Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of ^106.75 and 10^6.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c. Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10⁶c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×10⁸ TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10⁹ TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition. This revised version was published online in June 2006 with corrections to the Cover Date.     

On the Modeling of Some Anti-inflammatory and Anti-thrombotic Drugs by AM1

Two families of autacoids from cell membrane phospholipids have been identified. The first, the icosanoids, which are formed from arachidonic acid, include prostaglandins and leukotrienes. The other includes modified phospholipids, as the platelet aggregating factor (PAF). These compounds are related to inflammatory and cardiovascular diseases. We review in this paper some of the work that has been done in our laboratories in the last few years relating to the modeling of new potential thromboxane synthase (TXS) and 5-lipoxygenase (5-LO) and cyclooxygenase (COX) inhibitors, and TXA₂ receptor antagonists derived from nitrogenated heterocycles. We include the results of the modeling of a group of proposed PAF antagonists, and compare their structures with PAF itself and with a recently proposed PAF antagonist model. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analogues of tentoxin: Tools for mechanistic investigations

Tentoxin[cyclo-(MeAla¹-Leu²-MePhe³-Gly⁴] is a metabolite isolated from a phytopathogenic fungusAlternaria alternata, which induces chlorosis of many plants. This potentialnatural herbicide binds specifically to the soluble part CF₁of the chloroplastic coupling factor, which is a proton ATP-synthase. Theeffect of the toxin is concentration dependent: at low concentration it is apowerful inhibitor, while at higher concentration, it stimulates the enzyme.We synthesized tentoxin and we designed new analogues in order to vary thehydrophobicity on the side chain and to study the structure activityrelationships. Comparative activities suggest that it is possible toseparate inhibitory and activating effects using tentoxin analogues, showingsome evidence for the existence of two binding sites with different affinityconstant.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

The proline biosynthesis in living organisms

Summary In this article we review recent work on the physiology of proline and ¹-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK -glutamyl kinase - GPR -glutamyl phosphate reductase - P5CR ¹-pyrroline-5-carboxylate reductase - GSA glutamic--semialdehyde - P5C ¹-pyrroline-5-carboxylate - P₁ Inorganic phosphate - AMP, ADP, ATP Adenosine 5-mono-, di-, triphosphate - NAD⁺, NADH nicotinamide adenine dinucleotide, and its reduced form - NADP⁺, NADPH nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle - OAT ornithine amino transferase; CHO: Chinese hamster ovary - IGF-1 insulin-like growth factor-1 - P5CDH pyrroline 5carboxylate dehydrogenase - IMP inosine 5-monophosphate     

Synthesis of some racemicγ-fluoro-α-amino acids

Summary Versatile three-step procedures for syntheses of seven racemi-fluoro-a-amino acids are described. Alkylation oftert-butyl N-(diphenylmethylene) glycinate with 1-bromo-2-fluoroalkanes gave N-protected aminoacid esters both in anhydrous medium using lithium-diisopropylamide as base at low temperature or in a two phase system of 50% aqueous sodium hydroxide and methylene chloride with triethylbenzylammonium chloride as the phase transfer catalyst at room temperature. Subsequent two-step deprotection with citric acid and hydrochloric acid gave the title compounds in 13–33% overall yields.Dedicated to Professor Dr.mult., Dr.h.c. Alois Haas on the occasion of his 65th birthday     

Roles of ubiquitin-mediated proteolysis in cell cycle control

Selective degradation of cyclins, inhibitors of cyclin-dependent kinases and anaphase inhibitors is responsible for several major cell cycle transitions. The degradation of these cell cycle regulators is controlled by the action of ubiquitin—protein-ligase complexes, which target the regulators for degradation by the 26S proteasome. Recent results indicate that two types of multisubunit ubiquitin ligase complexes, which are connected to the protein kinase regulatory network of the cell cycle in different ways, are responsible for the specific and programmed degradation of many cell cycle regulators.     

The medical utility of genomics data in neuropsychiatry: mutational genetics versus association genetics

The long anticipated ‘genetic revolution’ in neuropsychiatry has yet to have an impact on the practice of clinical medicine. Excitement in the 1980s over major genetic breakthroughs in schizophrenia and manic depression, for example, has been replaced in the late 1990s by the sobering realization that most common neuropsychiatric disorders are multifactorial. Despite considerable effort and resources, no ‘causative’ genetic variation has been identified that plays a definitive major role in any common neuropsychiatric disorder.     

Immobilization of Haloferax mediterranei aldolase by cross-linking in a proteinic matrix: stability and halophilic characteristics

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) of Haloferax mediterranei was immobilized by treating the cell extract in the presence of 10% BSA, with the cross-linking reagent, 0.5% glutaraldehyde for 15min, with the retention of 60% of its original activity. The immobilized preparation exhibited a shift in the temperature optimum from 55°C to 65°C. The enzyme showed enhanced stability towards inactivation by radiation and storage (0–5°C) on immobilization. Immobilization also made the enzyme less halophilic, reducing its denaturation on prolonged storage in a non-salt medium, as well as exhibiting optimal activity at a lower KCl concentration (0.5m) as compared to the soluble enzyme (1–2m).