Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Quantitative 125J-Autoradiographie einzelner Zellen

Zusammenfassung Das nach der Energie seiner -Zerfälle zwischen Tritium und Kohlenstoff-14 liegende Isotop ¹²⁵J wurde auf seine Eignung zur quantitativen Autoradiographie geprüft. Absorption und Geometrie-Faktoren der radioaktiven Strahlung wurden untersucht. Hieraus ließen sich geeignete Meßbedingungen entwickeln. Durch gleichzeitige Exposition von radioaktiven Referenzquellen können absolute Radioaktivitätsmengen ermittelt werden. Als Referenzquellen sind membranmarkierte Standardzellen geeignet, die den physikalischen Eigenschaften des Isotopes Rechnung tragen. Hierzu wurden enzymatisch radiojodinierte Schaferythrozyten auf ihre Eignung geprüft. Die absolute Zahl von antigenen Stoffen auf den Oberflächen einzelner Zellen erhält man, wenn man die spezifische Aktivität der markierten Antikörpermoleküle bestimmt und die Silberkorndichte der zu untersuchenden Zellen und der Standardzellen mißt. Die Radioaktivität pro Standardzelle wird in herkömmlicher Weise ermittelt.Die neue Methode wurde zur Quantifizierung membrangebundener Immunglobulinmoleküle vom IgG-Typ auf einzelnen menschlichen Lymphozyten angewendet. Hierbei ist die Ermittlung einer immunologischen Sättigung des markierten Antikörpers wesentlich. Auf Lymphozyten von Normalpersonen und von chronisch-lymphatischen Leukämiepatienten konnten sehr unterschiedliche absolute Immunglobulinmengen bestimmt werden.

---

Quantitative ¹²⁵I-autoradiography of individual cells

Summary Iodine 125, an emitter of -radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures.The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.

---

---

Assoziation mit EURATOM 031-64 I BIAD.     

Increased activity of chondroitin sulfate-synthesizing enzymes during proliferation of arterial smooth muscle cells

Cultured arterial smooth muscle cells incorporate [35S]sulfate into the extracellular chondroitin sulfate/dermatan sulfate containing proteoglycans at a higher rate in the phase of logarithmic growth than do non-dividing cells. The cell growth-dependent decrease in 35S incorporation with increasing cell density is accompanied by a decrease in the activity of chondroitin sulfate-synthesizing enzymes. The specific activity of xylosyl transferase, N-acetylgalactosaminyl transferase I and chondroitin sulfotransferase declines as the cells proceed from low to high densities. The corresponding correlation coefficients are 0.86, 0.91 and 0.89. The ratio of C-6OH/C-4OH sulfation of chondroitin shows a cell proliferation-dependent decrease indicating an inverse correlation of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase activity. The observed changes in the expression of enzyme activities are thought to have some implications in the pathogenesis of arteriosclerosis, the initial stages of which are characterized by proliferation of arterial smooth muscle cells.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

An extraction method for nematodes in decomposition studies using the minicontainer-method

The minicontainer-method is a new method developed to study biological processes related to soil litter decomposition. An adaptation of the classical Baermann-funnel technique is described which can be used, in association with the minicontainer method, to investigate the role of Nematoda in litter decomposition. The use of the extraction method is illustrated in a study of the effects of different tillage systems on the decomposition of rye straw and on the nematode density in minicontainers with different mesh sizes of 20 µm, 500 µm and 2 mm. Three tilled plots (conventional deep plough, cultivator and two-layer plough) and an untilled control were compared after periods of 4 weeks and 38 weeks. On both sample dates there were significant main effects of treatment and mesh size on the nematode density, and additionally, after 38 weeks significant treatment x soil depth interactions. After 4 weeks, there were significant main effects of treatment and soil depth on the decomposition, but no mesh size effects, whereas after 38 weeks, all experimental factors had a significant effect on the decomposition of the straw. Due to the small volume of litter substrate used in the minicontainer method, the efficiency of nematode extraction is high and the lack of oxygen in the minicontainers presents no serious problem during the extraction process. The method also allows the simultaneous extraction of a large number of samples within a short period of time. Our results indicate that the method is suitable to study the microdistribution of nematode activity within the soil profile and improves the possible applications of the minicontainer-method.     

Presynaptic calcium and control of vesicle fusion

Vesicle fusion and transmitter release at synapses is driven by a highly localized Ca2+ signal that rapidly builds up around open Ca2+-channels at and near presynaptic active zones. It has been difficult to estimate the amplitude and the kinetics of this 'microdomain' signal by direct Ca2+-imaging approaches. Recently, Ca2+ uncaging at large CNS synapses, among them the calyx of Held, has shown that the intrinsic cooperativity of Ca2+ in inducing vesicle fusion is high, with 4-5 Ca2+ ions needed to trigger vesicle fusion. Given the Ca2+-sensitivity of vesicle fusion as determined by Ca2+-uncaging, it was found that a surprisingly small (10-25 microM) and brief (<1 ms) local Ca2+ signal is sufficient to achieve the amount, and the kinetics of the physiological transmitter release. The high cooperativity of Ca2+ in inducing vesicle fusion and the non-saturation of the Ca2+-sensor for vesicle fusion renders small changes of the local Ca2+-signal highly effective in changing the release probability; an insight that is important for our understanding of short-term modulation of synaptic strength.     

The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K⁺-transport system KdpFABC in response to K⁺ limitation or salt stress. Under K⁺ limiting conditions the Kdp system restores the intracellular K⁺ concentration, while in response to salt stress K⁺ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K⁺, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K⁺ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K⁺ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.