Genetic and biochemical characterization of a new chymotrypsin isozyme of the house mouse,CTRA-1

Genetic variation of a codominantly inherited pancreas protease, designated CTRA-1, was discovered in the house mouse by isoelectric focusing in polyacrylamide gels. Phenotype CTRA-1A was found in MOLH/Fre and in the majority of common laboratory mouse strains. Phenotype CTRA-1B was found in PWD/Ph. It was characterized by the absence of a corresponding protease band. A third phenotype, CTRA-1C, was observed in IS/Cam and a fourth phenotype, CTRA-1D, was detected in SEG/1. CTRA-1 was found only in the pancreas and may represent the A form of chymotrypsin. The enzyme was shown to be controlled by the presumed structural locus Ctra-1 located on chromosome 8. From two backcross series, including a total of 274 animals, the gene order (Es-1, Es-9)-3.9 +/- 1.7%-Got-2-3.9 +/- 1.7%-(Es-2, Es-7, Es-23)-0.7 +/- 0.5%- Ctra-1-6.3 +/- 2.2%-Prt-2 was established.     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

DNA-Analyse mittels Restriktionsenzymen: Bedeutung und m?gliche Anwendungen in der Ornithologie

Zusammenfassung Die Analyse von Sequenzunterschieden in mitochondrieller und genomischer DNA von Vögeln mittels Restriktionsenzymen eröffnet völlig neue Perspektiven für systematische, populationsgenetische und verhaltensökologische Forschung. Diese Methode ist der elektrophoretischen Untersuchung von Isoenzymen und der DNA-DNA-Hybridisierung vielfach überlegen. Das Prinzip, der technische Ablauf und die theoretischen Vorteile werden erläutert. Einige bisherige Untersuchungen dienen als Beispiele für vielversprechende Anwendungsmöglichkeiten in der Ornithologie. Die Einrichtung eines Schwerpunktlabors für solche Arbeiten wird vorgeschlagen, um technische und personelle Ausstattung optimal nutzen zu können.

---

Restriction enzyme analysis of DNA: principle and possible applications in ornithology

Summary The analysis of restriction fragment length polymorphisms in mitochondrial and genomic DNA of birds opens up a large new field of research for ornithologists. The method is in most contexts superior to electrophoretic analysis of allozymes and an important complement to DNA-DNA hybridization. Its principle, the technical procedures and theoretical advantages are briefly explained. Some recent studies are reviewed and potential applications outlined. Ornithological institutions in Germany should set up a central laboratory for such research to use personnel and technical equipment most efficiently.

     

Nonradioactive in situ nick translation combined with counterstaining: Characterization of C-band and silver positive regions in mouse testicular cells

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.     

N-hydroxymethylpentamethylmelamine,a major in vitro metabolite of hexamethylmelamine

N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.     

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10⁴ [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L -leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L -leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L -leucine. The kinetic constants V_max and K_m of enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L -leucine was produced continuously out of α-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.