Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid

Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5(S)-hydroperoxy, 15(S)-hydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid or 5(S),15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-1. Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A4 as is proposed for human and porcine PMNs.     

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight⁻¹ at D = 0.1 h⁻¹ to 2 U mg of cell dry weight⁻¹ at D = 0.8 h⁻¹. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.     

Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.     

Stereometry in very close-range stereophotogrammetry with non-metric cameras for human movement analysis

In this paper a stereophotogrammetric algorithm based on a black-box approach to the modelling of object to image spaces relationship is proposed. The algorithm is well suited for 'very close-range photogrammetry', with respect to experiments in which the measurement field is 0.5 X 0.5 X 0.5 m or smaller, as in the analysis of a few or small body segments movements. The attainable accuracy is high, better than 0.1% of the observation distance. Non-professional and even different cameras can be used. Consequently an inexpensive experimental set-up can be realized. A very simple, cheap and easily usable calibration object is needed. Computation time for the reconstruction of object-space co-ordinates of point body landmarks is one order of magnitude lower than in the case of the Direct Linear Transformation (DLT) (Abdel Aziz and Karara, Proceedings of the ASP/U1 Symposium on Close-Range Photogrammetry, pp. 1-18. American Society of Photogrammetry, 1971; Marzan and Karara, Proceedings of the Symposium on close-range Photogrammetric Systems, pp. 420-467. American Society of Photogrammetry, 1975). Computation time for calibration is two-fold in respect of the DLT. An example of application to the recording of the movements of the index finger with respect to the metacarpophalangeal joint is given.     

Rationalization of some genetic anticodonic assignments

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.     

Differentiation of keratinocytesin vitro: a new culture vessel mimicking thein vivo situation

A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO₂ in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO₂, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiationin vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.     

Preparation of h and m antigens ofHistoplasma capsulatum free of heterologous antigens

Salt gradient elution of crude histoplasmin on CM-Sepharose CL6B at pH 3.0 was used in essentially a one-step procedure to isolate the h, m, and non-m antigens ofHistoplasma capsulatum and free them of any c antigen common to other pathogenic fungi. The h antigen fraction was pure; the isolated m and non-m antigens contained less than 0.1% of the c antigen present in the original preparation. The residual c antigen in these fractions was removed by affinity chromatography on Affigel-10 coupled to aBlastomyces dermatitidis fungal antiserum. The final preparations of h, m, and non-m were pure when tested by immunodiffusion (ID), sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), or electrofocusing. The antigens were serologically stable at neutral pH for at least three months at 10°C. The relative molecular weights, isoelectric points, and amino acid composition of the antigens are described.