complexes is obtained from measurements with sine-modulated excitation employing the so-called multifrequency phase fluorometry. A torsional dynamics model developed by J. M. Schurr [(1984) Chemical Physics, Vol. 84, pp. 71-96] and translated into the frequency domain is found here to describe accurately DNA-ethidium fluorescence data collected under modulated excitation. At a low dye/DNA ratio (1:400) the value of the DNA torsional constant (alpha = 4.63 +/- 0.2 10(-12) dyne cm) fitting the data agrees very well with the known values of alpha. When the measurements are extended to a higher ethidium/DNA ratio, energy transfer effects between intercalated dyes are observed. A theoretical prediction of the donor and acceptor dye contributions to the fluorescence polarization anisotropy is made here, taking into account also dye-dye distance distributions.     

Analysis of ecdysteroids by fluorometry

All ecdysteroids are fluorescent when treated with sulphuric acid. The fluorescence can be used for the detection on thin-layer plates as well as for the quantitation of ecdysteroids in ethanolic solution. A standard procedure for the quantitative analysis of ecdysteroids by fluorometry is given. The assay is linear over two orders of magnitude of steroid concentration and is sensitive down to the range of 10⁻¹¹ mol ecdysteroid. The assay is group specific for ecdysteroids. Ecdysone gives the highest fluorescence and is therefore used as the standard. Other ecdysteroids exhibit a fluorescence value which is about 50% relative to ecdysone. Any interference in the assay by other steroids including cholesterol and 7-dehydrocholesterol is low. The fluorometric assay may be used to detect and quantify ecdysteroids in extracts of biological material after a chromatographic step. This is shown by the analysis of ecdysteroids from the blue blowfly, Calliphora vicina.     

A comparative study on bovine alpha-lactalbumin and lysozyme by nanosecond fluorometry.

Analysis of the time decay of fluorescence anisotropy of 1-dimethylaminoaphthalene-5-sulfonyl (DNS) and fluorescamine derivatives of bovine alpha-lactalbumin and lysozyme reveals that no significant differences in mean rotational relaxation times are present. While fluorescamine molecules appear to orient randomly on these proteins, DNS is bound with a preferential orientation. Other fluorescence characteristics of the labels are also cited.     

Nitrogen effects on the pelagic food web are modified by dissolved organic carbon

Global environmental change has altered the nitrogen (N) cycle and enhanced terrestrial dissolved organic carbon (DOC) loadings to northern boreal lakes. However, it is still unclear how enhanced N availability affects pelagic food web efficiency (FWE) and crustacean zooplankton growth in N limited boreal lakes. Here, we performed in situ mesocosm experiments in six unproductive boreal Swedish lakes, paired across a DOC gradient, with one lake in each pair fertilized with N (2011: reference year; 2012, 2013: impact years). We assessed how zooplankton growth and FWE were affected by changes in pelagic energy mobilization (PEM), food chain length (phytoplankton versus bacterial production based food chain, i.e. PP:BP), and food quality (seston stoichiometry) in response to N fertilization. Although PP, PEM and PP:BP increased in low and medium DOC lakes after N fertilization, consumer growth and FWE were reduced, especially at low DOC—potentially due to reduced phytoplankton food quality [increased C: phosphorus (P); N:P]. At high DOC, N fertilization caused modest increases in PP and PEM, with marginal changes in PP:BP and phytoplankton food quality, which, combined, led to a slight increase in zooplankton growth and FWE. Consequently, at low DOC (<12 mg L^?1), increased N availability lowers FWE due to mismatches in food quality demand and supply, whereas at high DOC this mismatch does not occur, and zooplankton production and FWE may increase. We conclude that the lake DOC level is critical for predicting the effects of enhanced inorganic N availability on pelagic productivity in boreal lakes.     

Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.     

Characterization of bioreactors by in-situ fluorometry

A new microfluorometer with a special fiberoptic for the simultaneous detection of two different wavelengths was developed. Several tracers were tested for reactor characterization at different wavelengths, and the influence of the pH value and culture medium components on fluorescence was studied. In CST bioreactors, the effect of aeration rate and stirrer speed on the fluorescence can be used for reactor characterization. Mixing time experiments were performed in two different bubble columns.     

Front-face fluorometry of liquid samples.

