Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

Structure-based 3-D-QSAR analysis of marine indole alkaloids

A 3-D-QSAR study has been performed on these indole alkaloid derivatives to correlate their chemical structures with their observed antitumor activity against IGROV1. Due to the absence of information on their active mechanism, comparative molecular field analysis (CoMFA) has been applied. A model able to well correlate the antitumor activity with the chemical structures of mono and bis(indole) alkaloids 1-18 has been developed which is potentially helpful in the design of novel and more potent antitumor agents.     

The effect of oligomycin on rat liver mitochondria respiring in state 4

It has been found that oligomycin inhibits up to at least 50% state-4 mitochondrial respiration. A time dependence of oligomycin inhibition has been shown. A titration curve for state-4 respiration of sigmoidal profile has been presented. The possibility of misreading this oligomycin effect, so far never reported, has been excluded by evaluating the quality of mitochondrial preparations used in respect to their morphological, functional and electrochemical properties. The conclusion has therefore been put forward that the most part of respiration in steady-state-4 is driven by ATP synthesis.     

Characterization of lipophilicity and antiproliferative activity of E-2-arylmethylene-1-tetralones and their heteroanalogues

A molecular library based on E-2-arylmethylene-1-tetralone has been designed and synthesized. A reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed and applied to separate them and to characterize their lipophilicity. The chromatographic method applied here was suitable to separate the structural (ortho and para) isomers of compounds and was sensitive enough to differentiate their lipophilicities. The measured (k') and computer calculated (CLOGP) lipophilicity values has been compared. Good linear correlation has been found in the case of these structurally related molecules. In vitro biological assay has been performed with Methylene blue dying to investigate the antiproliferative potency of the compounds synthesized in this work. The measured (k') and calculated (CLOGP) lipophilicities of the compounds were compared with the antiproliferative activities and an optimum value of lipophilicity has been found for these compounds.     

Two subforms of eukaryotic topoisomerase I. Purification and structure-function relationships.

A new method for isolation of eukaryotic topoisomerase I from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.     

Synthesis and biological evaluation of novel triazoles and isoxazoles linked 2-phenyl benzothiazole as potential anticancer agents

A new series of isoxazoles and triazoles linked 2-phenyl benzothiazole were synthesized and evaluated for their anticancer activity. These compounds have been tested for their cytotoxicity three cancer cell lines. Among the compounds tested, compound 5d showed good cytotoxicity against Colo-205 and A549 cells in comparison to standard control PMX 610(1). Further compound 5d has been tested for its apoptotic activity and its inhibitory activity against caspase and PARP proteins. Hence this compound has the potential that it can be selected for further biological studies.     

The electrophoresis of histones in polyacrylamide gel and their quantitative determination

1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2.4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel.     

Mathematical model study of megakaryocytopoiesis. 1. The simulation model, cell steady-state distribution and the number of divisions in a proliferating population

A method of simulation has been proposed for studying megakaryocytopoiesis. A model has been developed which takes into account the age structure of cell population, the stochasticity of their maturation, and the principles of megakaryocytopoiesis regulation (known from the experiments). The mean number of divisions in a proliferating pool has been determined, as well as the duration of development of the cells with a positive acetylcholine esterase reaction.     

Synthesis by high-efficiency liquid-phase (HELP) method of oligonucleotides conjugated with high-molecular weight polyethylene glycols (PEGs)

The chemical modification of synthetic oligonucleotides has recently been investigated to improve their pharmacological utilization. In addition to chemical alterations of the backbone and of the heterocyclic bases, their conjugation with amphiphylic moieties, such as the polyethylene glycol has been proposed. The large scale production of these molecules as demanded for commercial purposes is hampered by the heterogeneity of the solid-phase processes and by the low reactivity of high-molecular weight PEGs in solution. A new synthetic procedure based on the recently developed liquid-phase method (HELP), has been set up to overcome these limitations.     

Genetic control of migratory behaviour in birds

Although it has long been suspected that biannual migration in birds has a direct genetic basis, only in the last decade have details of the inheritance of behavioural traits such as migratory activity and directional preferences been demonstrated. A model has now been developed to estimate how inexperienced first-time migrants manage to reach their unknown winter quarters on the basis of inherited spatio-temporal programs. Furthermore, in obligate partial migrants the decision to migrate or not has been shown to have a strong genetic base. Migratoriness and sedentariness in partial migrants have been shown to have a high potential for rapid evolution. A recent set of results has suggested that novel migratory habits can evolve in less than 25 years. A possible consequence is that environmental changes, including 'greenhouse' effects, might considerably alter avian migration systems by acting on genetic variation for migratory tendencies.     

A new classification of resin-based aesthetic adhesive materials

The purpose of this article is to illustrate a new classification of resin based aesthetic materials laying on the characterization of their matrix and their filler morphology. Four samples per material have been prepared for SEM evaluation. Each sample has been treated with chloroform to dissolve its matrix in order to evidence the filler morphology. A general schema of four different matrix systems which characterize the material's level of hydrophobicity can be put in evidence. The subsequent filler analysis individuates a more complex schema based on filler size and construction. A new classification based on matrix nature and filler morphology has been proposed. Based on this concept mechanical and aesthetic characteristics of the materials can be presumed.     

