The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca.

Summary A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neo^r) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neo^r phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.     

Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria

Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222.     

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Metabolic products of microorganisms. 192. The anthraquinones of the Aspergillus glaucus group. II. Biological activity

From the mycelia of Aspergillus cristatus the following anthraquionic pigments were isolated: catenarin, emodin, erythroglaucin, rubrocristin, physcion, physcion-9-anthrone, questin, viocristin, and isoviocristin. The latter two do not belong to the 9, 10-anthraquinone series but to the 1,4-anthraquinones, and so far they have not been reported among naturally occurring quinones.Emodin, catenarin, viocristin, and isoviocristin snowed antibacterial activity with minimal inhibitory concentrations ranging from 1–10 g/ml. In Bacillus brevis catenarin and emodin inhibited the incorporation of uracil and leucine preferentially. At higher concentrations the incorporation of thymidine into the trichloroacetic acid-precipitable fraction of cells was also affected. In the presence of viocristin or isoviocristin all three macromolecular syntheses came to a halt. Rubrocristin, erythroglaucin, and physcion showed no significant inhibitory effects.In Ehrlich ascites carcinoma cells catenarin, emodin, and viocristin inhibited the incorporation of uridine and thymidine. The incorporation of leucine was hardly affected.In vitro, inhibition of DNA-dependent RNA polymerase from Escherichia coli by catenarin and to a lesser extent by emodin was observed, whereas rubrocristin (catenarin-8-methyl ether), physcion, and erythroglaucin were not active.Abbreviations MIC minimal inhibitory concentration - TCA trichloroacetic acid - ECA Ehrlich ascites carcinoma Metabolic Products of Microorganisms. 191. W. Keller-Schierlein und B. Joos; Über das 4-Oxohomotyrosin, ein Abbauprodukt des Echinocandins. Helv. Chim. Acta (in press)     

Oxygen solubilities in fermentation fluids

Summary Fermentation media consist of a large number of chemicals whose composition undergoes alteration during the course of fermentation. As a result of this, conventional methods and correlations for oxygen solubility measurement and prediction do not apply in these systems. Using a physical method, oxygen solubilities were measured in simulated chemical systems and in fermentation broths. Sugars, salts, and fermentation products were identified as major factors influencing oxygen solubility. Salt effect was correlated with electrical conductivity of the medium, which was easy to measure during fermentation. For mixtures and for fermentation medium, individual influences were found to be log-additive in accordance with Danckwerts (1970).     

Die Pulvillen vonCalliphora erythrocephala (Diptera,Brachycera) als Adhäsionsorgane

Zusammenfassung Die Pulvillen vonCalliphora erythrocephala wurden licht und fluoreszenzmikroskopisch, raster- und transmissions-elektronenmikroskopisch untersucht. Das Adhäsionssekret wurde dünnschichtchromatographisch mit dem Körperoberflächen-Lipid verglichen.Die Pulvillen tragen auf der Ventralseite mehrere Tausend langer dünner Hafthaare mit sohlenartigen Endverbreiterungen. Die Kutikula des Pulvillus besteht hauptsächlich aus elastischen Kutikulatypen (Endo- und Mesokutikula) in verschiedenen, spezifischen Ausbildungsformen.Die Pulvillen enthalten ein sekretorisches Epithel ohne Ausführgänge, das Protein- und Lipideinschlüsse zeigt.Das Pulvillensekret ist eine schwerflüchtige, ölige Flüssigkeit, die nicht identisch ist mit dem Körperoberflächen-Lipid. Das Sekret wird in der homogenen Kutikala des Pulvillus gespeichert und über das Porenkanalsystem der Ventralseite nach außen geleitet.Der Zusammenhang zwischen der Struktur der Pulvillen und ihrer Rolle beim Adhäsionsvorgang wird diskutiert.

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The pulvilli ofCalliphora erythrocephala (Diptera, Brachycera) as adhesive organs

Summary The pulvilli ofCalliphora erythrocephala were examined by light microscopy, fluorescent light microscopy, SEM and TEM. The adhesive secretion was compared with the general surface lipid layer by means of thinlayerchromatography.On the ventral surface the pulvilli bare several thousand long, slender tenent hairs with solelike spatulate tips. The pulvillar cuticle is composed on the whole of elastic cuticle types (endocuticle, mesocuticle) with specific modifications of structure. The pulvilli contain a secretory epithelium without transport ducts which has lipid and proteinaceous inclusions.The pulvillar secretion is a slowly volatile oily liquid which is not identical to the general surface lipid. The secretion is stored in the homogeneous cuticle within the pulvillus and transported to the ventral surface via the pore canal system.The correlation between the structure of pulvilli and their function in the adhesion process is discussed here.