Quantitative analytical spectrofluorometry is usually performed with solutions which are optically dilute at the excitation wavelength and employs square cuvettes for observation at right angles to the excitation beam. In many biochemical applications, particularly when studying fluorophores in blood, front-face fluorometry of optically dense samples offers certain advantages which include fluorescence intensities which are an order of magnitude larger. The features which characterize quantitative fluorometry employing these two geometries are compared and a design for a front-face cell and cell holder, suitable for use in any standard spectrofluorometer, is presented.     

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry.

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.     

Retarding effects of cyclodextrins on the decomposition of organic isothiocyanates in an aqueous solution

One- and two-dimensional (1)H nuclear magnetic resonance ((1)H NMR) spectra were measured in order to estimate the dissociation constants (K(diss)) and molecular geometries of cyclodextrin (CD) with three organic isothiocyanates (ITC), allyl-ITC, 3-butenyl-ITC, and 4-pentenyl-ITC, in an aqueous solution (pH 5.0, I(c)=0.75 M). In every ITC, the K(diss) values decreased in the order of alpha-CD>beta-CD>gamma-CD, and the three rod-like linear molecules of ITCs were accommodated with the best fit into the smallest cavity of alpha-CD. By rotating-frame nuclear Overhauser enhancement spectroscopy, cross peaks were found between the H-3 of alpha-CD and the H-1 of AITC. From the result, the molecular geometry of alpha-CD-AITC complex presume that the isothiocyanate group of AITC is located some where around the widening rim and a hydrophobic allyl group is included within the hydrophobic cavity of alpha-CD.     

Specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.

alpha-, beta-, and gamma-cyclodextrins (CDs) completely inhibited raw starch digestion by glucoamylase I (GA I, MW 90,000) from Aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of GA I at the CD concentrations of 1-5 mM. CDs at 1-5 mM did not inhibit gelatinized starch hydrolysis by GA I, but at the concentration of 50 mM, they inhibited such hydrolysis slightly. GA I was specifically adsorbed onto CD-Sepharose 6B, but glucoamylase I' (GA I', MW 73,000), which does not adsorb onto or digest raw starch, from the same strain was not adsorbed onto that gel. The adsorption of the glucoamylases onto raw starch and CD-Sepharose 6B was correlated to their digestion of raw starch. The hydrophobic adsorption of GA I onto CDs and raw starch occurred competitively at the Cp region, which is on the C-terminal side of Gp-I in the site for raw starch affinity of GA I, and inclusion complexes were formed.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.

We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.     

蒽和UV—B辐射对米氏凯伦藻生长的影响

为研究多环芳烃蒽（anthracene）和UV—B辐射对米氏凯伦藻（Kreniamikimotoi Hansen）的单独效应和联合毒性效应,采用实验生态学的方法,以米氏凯伦藻为实验材料,蒽质量浓度设为0．00、6．25、11．50、20．00、35．00、62．50μg／L,UV—B辐射剂量设为0．00、0．375、1．125、2．25、3．75、6．00J／m^2。实验结果表明：对米氏凯伦藻的生长,多环芳烃蒽具有抑制效应,小剂量的UV-B具有刺激作用,随着剂量的增加表现出抑制作用,蒽与UV—B的联合则表现出更强的抑制作用,二者表现为协同作用。蒽和UV—B对米氏凯伦藻的96h—EC。分别为15．35μg／L和2．843J／m^2,而蒽在UV—B辐射条件下的96h-EC50为7．376μg／L。     

Galactosylation at side chains of branched cyclodextrins by various beta-galactosidases.

The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various beta-galactosidases. Bacillus circulans beta-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor beta-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these beta-galactosidases seem to be different from those by B. circulans beta-galactosidase, judging from the retention times on high performance liquid chromatography.     

Reversal of optical induction in transamination by regioisomeric bifunctionalized cyclodextrins.

Two isomeric compounds have been synthesized carrying a pyridoxamine on C-6 of beta-cyclodextrin and an imidazole unit on C-6 of the neighboring glucose residue. Each one stereoselectively transaminates phenylpyruvic acid to produce phenylalanine, and with opposite stereochemical preferences. Structure determinations by X-ray crystallography and NMR spectroscopy indicate that the imidazole units serve to block proton addition from their side, rather than acting to protonate the transamination intermediates. Related cyclodextrin-pyridoxamine compounds had been reported carrying ethylenediamine units instead of imidazoles, and high enantioselectivities in transamination were claimed. Our work indicates that these claims are incorrect, and that only poor selectivities are seen that are often unrelated to the position of the ethylenediamine units. Neither of these transaminating systems yet approaches the enantioselectivity seen with a pyridoxamine carrying a chirally mounted internal base group.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.