Enzymatic activity and thermoresistance of improved microbial transglutaminase variants

Amino Acids - Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side...     

The temperature dependence of protein kinase A and C activity in variant mouse neuroblastoma cells differing genetically in their heat resistance]

The ability of the mouse neuroblastoma cell line NTR to proliferate at 40 degrees C correlates with the position of the temperature optimum of protein kinases A and C activities in the region of higher temperatures compared to those for cells of the original line N18AI, and with higher thermostability of protein kinase A after its heating at various elevated temperatures. The found changes in protein kinases A and C in the cells of NTR line mean that the selection of variants, capable of growing at elevated temperatures, is accompanied with conformational protein changes.     

The isolation of ovomucoid variants differing in carbohydrate composition

Three major and two minor species of ovomucoid were separated by chromatography on sulphoethyl-Sephadex. The predominant sialic acid-free species was further resolved into three fractions by DEAE-cellulose chromatography. Although all species of ovomucoid had closely similar trypsin-inhibiting activity, immunochemical properties and amino acid composition, they differ in carbohydrate composition. Wide variation was observed in the content of galactose, N-acetylglucosamine and sialic acid. Charge heterogeneity was related, in part, to variation in sialic acid content. The implications of variable carbohydrate composition for the structure and function of ovomucoid are discussed.     

The in vivo differentiation of murine neuroblastoma

C1300 neuroblastoma was implanted with regenerating skeletal muscle to study the role of tissue interactions during tumor cell differentiation. Combined tumor-muscle implants, placed subcutaneously or within diffusion chambers were compared with control tumors implanted without muscle. Neuroblastoma implanted with injured muscle undergoes a partial neuronal differentiation. The tumor cells lose their normal round cell configuration and develop numerous cytoplasmic processes. Accompanying these outward changes are an increased content of microtubules in the neuritic processes; the appearance of glial-like processes containing abundant microfilaments; and the occurrence of growth vesicles identical to those of the growth cones of normal neurites. Although the implants usually contain large numbers of regenerated myofibers, tumor cell differentiation is not dependent upon the presence of these newly formed fibers. Tumor differentiation occurs equally well on the surfaces of degenerating muscle fragments, fibrin deposits and on the membrane surfaces of the diffusion chambers. These observations suggest that non-specific cell surface phenomena, rather than neuromuscular interactions were primarily responsible for the tumor cell differentiation in vivo.     

Neurotransmitter metabolism in murine neuroblastoma cells.

Murine neuroblastoma cells in culture are able to synthesize the putative neurotransmitters--acetylcholine, dopamine, norepinephrine, tyramine, octopamine, histamine, serotonin and γ-aminobutyric acid (GABA). They possess not only synthetic, but also degradative enzymes involved in metabolism of these transmitters, and many of these enzymes increase in activity as the cells “differentiate”. Catecholamines, and perhaps other transmitters, appears to be stored within membrane-limited vesicles which accumulate within the process endings of these cells. Uptake of some transmitters, GABA, glycine, dopamine and norepinephrine, shows characteristics of the high affinity transport systems observed in other neuronal populations; uptake of choline and other amino acids is similar to that in non-neuronal populations. Cells show receptor sensitivities to acetyl-choline, dopamine, norepinephrine, prostaglandins E₁ and morphine, as demonstrated by electrophysiologic, toxin binding and cyclic nucleotide studies.     

Aluminum speciation and morphological differentiation in murine neuroblastoma cells

Aluminum is known as a neurotoxic metal ion in experimental animals as well as in humans. The present study was carried out to determine whether and how the physicochemical properties of the metal coordination sphere (metal speciation) can influence the differentiation of murine neuroblastoma cells as has been observed previously in this laboratory (1). Results herein reported indicate that while the aluminum lipophilic species—particularly aluminum acetylacetonate (Alacac₃) and aluminum maltolate (Almalt₃), both hydrolitically stable and differently lipophilic—are both rather cytotoxic, metal hydrophilic species show different neuritogenic properties indicating the ability of Al(III), when diversely coordinated, to produce different biological effects.     

Production and characterization of genetically modified human IL-11 variants

Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core α-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114–115 aa), the third loop (M3:160–161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11.     

Phenotypic characteristics of heat-resistant murine neuroblastoma cells

One-step selection of murine neuroblastoma (N18TG2 cell line) for the resistance to injuring action of higher temperature resulted in obtaining cell line NTR1 stably capable of proliferating at 40 degrees C. The thermoresistance is shown to be coupled with the changes in some phenotypic features of NTR1 cells: the multiplication rate, saturation density of cells, morphological features, inducibility of long neurite-like processes and adhesion. A possible significance of plasma membrane changes in genetically stable thermoresistance of NTR1 neuroblastoma cells is discussed.     

Retinoic acid induction of sialyltransferase activity in neuroblastoma cells of differing sialylation potentials

In order to determine how glycosylation changes associated with cellular differentiation may be influenced by the basal cellular sialylation potential, the effect of retinoic acid (RA)-induced differentiation was investigated in neuroblastoma cells expressing differing levels (and activities) of the 2,6(N) sialyltransferase (ST6N) enzyme. The increase in ST activity was proportional to the basal cellular sialylation potentials with the high activity clones showing the greatest increase. This was paralleled by an up-regulation of the level of overall sialoglycoprotein glycosylation level. An increase in the levels of the polysialic acid (PSA) epitope was associated with a parallel increase in the levels of the neural cell adhesion molecule (NCAM) protein backbone although there was no overall change in the PSA:NCAM ratio following RA treatment.     