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Abkürzungen bl Basales Labyrinth - cv coated vesicle - db dense body - dlk Dichter Lamellenkörper - dw Dorsalwand - e Epithel - ef Endokutikulafasern in der Übergangszone (meso 2b) - endo 1 lamelläre Endokutikula - endo 2 homogene Endokutikula - endo 3 gelatinöse Endokutikula - ep Empodium - epi Epikutikula - exo Exokutikula - fr Fersenregion - fs Faltensaum - hh Hafthaare - hs Haarschaft - k Kutikula - kr Kralle - ku Kutikulinschicht - l Lumen - li Lipid - lr Dorsale Längsrippen - m Mitochondrium - mb Mesokutikulabalken in der Übergangszone (meso 2b) - meso 1 helle homogene Mesokutikula - meso 2a dunkle homogene Mesokutikula, kompakt - meso 2b Übergangszone, in der dunkle homogene Mesokutikulabalken mit faseriger Endokutikula verzahnt sind - mt Mikrotubulus - mvbd Dunkler multivesikulärer Körper - n Kern - pc Porenkanal - ps Proximalsklerit - pt Prätarsus - rer Rauhes endoplasmatisches Retikulum - s Schulter - sd Sehnendrüse - se Sehne - so Sohle - sr Saumregion - t Tubulus - t₅ Tarsalglied 5 - ta Tasche - td Tarsaldrüse - te Tarsalepithel - tg Taschengrund - tk Terminalkanäle - utr Unguitractor - v Heller Vesikel - va Proteinvakuole - w Wachsschicht - z Zementschicht     

INFLUENCE OF COLD EXPOSURE ON THE ACTIVITIES OF SOLUBLE AND MEMBRANE-BOUND DOPAMINE β-HYDROXYLASE IN RAT HEART VESICLES

Abstract— A fraction containing noradrenaline storage vesicles of the sympathetic nerve terminals in the rat heart was obtained by differential centrifugation. In this preparation, 17% of the dopamine β-hydroxylase was present in a soluble form. Cold exposure (3°C) for periods from 5 to 30 min led to an increase in the activity of soluble dopamine β-hydroxylase by about 50%, while the activity of membrane-bound dopamine β-hydroxylase was simultaneously decreased by approx 30%. The nor-adrenaline content of the vesicles rose concomitantly with the increase in the activity of soluble dopamine β-hydroxylase. This rise in noradrenaline content was caused by an enhanced synthesis and not by an alteration in the subcellular distribution. The results are discussed with respect to the fate of dopamine β-hydroxylase during enhanced sympathetic nerve activity.     

Combined tazemetostat and MAPKi enhances differentiation of papillary thyroid cancer cells harbouring BRAFV600E by synergistically decreasing global trimethylation of H3K27

Clinical efficacy of differentiation therapy with mitogen-activated protein kinase inhibitors (MAPKi) for lethal radioiodine-refractory papillary thyroid cancer (RR-PTC) urgently needs to be improved and the aberrant trimethylation of histone H3 lysine 27 (H3K27) plays a vital role in BRAF^V600E-MAPK-induced cancer dedifferentiation and drug resistance. Therefore, dual inhibition of MAPK and histone methyltransferase (EZH2) may produce more favourable treatment effects. In this study, BRAF^V600E-mutant (BCPAP and K1) and BRAF-wild-type (TPC-1) PTC cells were treated with MAPKi (dabrafenib or selumetinib) or EZH2 inhibitor (tazemetostat), or in combination, and the expression of iodine-metabolizing genes, radioiodine uptake, and toxicity were tested. We found that tazemetostat alone slightly increased iodine-metabolizing gene expression and promoted radioiodine uptake and toxicity, irrespective of the BRAF status. However, MAPKi induced these effects preferentially in BRAF^V600E mutant cells, which was robustly strengthened by tazemetostat incorporation. Mechanically, MAPKi-induced decrease of trimethylation of H3K27 was evidently intensified by tazemetostat in BRAF^V600E-mutant cells. In conclusion, tazemetostat combined with MAPKi enhances differentiation of PTC cells harbouring BRAF^V600E through synergistically decreasing global trimethylation of H3K27, representing a novel differentiation strategy.