Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states

The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.     

Metabolism of the mutagen MeIQx in vivo: metabolite screening by liquid chromatography-thermospray mass spectrometry

2-Amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) is a potent mutagen found in cooked food. MeIQx and its isotopically labelled (13C, 15N2 and 14C) analogues were synthesised and used for metabolic studies in vivo. An equimolar mixture of MeIQx and its 13C, 15N2 stable isotope labelled analogue (containing tracer amounts of 14C-MeIQx) was given intraperitoneally to mice. Some 67% of the radioactivity was eliminated in urine and faeces within 24h. Four radiolabelled species were observed when urine was analysed by HPLC, corresponding to unchanged MeIQx and three more polar metabolites. Urine was analysed directly by HPLC-thermospray mass spectrometry. Four signals were observed containing the characteristic 1:1 isotopic doublet, corresponding to unchanged MeIQx, an MeIQx glucuronide, and two uncharacterized metabolites.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

De novo t(2;13)(p24.3;q14.2) and retinoblastoma

Summary The two probes H3-8 and H2-42, known to be located in 13q14, were mapped by in situ hybridization to either side of the 13 breakpoint of an apparently balanced de novo t(2;13)(p24.3;q14.2) detected in a patient with retinoblastoma as the only phenotypic manifestation.     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin