Covalent and noncovalent receptor-glucocorticoid complexes preferentially bind to the same regions of the long terminal repeat of murine mammary tumor virus proviral DNA

Dexamethasone 21-mesylate, an irreversible antiglucocorticoid in HTC cells, forms a covalent receptor-steroid complex which can be activated in cell-free systems. The molecular basis of its antiglucocorticoid activity is unknown; it might result from altered DNA sequence preferences and/or affinities of the covalent receptor-steroid complex. To test this hypothesis, the affinities of both covalent receptor-antagonist and noncovalent receptor-agonist complexes for defined DNA sequences were measured in a DNA binding competition assay. This assay requires neither purified complexes nor large quantities of DNA, yet it provides quantitative comparisons of the affinities of different double-stranded DNAs for binding receptor-steroid complexes. In this assay, activated covalent receptor-dexamethasone 21-mesylate complexes in crude cytosol bound to calf thymus DNA and cloned subregions of the long terminal repeat (LTR) of murine mammary tumor virus (MMTV) proviral DNA with approximately the same relative affinities as did noncovalent receptor-dexamethasone complexes. Both types of complex exhibited similar orders of preferential binding to DNA sequences. LTR subregions, as well as the entire LTR, were 2-20 times more potent competitors than calf thymus DNA. Cloned sequences from the 3' terminus of the LTR were more effective competitors than either the entire LTR or comparably sized DNAs from the 5' terminus. The DNA sequences with the greatest affinities for both covalent and noncovalent complexes are located within the region of -221 to -67. These studies support the theory that recognition by regulatory elements of specific DNA sequences upstream of responsive genes is an integral step of hormone action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships among negative life events, physiological reactivity, and health symptomatology

College undergraduates classified as high (n = 25) and low (n = 25) on recent life stress participated in an experiment involving a novel laboratory stressor. Heart rate and pulse arrival time (PAT) were measured during baseline, anticipation, testing, and recovery periods of the experiment. The results did not replicate those obtained by Pardine and Napoli in that high and low life stress subjects did not show differential physiological reactions. In addition, regression analyses failed to demonstrate that physiological reactivity moderated the relationship between life stress and subsequent self-reported psychiatric or physical health symptomatology. The present findings demonstrated neither the stress-buffering effects of physiological reactivity nor a relationship between life stress and reactivity when the latter was conceptualized as an outcome.     

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.     

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.     

The significance of primate paleontology for anthropological studies

The correct use of taxonomic names must become widespread if a clear understanding of primate paleontology is to exist among anthropologists. Physical anthropologists are urged to acquire genuine competence in the paleontology, systematics, and taxonomy of mammals. Examples are given of improper taxonomic procedure and of the perpetuation of invalid names. The need for a stable and correct nomenclature of the primates is emphasized. The importance of examining actual fossil specimens is stressed. The taxonomy of the Hominoidea is discussed and a summary of invalid names in current use is given. Recently discovered fossils from Oligocene strata in the Egyptian Fayum are figured and the pertinence of these to the origins of higher primates is suggested.     

Indolderivate im Apfel

Zusammenfassung Zur Identifizierung der Streckungswuchsstoffe in Apfelgeweben wurden Extrakte aus vegetativen und reproduktiven Organen in verschiedenen Entwicklungsstadien chromatographiert und mit Hilfe des Weizen-Koleoptilzylinder-Tests, verschiedener Farbreagentien und synthetischer Vergleichssubstanzen auf ihre Wuchsstoffwirkung und ihren Gehalt an Indolderivaten geprüft. Alle untersuchten Gewebe ergaben im biologischen Test dieselben Wuchsstoffe, allerdings mit erheblichen quantitativen Unterschieden.In der sauren Fraktion von Ätherextrakten aus dem Fruchtfleisch verschiedener Apfelsorten ließen sich Malonyltryptophan, Indol-3-carbonsäure, 2-Hydroxy-indol-3-essigsäure, Indol-3-essigsäure, Indol-3-aldehyd (vor allem in reifen Früchten) und Indol-3-essigsäure-äthylester (in unreifen Früchten) identifizieren. In Gegenwart von IES trat häufig auch Indol-3-acetamid auf; dieses, die Indol-3-essigsäure und 2-Hydroxy-indol-3-essigsäure und der Indol-3-essigsäure-äthylester zeigten im Weizen-Koleoptilzylinder-Test Wuchsstoffwirkung.Nach Hydrolyse des mit Äther extrahierten Materials fanden sich neben einer größeren Anzahl unbekannter Substanzen, die im Farb-Test Indolreaktionen ergaben, drei weitere, im biologischen Test aktive Indolderivate, von denen das eine als Indol-3-acetylasparaginsäure identifiziert werden konnte; die anderen beiden sind möglicherweise ebenfalls Verbindungen der IES mit Aminosäuren.Im Weizen-Koleoptilzylinder-Test konnte eine Überlagerung der Indolderivate mit unbekannten, ebenfalls aktiven Substanzen nicht ausgeschlossen werden.Mit 3 Textabbildungen     

