Steroid-bovine serum albumin conjugates: molecular characterization and their interaction with androgen and estrogen receptors

Conjugates of testosterone-3-carboxymethyloxime (T-3-CMO), testosterone-17-hemisuccinate (T-17-HS), 17 beta-estradiol-6-carboxymethyloxime (E-6-CMO), or 17 beta-estradiol-17-hemisuccinate (E-17-HS) and bovine serum albumin (BSA) with varying steroid:protein ratios were prepared using the mixed anhydride method. Dialysis followed by molecular filtration yielded monomer steroid-BSA conjugates with a molecular weight of 70,000 dalton, and polymer conjugates with molecular weights of 140,000 dalton and higher. When conjugates were prepared with increasing initial steroid:BSA molar ratios the ratio of the obtained conjugates increased, in parallel with a decrease in the relative amount of monomers and an increase in the mean molecular size of polymers. The molecular properties of these conjugates were studied further by polyacrylamide gel electrophoresis (PAGE) in native and denaturing conditions. In native PAGE the monomer fractions showed one main band with a mobility slightly lower than BSA and a faint band corresponding with BSA-dimers. The polymer fractions consisted of a heterogeneous population of protein oligomers with molecular weights varying from 140,000 to over a million dalton. In the presence of sodium dodecylsulphate part of the polymers dissociated into monomers. In buffered aqueous solutions the bulk of the conjugate preparation retained its molecular size and composition, although the generated covalent bonds were found to be liable to spontaneous hydrolysis. Steroid-protein conjugates were shown to contain appreciable amounts of non protein-bound steroids. Binding of T-BSA to androgen receptors in rat ventral prostate cytosol was assayed using LH-20 chromatography and sucrose gradient centrifugation analysis. Binding of E-BSA to estrogen receptors was analysed with rat uterus cytosol using the dextran coated charcoal assay and the sucrose gradient centrifugation technique. Relative binding affinities (RBA) were analyzed in competition experiments using radiolabeled ligands. It was found that the molecular size of the conjugate does not influence its interaction with steroid receptors. Steroid coupled via the 17-position show a higher RBA to receptors than the T-3 or E-6 derivatives. The RBA of T-3-BSA, T-3-CMO, T-17-BSA and T-17-HS appeared to be very low, i.e. between 0.1 and 1.7% of the RBA of dihydrotestosterone. Consequently, high concentrations of conjugate are required to saturate androgen receptor binding sites. Under these conditions involvement of type II and eventually type III binding sites, which show less ligand specificity and lower affinity, may be anticipated preventing exclusive detection of androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

The isolation and genetic analysis of aCaenorhabditis elegants translocation (szT1) strain bearing an X-chromosome balancer

A single X-chromosome balancer-bearingCelegans ♂+, as a founder of a strain (AF1), was isolated directly from Fl progeny of irradiated+dpy-8unc-3/lon-2++ hermaphrodites on the basis of the absence of recombinant F2 categories. The balancer chromosome (Bal-X-1) suppresses recombination over a two-thirds section of the X chromosome (between genesdpy-8 andlet-2) and is associated with a reciprocal translocation between linkage groups (LG) X and I. Animals homozygous for the translocation (szT1(X:1)) are nonviable. Hermaphrodites heterozygous for the translocation segregate male selfprogeny at a frequency of 0.08-0.12.Bal-X-l carries the marker mutationlon-2(e678) and can be detected cytologically. This balancer chromosome proved useful for rnaimaininga number of X-linked lethal mutations and deficiencies inC. elegans.     

Local names for vascular plants in the John Crow mountains,Jamaica

Local names of vascular plants, accompanied by botanical specimens, were collected in the John Crow Mountains, Portland Parish, Jamaica. Each of the names was volunteered by one local informant; many were independently corroborated by others. We recorded 91 names of forest trees and shrubs, 20 names of climbers, and 33 names of other plants. The uneven distribution of local names among different categories of plants is discussed. The names are mostly of English origin, with a scattering from Spanish, Amerindian and West African languages. Many names are not listed in the standard floras; 20 names are apparently hitherto unrecorded in print. The same name was often found to be applied to a different species from the usage given in the floras, showing the variation in vernacular names from one part of the island to another.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.     

Teratogenic and goitrogenic activity of propineb and propylenethiourea in the rat

The teratogenic and goitrogenic effects of Propineb, dithiocarbamate pesticide and Propylenthiourea (PLTU), its metabolite and degradation product have been studied. The aim of this study was to show the possible correlation between the two activities. Female Sprague-Dawley rats were treated with Propineb and PLTU starting from 6th to 16th day of pregnancy. The functional state of maternal and foetal thyroid, the toxicity of products versus dams and embryotoxic and teratogenic effects were examined. The observed goitrogenic effect may be compared to that reported in the previous studies of the authors, if considering time of sacrifice. In fact, the lesion quickly rises and as rapidly regresses when treatment is stopped. The foetal thyroid has not been affected by the product administered to the dams. PLTU showed a clear teratogenic activity at doses that did not show any maternal toxicity (45 and 90 mg/k).     

The use of microcomputers for the quantitation of light intensity patterns using digitized video signals

We have developed an inexpensive yet versatile microcomputer-basedsystem for quantitating light intensity levels in autoradiographs.This system employs a standard video camera interfaced to ananalog-to-digital convertor. A program has been written forthis system which can measure intensities within a defined regionof an autoradiograph, permitting an easy and accurate quantitationof spots or bands of irregular shape. Received on June 18, 1985; accepted on September 3, 1985     

Oral Ultrastructure and Oral Development of the Misaligned Undulating Membrane Mutant of Tetrahymena thermophila1

The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation. The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA). The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs. Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5. The effect of the mutation involves a proliferation of excess basal bodies in the UM field. Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs. This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs. This level of regional control is not clearly evident when a single UM is present. The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development. On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.     

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.