Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Versuche zum Herbizideinsatz in gesäten Kohlarten

In den Jahren 1987 bis 1989 wurden die Auswirkungen und Effekte eines kombinierten Befalls des Winterweizens mit Sitobion avenae (Fabr.) und Puccinia recondita Rob.ex Desm. f.sp.tritici Erikss. untersucht. Dabei wurde das Verhalten der Schaderregerpopulationen und die Einflußnahme auf das Ertragsgeschehen bei separatem und simultanem Befall erfaßt. Während bei starkem Befall der oberen Blattetagen mit P.recondita eine Förderung der Aphidenpopulation an den Ähren der befallenen Pflanzen registriert werden konnte, war bei schwachem Braunrostbefall keine Einflußnahme auf die Blattläuse nachweisbar. Durch das kombinierte Auftreten beider Schaderreger kann es zu Reduktionen bei der Kornmasse/Ähre und bei der Tausendkornmasse kommen, die größer sind, als die Summe der Verluste bei Einzelbefall. Ähren stark braunrostbefallener Winterweizenpflanzen wurden zeitiger von S.avenae angeflogen und schneller besiedelt als diese von gesunden Kontrollpflanzen. Als Ursache für die Förderung der Populationsentwicklung von S.avenae werden durch P.recondita induzierte Veränderungen im Angebot an freien Aminosäuren in der Wirtspflanze diskutiert.     

Testis size,epididymis weight,and sperm competition in rhesus macaques

The intensity of sperm competition is often measured using the gonadosomatic index (testes/body weight). But sperm competition could be mediated more by size of the epididymis than by size of the testicles, and little information is available on the relationship between testicular and epididymal size. We found that both organs were positively correlated in size among male rhesus macaques. Body weight accounted for over 70% of the variance in testicle size and volumetric estimates of testicle size accurately reflected testicle weight. We conclude that methods for ascertaining testicle size are accurate, but the covariation in size between testicles and epididymis will hamper understanding of the physiological mechanisms involved in sperm competition in primates. © 1993 Wiley-Liss, Inc.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Immunofluorescent localization of collagen types I,III and IV,fibronectin, laminin,and basement membrane proteoglycan in developing mouse skin

Summary The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14–18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components — type IV collagen, laminin and heparan sulphate proteoglycan — were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components — types I and III collagen, and fibronectin — was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.