Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase,as observed in a new crystal form: Implications for the reaction mechanism

The structure of trypanosomal triosephosphate isomerase (TIM)has been solved at a resolution of 2.1Å in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 Å, 48.3 Å, 131.0 Å, 90.0°, 100.3°, 90.0°), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the “Open” conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 Å resolution), and with 40 μM phosphoglycolohydroxamate (studied at 2.5 Å resolution). It is found that transfer to 0.4 M ammonuum sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a “closed” conformation. In a mother liquor containing 40 μM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding. © 1993 Wiley-Liss, Inc.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Anergic TH1 clones specific for hepatitis B virus (HBV) core peptides are inhibitory to other HBV core-specific CD4+ T cells in vitro.

A strong and transient hepatitis B virus core (HBc)-specific CD4+ T-cell response has been shown to be associated with viral elimination in acute self-limited hepatitis B but to be absent in chronic hepatitis B. So far, little is known about immunological mechanisms involved in the regulation of the HBc-specific CD4+ T-cell response. We studied 28 patients with acute hepatitis B, and frequently a sudden decrease in the HBc-specific CD4+ T-cell response was found between 4 and 8 weeks after disease onset. Thirty-two CD4+ T-cell clones specific for amino acids 50 to 69, 81 to 105, 117 to 131, or 141 to 165 of HBc were isolated from a patient shortly before the peripheral blood mononuclear cell response to most HBc-derived peptides abruptly disappeared. TH1 clones, but not TH0 clones, could be anergized in vitro by stimulation with specific peptides even in the presence of costimulatory cells. Moreover, when anergic cells were mixed with responsive cells, the proliferation of HBc-specific TH1 or TH0 clones was inhibited antigen specifically by anergic cells. The unusual susceptibility of HBc-specific TH1 clones to anergy induction in vitro as well as their potential to inhibit other HBc-specific TH1 and TH0 clones suggests that anergy induction may be involved in the downregulation of the virus-specific immune response during acute hepatitis B in vivo.     

Increased sensitivity of antithymocyte globulin-treated newborn rat assay for metastasis

Tumorigenic and metastatic abilities of FL, transformed human amnion, Vero and LLC-MK2, continuous monkey kidney cell lines (CCLs), none of which produced lung metastases in nude mice, were studied in ATG-treated newborn Wistar rats. Effects of subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10(6) and 10(7) cells were compared. l.p. inoculation of the same number of FL cells produced larger primary tumors, increased incidence of lung metastasis and more metastases per rat than s.c. inoculation. The same was true of the higher inocula administered by either route as compared to the smaller inocula. The CCLs (Vero at passages 320 and 323, LLC-MK2 at passage 306) inoculated either s.c. or i.p. induced small tumors regressing in all animals. After s.c. inoculation of 10(6) Vero or LLC-MK2 cells lung metastases did not develop in any rats, but 10(7) inoculum and i.p. administration of both size inocula caused single lung metastases. Thus, increased sensitivity of the rat system was achieved by larger size of the s.c. inoculum or the i.p. route of inoculation. Highly sensitive assay is especially important for evaluation of possible metastatic abilities of CCLs.     

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Characterization of a cycling murine pluripotent stem cell population

In the present report we describe the properties of pluripotent stem cells isolated with elutriation and Percoll density gradient centrifugation techniques from spleens of recovering thiamphenicol pretreated anemic mice. Cells with a diameter between 7.2 and 8.4 microns and sedimenting at a density of 1.065 g/ml had a high capacity to repopulate lethally irradiated mice with all the hemopoietic precursor cells. The spleen colonies formed on day 8 and day 12 after inoculation in irradiated recipients showed no morphological differences in mixed and lineage-specific composition. Day 12 colonies were much larger than day 8 colonies. Pluripotent stem cells isolated from regenerating spleens were very susceptible to 3'-thymidine; 65% of the cells were killed. Only 2% kill was found in stem cells present in vivo 4 days earlier in the bone marrow. The purified stem cells were probably all in cycle and possibly partly synchronized.