Equilibrium hormone binding to human estrogen receptors in highly diluted cell extracts is non-cooperative and has a Kd of approximately 10 pM

It is generally accepted that the K_d for hormone binding to estrogen receptors in extracts ranges between 0.1–1 nM and that binding displays positive cooperativity due to formation of homodimers. After carefully optimizing assay procedures, to diminish ligand depletion phenomena and to fully control recoveries, we find a single class of non-interacting high affinity hormone binding sites with a K_d of approx. 10 pM. Ligand depletion was avoided by decreasing receptor concentrations to 5–8 pM. We were therefore obliged to employ radioiodinated estradiol as a probe as the specific radioactivity of tritiated estradiol was too low to maintain the accuracy of the binding assay. Human estrogen receptor extracted from the MCF7 cell line and recombinantly produced (in yeast) wild-type human receptor have identical equilibrium hormone binding characteristics.     

Ovarian superstimulatory response relative to follicular wave emergence in heifers

Two experiments were designed to evaluate the responsiveness of beef heifers to superstimulatory treatments administered during the first follicular wave. Heifers were examined daily (Experiment 1) or twice daily (Experiment 2) by ultrasonography to determine the status of follicular wave development and the day of initiation of superstimulatory treatment. Heifers in both experiments were superstimulated with a total dose of 10 ml Folltropin (equivalent to 200 mg of NIH-FSH-P1), divided into 10 equal intramuscular injections over 5 days. On the last day of treatment, heifers received 500 mug of cloprostenol after each injection of Folltropin to induce luteolysis. In the respective groups, superstimulatory treatments were initiated on Day -1, Day 0 (day of ovulation) or Day +1 for Experiment 1, and on Day -1, Day 0, Day +1 or Day +2 for Experiment 2. In Experiment 1, the number of ovulations in each ovary was assessed by ultrasonography and by counting the number of corpora lutea (CL) in each ovary at slaughter. The correlation between both techniques for assessing ovulatory response was high (r= 0.98; P< 0.0001), and there was no significant difference in the mean number of ovulations detected by ultrasound (5.7+/-1.1) versus the mean number of CL counted at slaughter (6.2+/-1.2). In Experiment 1, the mean (+/- SEM) number of CL counted at slaughter in heifers treated on Day -1 (9.4+/-3.8) and Day 0 (7.3+/-1.6) was higher (P< 0.05) than that of heifers treated on Day +1 (0.7+/-0.3). The mean number of follicles >/=7 mm in diameter on the last day of treatment was also higher (P<0.05) in the Day -1 group compared with the Day +1 group; the Day 0 group was intermediate. In Experiment 2, the mean number of ovulations was higher (P< 0.05) in the Day 0 group (18.4+/-3.4) than the Day -1 (9.5+/-2.3), Day +1 (6.7+/-2.2) or Day +2 (6.5+/-2.3) groups. Heifers in the Day -1, and Day 0 groups had more (P< 0.05) follicles >/=7 mm at the end of treatment compared with heifers in the Day +1 or the Day +2 group. The stated hypothesis was supported: exogenous FSH treatment initiated at the time of wave emergence, near the expected time of the endogenous wave-eliciting FSH surge, has a positive effect on the superstimulatory response. A higher superstimulatory response was elicited when treatments were initiated on the day of, or the day before, wave emergence compared with that of later treatments.     

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS 200 . The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55°C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 μl of 1 mmol 1^-1 AttoPhos^TM (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1–10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR. assay described here can be useful to screen a large number of food samples for contamination by salmonellas.     

Nature and metabolism of a thymus cyst studied by histochemistry and autoradiography

Summary A thymus cyst was discovered in connection with autoradiographical studies on sulphur metabolism of the rat. The coincidence must be considered unique and has motivated amplifying histochemical investigations.The cyst-content showed a strong positive PAS-reaction and after toluidine blue -metachromasia, which along with the incorporation of S³⁵ makes the presence of acid mucopolysaccharides likely. A strong blackening was noticed on the autoradiogram over the greater part of the cyst. This infers that the content has been metabolized here, in contradistinction to the centre with inactive colloid.     

Recoverability of heat-injured Bacillus spores by lysozyme and EDTA or alkaline thioglycollate

The D_95°C value of Bacillus thuringiensis spores plated in the presence of lysozyme increased from 3.0 min to 3.6 min by post-treatment of heat-injured spores with 50mm EDTA. In the case of Bacillus alvei and Bacillus polymyxa spores D-values decreased from 4.9 to 4.3 min and from 4.7 to 4.1 min respectively. Post-treatment of heat-injured spores treated with alkaline thioglycollate increased D_95°C values of Bacillus alvei from 4.2 to 5.3 min, B. thuringiensis 3.6 to 4.7 min, and Bacillus polymyxa from 4.2 to 5.0 min when spores were plated in the presence of lysozyme. Electron micrographs of heat-injured B. alvei spores treated with sodium thioglycollate indicated that the coat layers of the treated spores were granulated and less intact than the control spores.     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Tetrahymena. Adaptation to high iron

Fe-starved ciliates Tetrahymena thermophila cease to multiply at Fe(III) concentrations above 10 microM in a synthetic nutrient medium lacking a good iron chelator such as citrate. If, however, the Fe(III) concentration is gradually increased over a series of subcultivations the cells will tolerate up to 300 microM Fe(III). Our experiments rule out the possibility of genetic selection of Fe-tolerant clones and suggest a physiological type of adaptation.     

The fluctuations in the level of endogenous growth regulators in seeds ofAcer pseudoplatanus L. in the course of stratification

In the course of cold stratification ofAcer pseudoplatanus L. fruits a statistically significant alternation occurs in their seeds of a rise and fall in the level of endogenous growth regulators. In the initial weeks the inhibitory effect slightly declines, or, on the contrary, the stimulatory effect slightly increases; in the middle phase of stratification a marked increase in inhibitions and reduction of stimulations appears, and towards the end of stratification the stimulatory effect of isolated substances in the individual biotests rises again, or their inhibition effect is decreased. No direct dependence was found between the decrease of the degree of dormancy and the drop of inhibitor, or increase of promotor levels. However, a certain analogy was observed between the time course of fluctuations in the level of growth regulators and the germination energy of the seeds investigated. An enhancement of the growth activity of the substances isolated (e.g. those of a gibberellin-like nature) in the last weeks of stratification can already be considered as the result of the release of fruits from dormancy.     

Growth of Streptomyces bambergiensis and glucose isomerase biosynthesis

Summary Growth and glucose isomerase biosynthesis in Streptomyces bambergiensis ATCC 13879 have been studied under different conditions. Some data concerning correlation between cultivation conditions and elemental analysis of the cells are also presented.