N-mineralization in Löss-Parabrownearthes: Incubation experiments

Summary Nitrogen mineralization in Löss-Parabrownearthes was studied in long-term incubation experiments at different temperatures. Results obtained were used to optimize the parameters of a first-order kinetic model for N-mineralization of these soils: average reaction coefficients obtained by incubationat 35°C are 0.00737±0.00081 per day for the old organic material, and 0.25±0.07 per day for the fresh organic material.The mineralizable nitrogen of the fresh organic material at the beginning of march amounts to about 90±20 kg N/(ha*30 cm), independent of the kind of preceding crop. Temperature dependence of mineralization was estimated roughly. Attempts to simplify the procedure of evaluation of these parameters are described.Aqualfs and Udalfs according to the 7th approximation.     

Detection of hepatitis B surface antigen using passive hemagglutination

The passive hemagglutination test (Sero-Test CCB) for the detection of hepatitis B surface antigen (HBsAg) has been developed. The comparative study of the sensitivity of Sero-Test CCB, the passive hemagglutination test Hepanostikon (developed by Organon, the Netherlands) and the radioimmunoassay (with the use of an experimental assay kit provided by the Institute of Vaccines in Dessau, GDR) has been carried out by the determination of HBsAg in 100 coded sera from viral hepatitis patients and hepatitis virus carriers. Both passive hemagglutination tests (Sero-Test CCB and Hepanostikon) have yielded coinciding results (r = 0.90). The sensitivity of Sero-Test CCB has been found to exceed that achieved with the use of electrophoretic techniques 30-150 times, though it is 8 times lower than the sensitivity of the radioimmunoassay. The test kits Sero-Test CCB HBsAg are used for the examination of donor blood and for the survey of groups of persons subjected to a high risk of contacting hepatitis B infection in hemodialysis and transplantation centers, surgical wards, etc.     

The subcellular location of the higher acetylcholine content in rat brain found after killing by the near-freezing method as compared with decapitation

Abstract— We have confirmed the finding of Takahashi & Aprison (J. Neurochem. 11 , 887-898, 1964) that more acetylcholine is found in brains of rats killed by near-freezing compared to decapitation. The radioenzymic assays for acetylcholine and choline of Shea & Aprison (Analyt. Biochem. 56 , 165-177, 1973) and Goldberg & Mc Caman (J- Neurochem. 20 , 1 8, 1973) were used to measure both compounds and gave very similar results. The larger amount of ACh was observed both in powders of frozen rat brain and in homogenates prepared from animals killed by near-freezing. When subcellular fractionation of the homogenates was done in the presence of eserine, the larger amount of ACh was found in the soluble fraction (S₂). These results indicated that with the near-freezing method, an extra amount of ACh is preserved in a form that is originally protected from acetylcholinesterase but that becomes esterase-sensitive on fractionation since no differences were observed in P₁. P₂ or S₂ fractions when no eserine was present. The amounts of choline in homogenates and subcellular fractions were also measured after both methods of killing. Differences in the method of killing and postmortem changes which affect the choline values obtained are described.     

Die Nucleinsäuren grüner und gebleichter Zellen von Euglena gracilis

Zusammenfassung Bei der Isolierung der Gesamtnucleinsäuren aus autotroph gewachsenen Zellen von Euglena gracilis und ihrer säulenchromatographischen Auftrennung an methyliertem Serumalbumin nach den Verfahren, welche für Blau- und Grünalgen optimale Ergebnisse erbrachten, konnten nur drei Komponenten der löslichen RNA und zwei der hochmolekularen RNA nachgewiesen werden. Zellaufschluß in Gegenwart von Na-Dodecylsulfat (SDS) führte auch zur Extraktion der nativen DNA, welche im Eluierungsdiagramm als zusätzliches Absorptionsmaximum erschien. Mixotroph gewachsene Zellen besaßen die gleiche Zusammensetzung an Nucleinsäuren wie autotrophe. Mittels Dichtegradienten-Zentrifugation ließen sich die Masse der DNA in einem Sediment, die der Plastiden in einer grünen Bande anreichern. Letztere enthielten je eine Komponente DNA und hochmolekulare RNA; die Komponenten der löslichen RNA, sowie die Hauptmenge der hochmolekularen RNA blieben dabei in dem Überstand, welcher bei der ersten niedertourigen Zentrifugation des Ausgangshomogents angefallen war.In permanent gebleichten Zellen (durch Streptomycin, erhöhte Temperatur) ließen sich analog die Komponenten der löslichen RNA und die DNA nachweisen, jedoch fehlte bei der hochmolekularen RNA die typische Aufteilung in die zwei Komponenten (IV und V). Die Basenzusammensetzung derselben war identisch, unabhängig von autotropher oder mixotropher Anzucht der Zellen. Auch zwischen den beiden Teilfraktionen der hochmolekularen RNA aus gebleichten Zellen bestand kein Unterschied hinsichtlich des Basenverhältnisses; ein Vergleich mit den grünen Zellen zeigte jedoch, daß der Gehalt an Cytosin höher, der an Adenin niedriger lag.

