Absence of 7-acetyl taxol binding to unassembled brain tubulin

The effect of taxol on microtubule proteins at 0 degrees C is controversial. In order to determine if taxol is unable to bind to unassembled tubulin, as has been hypothesized, the binding of [3H]acetyl taxol has been studied using equilibrium microdialysis. Ac-taxol bound to microtubules at 37 degrees C and the binding remained stable when the temperature was lowered to 0 degrees C. Ac-taxol bound also at 0 degrees C to microtubules stabilized with rhazinilam. In contrast, there was no binding of Ac-taxol to unassembled tubulin, either free tubulin at 0 degrees C or tubulin, complexed with several microtubule poisons, at 0 and 37 degrees C.     

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

The error in the cryptic stereospecificity of methylmalonyl-CoA mutase. The use of carba-(dethia)-coenzyme A substrate analogues gives new insight into the enzyme mechanism

A preparation containing 80.0 +/- 0.5% (2RS)-methylmalonyl-carba-(dethia)-CoA and 20.0 +/- 0.5% propionyl-carba-(dethia)-CoA was reacted in buffered deuterium oxide with catalytic amounts of coenzyme B12, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase. The rearrangement of the methylmalonyl-carba-(dethia)-CoA to succinyl-carba-(dethia)-CoA was monitored by recording 500-MHz 1H-NMR spectra in short time intervals. After reaching equilibrium (approximately equal to 28 min) the products showed chemical stability for about 17 h, i.e. succinyl species did not undergo the spontaneous hydrolysis encountered with normal succinyl-CoA. In the pre-equilibrium stage only about 66% of the produced succinyl-CH2CoA was the expected monodeuterated species. The remainder was 15.5% unlabelled and 18.3% 3,3-dideuterated. After reaching equilibrium a continuous deuterium incorporation (washing-in) from the solvent to the products was observed and quantified. The time course of the appearance of unlabelled, mono-, di- and trideuterated succinyl-CH2CoA species was determined by assigning and integrating the isotope-shifted 1H signals from the various species. Furthermore, mutase catalyses slow deuterium incorporation into first the methylene and then the methyl group of propionyl-CH2CoA. On the basis of these data it was concluded that methylmalonyl-CoA mutase and epimerase are responsible for continuous deuterium incorporation and multiple incorporation occurs when the backward reaction (succinyl-CH2CoA----methylmalonyl-CH2CoA) becomes important. To account for all of the results obtained with dethia and natural substrates we propose a new mutase mechanism whereby the enzyme can retain full stereospecificity at C-3 of succinyl while an internal 1,2-H shift to give a C-2 succinyl radical is responsible for partial scrambling of diastereotopic protons at C-3. This mechanism successfully predicts the observed deuterium disproportionation in succinyl species and the order of appearance of di- and trideuterated products via the washing-in process.     

Acetylcholinesterase from Drosophila melanogaster. Identification of two subunits encoded by the same gene

Purified acetylcholinesterase from Drosophila melanogaster is composed of a 55 kDa and a 16 kDa noncovalently associated subunit. Cleavage of disulfide bonds reveals that two 55 kDa polypeptides are linked together in native dimeric AChE. Western blots with two antibodies directed against the N- and C-termini of the predicted AChE primary sequence show that the 55 and 16 kDa polypeptides originate from proteolysis of the same precursor encoded by the Ace locus.     

The R-banding pattern of the Chinese hamster Don cell line

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.     

The relation between the variability of polygenic morphological and monogenic biochemical traits as exemplified by Karakul sheep

A quantitative method for investigation of relationship between polygenic and monogenic traits has been proposed. It is based on examination of relationship between frequencies of distribution classes of an adaptive quantitative trait and frequencies of certain genetic character in the same classes. The method permits to locate a gene marker within a space of quantitative trait values. Using adaptively significant morpho-anatomic traits, it is possible to estimate indirectly adaptive values of gene markers under consideration, since, in accordance with the concept of adaptive norm, "average" phenotypes have maximal fitness, whereas deviative phenotypes transgress the bounds of the optimum. As a genetical character, genotype of certain biochemical locus, individual heterozygosity range or interlocus combinations of alleles could be used. The method has been applied to newborn Astrakhan lambs. Principal component analysis has been used to obtain complex characterization for six constitutional characters. Some regularities in location of homo- and heterozygous genotypes of the transferrin locus within a space of morphological characters' values have been revealed.     

Phosphorus in sediments — speciation and analysis

Characterization of sediment phosphorus is commonly based on sequential chemical extractions, in which phosphorus is supposed to be selectively removed from different compounds in the sediments. The first extraction schemes were designed to quantify discrete chemical or mineralogical compounds. As extraction schemes have been tested on different sediments, several systematic errors have been detected and the schemes have been modified and simplified accordingly. Other chemical extractions or treatments have attempted to determine phosphorus bound to particles with a certain strength or binding energy, the purpose being to determine the labile, loosely bound, exchangeable, mobile or algal-available fraction of sediment phosphorus. All extraction procedures yield operationally defined fractions and cannot be used for identification of discrete phosphorus compounds. The many methodological modifications make it necessary to be cautious when comparing results from the literature in this field.     

Demonstration of correlations between the 8 and 10 kHz atmospherics and the inflammatory reaction of rats after carrageenan injection

Between the mean daily density of 28 kHz atmospherics and the onset of epileptic fits there is a highly significant correlation coefficient (r) of 0.30; there is a negative coefficient of –0.20 between the fits and the mean daily density of 10 kHz atmospherics. The onset of heart infarction is correlated with 28 kHz atmospherics (r=0.15). Furthermore, we have discovered that sudden deafness is also correlated with certain configurations of atmospherics. In this paper we report the following correlation coefficients between the inflammatory reaction of rats to a carrageenan injection (rci) into a hind paw and the mean daily pulse rate of atmospherics of the same day:r=0.49 for the 8 kHz atmospherics (P<0.02) andr=0.44 for the 10 kHz atmospherics (P<0.04). The correlations between rci reaction and other atmospherics (12 and 28 kHz) are smaller and not significant. By the method of multiple linear regression we found a multipleR=0.54 between rci reaction and the 8 and 10 kHz atmospherics (the regression function for the rci reaction is 0.15+0.004×8 kHz+0.002×10 kHz,P<0.05).     

Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of X-rays

The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.