Surface distribution of the mannose 6-phosphate receptors in epithelial Madin-Darby canine kidney cells

We have analyzed the surface polarity of both the cation-independent (CI-MPR) and the cation-dependent (CD-MPR) mannose 6-phosphate receptors in the epithelial Madin-Darby canine kidney (MDCK) cell line grown on polycarbonate filters. The surface localization was studied by plasma membrane domain-specific surface labeling methods and by confocal microscopy using MPR-specific antibodies. The CI-MPR was shown to be exclusively present on the basolateral cell surface. In contrast, the CD-MPR was expressed neither apically nor basolaterally. However, an intracellular pool of CD-MPR could be detected. In MDCKII-RCAr cells, cell surface CI-MPR was shown to recycle between the basolateral plasma membrane and the trans-Golgi network. After exogalactosylation, cell surface CI-MPR acquired sialic acid residues in a time-dependent manner. Furthermore, the basolateral CI-MPR was shown to be functional. Lysosomal enzymes, bearing the mannose 6-phosphate recognition marker, were taken up from the basolateral medium and endocytosed into the cells. Uptake of lysosomal enzymes from the apical side was insignificant and not MPR mediated. These results extend previous immunoelectron microscopic studies on the intracellular polarity of the CI-MPR (Parton, R. G., Prydz, K., Bomsel, M., Simons, K., and Griffiths, G. (1989) J. Cell Biol. 109, 3259-3272) which showed that the CI-MPR was present in basolateral early endosomes and in late endosomes but absent from apical early endosomes.     

Amplification of small signals by voltage-gated sodium channels in drone photoreceptors

Summary Photoreceptor cells of the drone,Apismellifera , have a voltage-gated Na⁺ membrane conductance that can be blocked by tetrodotoxin (TTX) and generates an action potential on abrupt depolarization: an action potential is triggered by the rising phase of a receptor potential evoked by an intense light flash (Autrum and von Zwehl 1964; Baumann 1968). We measured the intracellular voltage response to a small (9%), brief (30 ms) decrease in light intensity from a background, and found that its amplitude was decreased by 1M TTX. The response amplitude was maximal when the background intensity depolarized the cell to –38 mV. With intensities depolarizing the cell membrane to –45 to –33 mV the average response amplitude was decreased by TTX from 1.2mV to 0.5mV. TTX is also known to decrease the voltage noise during steady illumination (Ferraro et al. 1983) but, despite this, the ratio of peak-to-peak signal to noise was, on average, decreased by TTX. The results suggest that drone photoreceptors use voltage-gated Na⁺ channels for graded amplification of responses to small, rapid changes in light intensity.Abbreviations TTX tetrodotoxin - V _i intracellular potential with respect to the bath - V _o extracellular potential - V _m,V _i-V _o approximate transmembrane potential - S amplitude of the voltage response to an 8.9% decrease in light intensity - N voltage noise, usually measured as root mean square voltage deviation as described in Methods     

Tarsius delta- and beta-globin genes: conversions, evolution, and systematic implications

Comparisons between duplicated genes have shown that gene conversions play an important role in the evolution of multigene families. Previous comparisons have documented in the recently duplicated gamma-fetal globin genes of catarrhine primates, over 15 separate conversions affecting extensive stretches of coding and noncoding sequences. In the present study, delta- and beta- globin genes from a lower primate Tarsius syrichta, and the delta-globin gene of the Asian great ape, Pongo pygmaeus, have been isolated and sequenced. Comparisons of these sequences with other primate delta and beta sequences confirmed a previously reported conversion in an anthropoid ancestor and revealed additional conversions in basal primate, stem haplorhine, tarsier, and early lemur lineages. Conversions found between primate delta- and beta-globin genes contrast with those found in the gamma-genes in that delta-beta conversions appear much less frequently and are more restricted to regions conserved by selection (i.e. coding and 5'-regulatory sequences). These differences indicate that soon after a duplication occurs, conversions can be quite frequent and encompass extensive portions of the duplicated region. With time, sequence differences accumulate, particularly in noncoding regions, and limit both the frequency and size of the conversions. Sequences conserved by selection accumulate differences more slowly and are therefore subject to gene conversions for a longer period of time. Both unconverted and converted sequences were consistent in supporting the placement of tarsier with anthropoids.