Social enrichment of the environment with infants for singly caged adult rhesus monkeys

The practicability of social enrichment for singly caged adult rhesus monkeys was examined. Twenty-nine weaned rhesus monkey infants were removed from breeding troops to avoid overcrowding and were placed with unfamiliar singly caged adults. An adult-infant pair was considered compatible when (1) the two animals started huddling with each other within the first 5 days after pair formation and (2) the infant showed no signs of depression and took its share from a limited amount of favored food. Adult-infant pairs were compatible in 90% (26/29) of cases. Compatibility depended neither on the sex, age, and origin of the adult nor on the sex of the infant. There was no evidence that partners lost interest in each other during 7–11 months of follow-up observations. Three adults exhibiting stereotypical behavior abandoned their peculiar habits after they had lived with their young companions for 4 months. It was concluded that the often-heard notion that rhesus monkeys are highly aggressive should not prevent simple attempts to provide singly caged animals with a companion.     

In vivo 19F-NMR study of halothane distribution in brain

Halothane distribution and elimination from rabbit brain was studied in vivo using 19F-NMR spectroscopy. Two exponential decay functions for the anesthetic were observed in the clearance curve. They are assigned to halothane in brain held in two distinct chemical environments characterized by different chemical shifts, and half-lives (25 and 320 min). A nonvolatile halothane metabolite with a half-life of several days was found to be present in rabbit brains. The in vivo results were corroborated by ex vivo experiments on excised brain tissue. Halothane was distributed in all of the major cell subfractions, whereas the metabolite was present predominantly in the cytoplasm.     

cAMP binds with high affinity and specificity to the androgen receptor from murine skeletal muscle

cAMP binding of the androgen receptor (AR) from murine skeletal muscle was studied. Testosterone affinity chromatography yielded androgen receptor with about 4000-fold purification. Determination of the cAMP binding in the affinity eluate, by adsorption of protein-cAMP complexes to cellulose ester filters or removal of unbound cAMP by dextran-coated charcoal, was not possible, as the observed binding was not stable during the assays. Displacement studies suggest that this is due to a very fast dissociation kinetics of the binding. The problem could be solved by assaying the components of affinity eluate immobilized to a testosterone affinity resin that stabilizes the cAMP-protein complexes. The cAMP binding found in the affinity eluate shows an upward concave Scatchard plot and is compatible with a model containing two independent binding sites with dissociation constants of 7 and 58 nM. However, a larger number of binding sites or negative cooperativity cannot be excluded. Sixteen cAMP binding sites were observed per testosterone binding site. The binding affinity of cAMP exceeds that of cGMP 200-fold, that of cCMP 2000-fold, and that of AMP and 2',3'-cAMP more than 10,000-fold. Results indicate that cAMP is bound by the AR, although it only represents about 1% of the total protein in the affinity eluate: (i) Specific testosterone and cAMP binding of affinity eluate was copurified by affinity chromatography, density gradient centrifugation, and gel filtration. The ratio of cAMP to testosterone binding in each peak was about 16:1, identical with that found in the total affinity eluate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Die Innervation der Lungenarterien in reimplantierten Hundelungen

Zusammenfassung Elektronenmikroskopische Untersuchungen am Nervenplexus der Pulmonalarterien reimplantierter Lungen von Hunden, deren Überlebenszeiten zwischen 20 Tagen und 5 Monaten lagen, haben zu folgenden Ergebnissen geführt: Im distal von der Arterienanastomose gelegenen Abschnitt der A. pulmonalis ist der größte Teil der Nervenfasern degeneriert. Diese degenerierten Fasern gehören zu extrapulmonalen Ganglienzellen (Grenzstrang des Sympathicus, N. vagus, extrapulmonaler Plexus peribronchialis). Zwischen Adventitia und Media der mittleren und kleinen Äste der A. pulmonalis können reichlich intakte Nervenfasern beobachtet werden, und im Bereich der Arteriolen fallen nur intakte Axone auf. An den terminalen Axonen sind synaptische Anschwellungen mit den feinstrukturellen Merkmalen adrenergischer oder cholinergischer Nerven nachzuweisen.In biochemischen Untersuchungen wurden Gehalt und Aufnahme von Noradrenalin (NA) in Stamm-, Lappen- und Segmentarterienwänden von normalen und reimplantierten Hundelungen quantitativ bestimmt. Die Reimplantationszeiten betrugen hierbei 6, 15 und 20 Tage. In teilweiser Übereinstimmung mit den elektronenmikroskopischen Befunden haben diese biochemischen Untersuchungen ergeben, daß im distal von der Arterienanastomose gelegenen Teil der A. pulmonalis praktisch keine NA-Aufnahme oder -Speicherung mehr nach-weisbar ist.Die Befunde werden wie folgt interpretiert: Die Innervation der mittleren Pulmonalarterienäste erfolgt zum Teil, die der kleinen und kleinsten Arterienäste ausschließlich von intrapulmonalen Ganglienzellen des Plexus peribronchialis aus, während die A. pulmonalis mit ihren großen Ästen im wesentlichen von extrapulmonalen Ganglienzellen innerviert wird.

