11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.     

A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.

A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Structures of the tetrahydrogenated menaquinones fromActinomadura angiospora,Faenia rectivirgula,andSaccharothrix australiensis

The structures of the tetrahydrogenated menaquinones fromActinomadura angiospora, Faenia rectivirgula, andSaccharothrix australiensis were determined by mass spectrometry and proton nuclear magnetic resonance spectrometry. The positions of saturation of the tetrahydrogenated menaquinones fromFaenia rectivirgula andSaccharothrix australiensis were units II plus III (counting from the ring system), whereas that ofActinomadura angiospora had units III and VIII hydrogenated. The tetrahydrogenated menaquinones fromFaenia rectivirgula andSaccharothrix australiensis are similar to those characterized from other Gram-positive taxa to date, whereas that fromActinomadura angiospora represents a hitherto unknown isomer.     

Differential expression of the human glucocerebrosidase-coding gene

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.