Effect of GABA-Administration on murine neuroblastoma cells in culture

Summary (Gamma aminobutyric acid) GABA was applied to cultures of mouse neuroblastoma cells of different ages at concentrations ranging from 10^-4 to 10^-6 M. The cultures were exposed to GABA either in short term experiments for 2 h to 2 days or for longer periods by adding the substance twice within 10 days at 5-day intervals. The following effects were observed: (1) There was a strong proliferation of coated vesicles, appearing to derive from the Golgi complex and the rough endoplasmic reticulum (RER), and also showing all intermediate stages of fusion and pinching off from the plasma membranes. (2) In numerous areas, electron-dense material aggregated at the inner aspect of the plasma membrane and around small invaginations of the plasmalemma. (3) The number and area of specialized contacts increased between cells and their processes. (4) Similar to cultures free of GABA, varicosities and terminal swellings of the cells and their processes were filled with small round vesicles, 40–60 nm in diameter, or with smooth, very large, empty-appearing vesicular inclusions, or with flat pleiomorphic vesicles. In addition, mitochondria and some formations of the smooth endoplasmic reticulum (SER) appeared, and primitive contacts (symmetrical densities) were formed. (5) Dense-cored vesicles were found peripherally and linearly arranged, surrounded by an electron-dense substance. (6) Electron-dense material of unknown origin was seen between cells or their processes near the peripherally arranged dense-cored vesicles. Exogenous GABA may play a specific role in the early stages of synaptogenesis, since it showed a positive effect on the neuroblastoma cells, which in the absence of GABA are only capable of forming primitive or immature presynaptic elements. The significance of the peripheral accumulation of dense-cored vesicles, accompanied by an amorphous, electron-dense substance occurring both intra- and extracellularly is discussed.     

Spermine supports catalysis of hairpin ribozyme variants to differing extents

Because of the ability to cleave RNA substrates in trans, the hairpin ribozyme has great potential for therapeutic application. Activity of a three-stranded version of the minimal truncated form is enhanced by the presence of the polyamine spermine. Since spermine is the most abundant polyamine in eucariots, improved prospects for the hairpin ribozyme as therapeutic agent were predicted. We have found that not all hairpin ribozyme variants accept spermine equally well as counter-ion. Particularly the two-stranded versions commonly used for therapeutic studies show rather decreased activity when spermine is present. We have investigated a number of hairpin ribozyme derivatives regarding their ability to carry out spermine supported catalysis. Among the studied structures a two-stranded reverse-joined hairpin ribozyme displayed the highest cleavage rates in a synergistic mixture of magnesium ions and spermine. The specific features of this ribozyme along with its potential for in vivo application are discussed.     

Investigations on OP-resistance of two genetically differing populations of Tetranychus urticae K.

In the work presented two genetically different populations of Tetranychus urticae Koch and their reaction to M-Systox-R were characterized. The Leverkusen-R population shows a pronounced dynamic resistance on the basis of vitality depressing r-factors: dominant semilethals and a major, recessive, mendelian factor.Contrary to this is the static resistance in the Blauvelt population, in which an intermediate resistance level even under strong and longlasting selection pressure could not be increased more than two to four fold. This stability is interpreted by the possibility of a major r-gene present in homozygous condition. Obviously it does not decrease viability and is hardly influenced by modifying genes.D.m. determinations over a period of one year showed fluctuations in response to M-Systox-R in both populations which apparently were due to seasonal influences through nutrition. The term vigor tolerance can be applied correctly to classify these fluctuations. For future toxicological and genetical work on spider mites the Slide-Dip Method used in this work was proposed to standardize results.

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Zusammenfassung In vorliegender Arbeit wurden zwei genetisch verschiedene Populationen von Tetranychus urticae K. und ihre Reaktion, gegenüber M-Systox-R charakterisiert. Die Leverkusen-R-Population zeigte eine ausgesprochen dynamische Resistenz auf der Basis von vitalitätsmindernden R-Faktoren, und zwar dominanten Semilethalfaktoren und einem größeren, rezessiven und mendelnden Gen.Im Gegensatz dazu steht die statische Resistenz in der Blauvelt-Population, bei der ein mittlerer Resistenzgrad auch bei starker und langdauernder Selektion nicht über das Doppelte bis Vierfache zu steigern war. Diese Stabilität kann so erklärt werden, daß ein größeres R-Gen in homozygotem Zustand in der Population vorliegt. Offenbar setzt es die Vitalität nicht herab und wird kaum durch modifizierende Gene beeinflußt.Dosis-Mortalitäts-Bestimmungen über den Zeitraum von einem Jahr zeigten in beiden Populationen Schwankungen in der Reaktion gegenüber M-Systox-R, die offenbar auf jahreszeitliche Einflüsse über die Ernährung zurückzuführen sind. Die Bezeichnung vigor tolerance kann zur Klassifizierung dieser Schwankungen richtig verwandt werden.Für zukünftiges toxicologisches und genetisches Arbeiten mit Spinnmilben wird die in vorliegender Arbeit verwandte Slide-Dip-Methode zur Vereinheitlichung der Versuchsergebnisse vorgeschlagen.

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Abbreviations used r resistant (ce) - d.m. dosage mortality - OP organophosphate - a.i. active ingredients     

Histone variants in rat spermatogonia and primary spermatocytes

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.