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The innervation of the pulmonary arteries in reimplanted canine lungsStudies of ultrastructure, noradrenalin content and uptake

Summary Electron microscopic studies on pulmonary arteries 20 days to 5 months after lung reimplantations (dogs) yielded the following results: In the plexus arterialis of the stem of the pulmonary artery most of the nerve fibres were found to be degenerated. These fibres belong to extrapulmonary ganglionic cells (i.e., sympathetic chain, vagus nerve, extrapulmonary peribronchial nerve plexus). Whereas in the nerve plexus of some of the branches of the pulmonary arteries and at the site of the arterioles no degenerated nerve fibres could be detected. The fibres which survived showed synaptic swellings with the fine structural characteristics of either adrenergic or cholinergic nerve terminals.Further investigations on the content and uptake of noradrenalin (NA) are consistent with the assumption that a part of the adrenergic nerves survived in the walls of the pulmonary arteries of reimplanted lungs, 6, 15, and 20 days after reimplantations. The highest decrease in the NA-content as well as in the NA-uptake was found in the stem of the artery in reimplanted lungs. Our present and previous findings lead to the following conclusion: in reimplanted lungs autonomic innervation of middle-sized, smaller, and smallest branches of the pulmonary artery is mainttained. The innervating nerve terminals belong to ganglionic cells of the plexus peribronchialis.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Differential effects of alpha subunit Asparagine56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity

The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG), are cysteine-knot growth-factor superfamily glycoproteins composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. The cysteine-knot motifs in both subunits create two hairpin loops, designated L1 and L3, on one side of the knot, with the intervening long loop, L2, on the opposite side. As the average alpha-subunit loop 2 oligosaccharide mass increased from 1482 to 2327, LH and FSH receptor-binding affinities of the dual-specificity eLH declined significantly, while the decrease in FSH receptor-binding affinity for eFSH was not significant. In the present study, we characterized hormone-specific glycosylation of alphaL2 oligosaccharides in eLHalpha, eFSHalpha, and eCGalpha preparations. MALDI mass spectrometry revealed 28-57 structures, including high mannose, hybrid, bi-, and triantennary oligosaccharides. The same intact subunit preparations and their alphaL2 loop-deglycosylated derivatives were combined with either eLHbeta or eFSHbeta, and the circular dichroism (CD) spectrum for each preparation was determined. We predicted that hybrid hormone preparations obtained by combining intact eLHalpha, eFSHalpha, and eCGalpha preparations with eLHbeta might exhibit differences in conformation that would disappear when the alphaL2 oligosaccharide attached to alphaAsn(56) was removed by selective peptide-N-glycanase digestion (N(56)dg-alpha). CD data supported the first prediction; however, elimination of alphaL2 oligosaccharide actually increased the conformational differences. The intact alpha subunit:eFSHbeta hybrids had virtually identical CD spectra, as expected. However, the N(56)dg-alpha:eFSHbeta hybrid spectra differed from each other. Oligosaccharide removal altered the conformation of most hybrids, suggesting that alphaAsn(82) oligosaccharide (located in alphaL3) also influenced gonadotropin conformation.     

Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells.

It was shown previously [Riedinger, H. J., van Betteraey-Nikoleit, M & Probst, H. (2002) Eur. J. Biochem.269, 2383-2393] that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.     

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.