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Summary The extraction of total nucleic acids from autotrophically grown cells of Euglena gracilis and their chromatographic separation on methylated serum albumin recently successfully applied to blue-green and green algae cells yielded 3 fractions of soluble RNA and two of high molecular RNA only. Cell disruption in the presence of SDS, however, made the native DNA extractable which appeared consequently as an additional peak in the chromatographic pattern. Cells from mixotrophic cultures had an analogous set of nucleic acids compared with those from autotrophic ones. Density gradient centrifugation gave rise to a sediment containing the bulk DNA, and to a band in which the plastids had accumulated. From ther latter a fraction of DNA and high molecular RNA was isolated; the soluble RNA and the bulk high molecular RNA was associated with a supernatant resulting from an initial low speed centrifugation of the original homogenate.Cells permanently bleached (by Streptomycin, increased temperature) had the typical set of soluble RNAs and DNA; the high molecular RNA, however, was eluted in a more or less uniform peak. The base composition of the fractions IV and V of high molecular RNA was identical, independent of autotrophic or mixtrophic growth of the cells. The two partial fractions of the high molecular RNA of bleached cells were identical, too, as far as the base ratio is concerned; compared with the green cells, however, the content of cytosine tended to increase, that of adenine to decrease.

     

CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity,transcriptional gene silencing and plant growth

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.     

Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation

Stochastic effects from demographic processes and selection are expected to shape the distribution of genetic variation in spatially heterogeneous environments. As the amount of genetic variation is central for long‐term persistence of populations, understanding how these processes affect variation over large‐scale geographical gradients is pivotal. We investigated the distribution of neutral and putatively adaptive genetic variation, and reconstructed demographic history in the moor frog (Rana arvalis) using 136 individuals from 15 populations along a 1,700‐km latitudinal gradient from northern Germany to northern Sweden. Using double digest restriction‐site associated DNA sequencing we obtained 27,590 single nucleotide polymorphisms (SNPs), and identified differentiation outliers and SNPs associated with growing season length. The populations grouped into a southern and a northern cluster, representing two phylogeographical lineages from different post‐glacial colonization routes. Hybrid index estimation and demographic model selection showed strong support for a southern and northern lineage and evidence of gene flow between regions located on each side of a contact zone. However, patterns of past gene flow over the contact zone differed between neutral and putatively adaptive SNPs. While neutral nucleotide diversity was higher along the southern than the northern part of the gradient, nucleotide diversity in differentiation outliers showed the opposite pattern, suggesting differences in the relative strength of selection and drift along the gradient. Variation associated with growing season length decreased with latitude along the southern part of the gradient, but not along the northern part where variation was lower, suggesting stronger climate‐mediated selection in the north. Outlier SNPs included loci involved in immunity and developmental processes.     

Effect of a high-intensity exercise training on the metabolism and function of macrophages and lymphocytes of walker 256 tumor bearing rats

Epidemiologic studies suggest that moderately intense training promotes augmented immune function, whereas strenuous exercise can cause immunosupression. Because the combat of cancer requires high immune function, high-intensity exercise could negatively affect the host organism; however, despite the epidemiologic data, there is a lack of experimental evidence to show that high-intensity training is harmful to the immune system. Therefore, we tested the influence of high-intensity treadmill training (10 weeks, 5 days/week, 30 mins/day, 85% VO(2)max) on immune system function and tumor development in Walker 256 tumor-bearing Wistar rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, in addition to some functional parameters such as hydrogen peroxide production, phagocytosis, and lymphocyte proliferative responses. The metabolism of Walker 256 cells was also investigated. Results demonstrated that high-intensity training increased the life span of tumor-bearing rats, promoted a reduction in tumor mass, and prevented indicators of cachexia. Several changes, such as a reduction in body weight and food intake and activation of glutamine metabolism in macrophages and lymphocytes induced by the presence of Walker 256 tumor, were prevented by high intensity training. The reduction in tumor growth was associated with an impairment of tumor cell glucose and glutamine metabolism. These data suggest that high-intensity exercise training may be a viable strategy against tumors.     

Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-beta1-signaling and upregulation of cathepsin B

The TGF-beta signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-beta1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-beta1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-beta signaling pathways showed no alterations in TGF-beta/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-beta1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-beta1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.