Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Histochemical demonstration of purine nucleoside phosphorylase (PNPase) in microglial and astroglial cells of adult rat brain

The histochemical localization of enzymes associated with purine nucleoside metabolism indicates that glial cells might participate in the regulation of these compounds in the central nervous system. In the present study we examined the histochemical localization of purine nucleoside phosphorylase (PNPase) in sections from adult rat brain. Some sections were also sequentially stained immunocytochemically for astroglial or microglial cells utilizing glial fibrillary acidic protein (GFAP) or OX-42 antibodies, respectively. Our observations showed that PNPase was restricted to glial cells, whereas neurons always remained negative. Brain sections stained for both PNPase and GFAP showed that the GFAP-positive astroglial cells were always PNPase positive. Other PNPase-positive but GFAP-negative cells were also observed. These cells resembled microglial cells, and brain sections reacted for both PNPase and OX-42 confirmed this by showing that the major part of OX-42-positive microglial cells were PNPase positive. In these sections, the PNPase-positive but OX-42-negative cells present resembled astroglial cells. From our double staining experiments, we conclude that PNPase is present in both astroglial and microglial cells in normal adult brain.     

Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites

Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B4.     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

The nervous system of Tricladida. III. Neuroanatomy ofDendrocoelum lacteum andPolycelis tenuis (Plathelminthes,Paludicola): an immunocytochemical study

The organization of the nervous system ofDendrocoelum lacteum (Tricladida, Paludicola, Dendrocelidae) andPolycelis tenuis (Tricladida, Paludicola, Planariidae) was studied by immunocytochemical double staining, using neuropeptide RFamide and serotonin (5-HT) antisera on cryosections. The study confirmed the status of the main nerve cords (MCs) as the most important and stable of the longitudinal cords and supported the hypothesis of a common phylogenetic origin of the MCs in flatworms. The ganglion-like structures along the MCs at the beginning of transverse commissures and laterla branches showed a close contact with ventral fibres of the submuscular nerve plexus indicating an origin from crossing points of insunken ring commissures. The distributional pattern and morphology of the RFamide-IR cell bodies inD. lacteum corresponded to that of neurosecretory cells. Most RFamide-IR cells were unipolar and rounded while 5-HT-IR cells were uni- bi- and multipolar. The neutropile consisted of a dense RFamide-IR and a loose 5-HT-IR network. RFamide dominated in all parts of the genital plexus.     

Regulation of and intervention into the oxidative pentose phosphate pathway and adenine nucleotide metabolism in the heart

The capacity of the oxidative pentose phosphate pathway (PPP) in the heart is limited, since the activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of this pathway, is very low. Two mechanisms are involved in the regulation of this pathway. Under normal conditions, G-6-PD is inhibited by NADPH. This can be overcome in the isolated perfused rat heart by increasing the oxidized glutathione and by elevating the NADP⁺/NADPH ratio. Besides this rapid control mechanism, there is a long-term regulation which involves the synthesis of G-6-PD. The activity of G-6-PD was elevated in the rat heart during the development of cardiac hypertrophy due to constriction of the abdominal aorta and in the non-ischemic part of the rat heart subsequent to myocardial infarction. The catecholamines isoproterenol and norepinephrine stimulated the activity of myocardial G-6-PD in a time- and dose-dependent manner. The isoproterenol-induced stimulation was cAMP-dependent and due to increased new synthesis of enzyme protein. The G-6-PD mRNA was elevated by norepinephrine. As a consequence of the stimulation of the oxidative PPP, the available pool of 5-phosphoribosyl-l-pyrophosphate (PRPP) was expanded. PRPP is an important precursor substrate for purine and pyrimidine nucleotide synthesis. The limiting step in the oxidative PPP, the G-6-PD reaction, can be bypassed with ribose. This leads to an elevation of the cardiac PRPP pool. The decline in ATP that is induced in many pathophysiological conditions was attenuated or even entirely prevented by i.v. infusion of ribose. In two in vivo rat models, the overloaded and catecholamine-stimulated heart and the infarcted heart, the normalization of the cardiac adenine nucleotide pool by ribose was accompanied by an improvement of global heart function. Combination of ribose with adenine or inosine in isoproterenol-treated rats was more effective to restore completely the cardiac ATP level within a short period of time than either intervention alone. (Mol Cell Biochem 160/161: 101–109, 1996)     

Significance of Ca2+ and K+ in Micrasterias Growth and Morphogenesis

Significance of Ca²⁺ and K⁺ for the complex morphogenesis ofMicrasterias, which takes place through multipolar tip growth,was investigated. Studies with different external Ca²⁺ concentrationsand Ca²⁺ channel inhibitors LaCl₃ and verapamil indicate thatCa²⁺ and Ca²⁺ channels are essential in the development, whiletreatments with different K⁺ concentrations and K⁺ channel inhibitorTEA demonstrate that potassium or K⁺ channels are not neededin the process, albeit the existence of K⁺ channels. K⁺ is notneeded even for the regulation of turgor pressure, which wasfound to decrease clearly during cell development. The plasmamembrane ATPase inhibitors diethylstilbesterol (DES) and Na-orthovanadatestop morphogenesis and indicate the importance of ion pumpsin the developmental process. Both supraoptimal, external K⁺and Ca²⁺ cause abundant Ca²⁺ precipitate formation in chloroplasts,which shows that chloroplasts are important in regulation ofcytoplasmic Ca²⁺ metabolism and that K⁺ activates the uptakeof Ca²⁺ through Ca²⁺ channels. (Received June 13, 1995; Accepted September 13, 1996)     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.