The glycerophosphoinositols and their cellular functions

Interest in the glycerophosphoinositols has been increasing recently, on the basis of their biological activities. The cellular metabolism of these water-soluble bioactive phosphoinositide metabolites has been clarified, with the identification of the specific enzyme involved in their synthesis, PLA2IVα (phospholipase A2 IVα), and the definition of their phosphodiesterase-based catabolism, and thus inactivation. The functional roles and mechanisms of action of these compounds have been investigated in different cellular contexts. This has led to their definition in the control of various cell functions, such as cell proliferation in the thyroid and actin cytoskeleton organization in fibroblasts and lymphocytes. Roles for the glycerophosphoinositols in immune and inflammatory responses are also being defined. In addition to these physiological functions, the glycerophosphoinositols have potential anti-metastatic activities that should lead to their pharmacological exploitation.     

A comparison of 2 methods for determining the hydrophobicity of Streptococcus groups A and B

A simple method for the evaluation of the hydrophobic properties of streptococci, pathogenic for humans, by their adherence to polystyrene and a modified method for measuring their hydrophobic properties by their sorption on hexadecane have been developed. The results obtained in the evaluation of the hydrophobic properties of streptococci by these two methods have been compared and the complete correlation of these results in classifying the cultures as hydrophobic and hydrophilic has been shown. For the first time the differences in the hydrophobic properties of different strains of group B streptococci have been established.     

Choline acetyltransferase.

Acetylcholine is essential to neural function. It synthesis is catalyzed by choline acetyltransferase, the enzyme responsible for the acetylation of choline by acetyl coenzye A, a reaction favored slightly thermodymodynamically and not at all kinetically. An analytically pure enzyme still has not been obtained; however, method of purification have been greatly improved recently. Numerous inhibitors of the enzyme have been synthesized and their structure-action relationships examained. Evidence has been accumulated showing the essential involvement of an imidazole group in the active site of choline acetyltransferase. The literature regarding the controversial role to thiol groups in choline acetyltransferase is reviewed. Recently, derivatives of coenzyme A have been introduced as inhibitors of this enzyme and the specificity of coenzyme A binding has been examined. Possible mechanisms responsible for the control fo acetylcholine synthesis are discussed.     

Bioconversion of substituted naphthalenes to the corresponding 1,2-dihydro-1,2-dihydroxy derivatives. Determination of the regio- and stereochemistry of the oxidation reactions

A mutant (TTC1) derived from Pseudomonas fluorescens N3 has been obtained for use in the bioconversion of several naphthalene derivatives to the corresponding optically active cis-dihydrodiols on a milligrams-to-grams scale. All main compounds have been characterized, their relative and absolute configuration assigned, and their enantiomeric purity determined. The regio- and stereoselectivity of the transformation has been established. The procedure therefore represents a valid method for the convenient preparation of a pool of valuable chiral syntons and auxiliaries.     

A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus.

Unicellular cyanobacteria of the genus Synechococcus are among the most abundant members of the picoplankton in the open ocean, and their contribution to primary production is considerable. While several isolates have been used for physiological, biochemical, and molecular studies of their unique adaptations to the marine environment, it has become necessary to develop molecular genetic methods for one or more model open-ocean cyanobacteria in order for studies of these organisms and their unique properties to progress. A number of molecular tools for the genetic manipulation of Synechococcus sp. strains WH7803, WH8102, and WH8103 have been developed. These include a plating technique for obtaining isolated colonies at high efficiencies and a conjugation method for introducing both a replicative vector and a suicide vector. In addition, a method for the generation of random, tagged chromosomal insertions (N. Dolganov and A. R. Grossman, J. Bacteriol. 175:7644-7651, 1993; N. F. Tsinoremas, A. K. Kutach, C. A. Strayer, and S. S. Golden, J. Bacteriol. 176:6764-6768, 1994) has been applied to these organisms.     

Gel chromatography on a Sepharose 4B column: earlier elution of protein-sodium dodecyl sulfate complexes of low Stokes radii

A procedure is described for the determination of the Stokes radius of a detergent micelle by gel chromatography. It was observed that different lots of Sepharose 4B can exhibit a wide variation in the permeation of their gel pores. It is shown that this variation is due to differences in their pore size distribution. It has been observed that protein-sodium dodecyl sulfate (SDS) complexes of high Stokes radii eluted on a Sepharose 4B column with Stokes radii lower than the theoretical, as it has been previously reported but that protein-SDS complexes of low Stokes radii (less than 70 A), contrary to what might have been expected, eluted with Stokes radii higher than the theoretical. Evidence was obtained that their anomalous elution is due to an interaction of the detergent SDS with the gel pores of small diameter.     

The amino acid sequence of myoglobin from skeletal muscles of red deer(Cervus elaphus)

Red deer myoglobin has been fragmented by restricted tryptic digestion and by treatment with cyanogen bromide. The fragments have been separated by gel permeation. The core peptide derived from cyanogen bromide cleavage have been further digested with trypsin and the resulting peptides have been separated on Dowex 1X2. All fragments have been characterized by their amino acid composition, by determination of their N-terminal sequence using automatic Edman degradation and of their C-terminal sequence following the kinetics of amino acid cleavage by carboxypeptidases A and B. The complete sequence has been found to be identical with the already known sequence of sheep myoglobin except for residue 145 which is Gln in red deer globin and Glu in sheep globin. Reinvestigation of the corresponding sequence in sheep globin has shown that residue 145 of sheep globin is also Gln.