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PubMed2001

X-ray studies on crystalline complexes involving amino acids and peptides. XXXVII. Novel aggregation patterns and effect of chirality in the complexes of DL- and L-lysine with glutaric acid.

The complexes of glutaric acid with DL-lysine contain singly positively charged zwitterionic lysinium ions and singly negatively charged semi-glutarate ions. Both the ions exhibit different conformations in the two complexes. The structures contain head-to-tail sequences of amino acids. However, the aggregation patterns in the two complexes are entirely different, demonstrating the effect of chirality on molecular aggregation. These patterns also turn out to be different from those so far observed, in structures containing amino acids. The structures contain characteristic interaction patterns involving linear arrays of alternating amino and carboxylate groups.

Crystallography, X-Ray↗
PubMed2005

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin.

Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 A, alpha = beta = 90, gamma = 120 degrees. This indicated a Matthews coefficient (VM) of 3.3 A3 Da(-1), corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 A resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

Animals↗
PubMed2005

Accessory molecules in the assembly of major histocompatibility complex class I/peptide complexes: how essential are they for CD8(+) T-cell immune responses?

Assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum is a highly coordinated process that results in abundant class I/peptide complexes at the cell surface for recognition by CD8(+) T cells and natural killer cells. During the assembly process, a number of chaperones and accessory molecules, such as transporter associated with antigen processing, tapasin, ER60, and calreticulin, assist newly synthesized class I molecules to facilitate loading of antigenic peptides and to optimize the repertoire of surface class I/peptide complexes. This review focuses on the relative importance of these accessory molecules for CD8(+) T-cell responses in vivo and discusses reasons that may help explain why some CD8(+) T-cell responses develop normally in mice deficient in components of class I assembly, despite impaired antigen presentation.

ATP-Binding Cassette Transporters↗
PubMed1977

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin.

A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.

Amino Acid Sequence↗
PubMed1979

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Amino Acyl-tRNA Synthetases↗
PubMed1980

Analysis of binding interactions between histone core complex and simian virus 40 DNA. A comparison of acetylated versus non-acetylated histone core complexes.

The interactions of acetylated and non-acetylated core complex histones with simian virus 40 (SV40) DNA 1 have been analyzed. A modified filter-binding assay utilizing micrococcal nuclease, which allows quantification of histone octamer binding to DNA has been developed. Using this assay it was determined that both non-acetylated core complex histones ad core complex histones acetylated with acetyl adenylate to levels existing in vivo bind cooperatively to SV40 DNA 1. Although both interactions are cooperative, the magnitude of the cooperativity parameter, omega, is significantly less in the acetylated case. This difference in cooperativity is in contrast to the nearly identical intrinsic association constant, K, observed in both cases.

Acetylation↗
PubMed1996

Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.

T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.

Amino Acid Sequence↗
PubMed1997

Information transfer in multienzyme complexes--1. Thermodynamics of conformational constraints and memory effects in the bienzyme glyceraldehyde-3-phosphate-dehydrogenase-phosphoribulokinase complex of Chlamydomonas reinhardtii chloroplasts.

Oxidized phosphoribulokinase is almost inactive in its isolated state but becomes active when associated with glyceraldehyde-3-phosphate dehydrogenase. There is therefore an information transfer that takes place between these two enzymes. However, when the complex dissociates, free oxidized phosphoribulokinase is even more active than when it is associated with glyceraldehyde-3-phosphate dehydrogenase. This means that glyceraldehyde-3-phosphate dehydrogenase exerts an imprinting effect upon phosphoribulokinase which persists for a while after the parting of the two proteins. Various methods derived from statistical thermodynamics can be used to estimate the fraction of energy transferred from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase and which alters the kinetic parameters of the latter enzyme. In the complex, the decrease of the free energy associated with the binding of ribulose 5-phosphate is larger than that of ATP. This implies that the mutual association of the two enzymes facilitates the binding of the former substrate but is without effect on that of the latter. The main effect exerted by the association of the two enzymes is to decrease by about 10 kJ/mol the height of the energy barrier of the catalytic process. Phosphoribulokinase keeps an imprinting effect exerted by glyceraldehyde-3-phosphate dehydrogenase after the parting of the two enzymes. Part of the energy transferred from one protein to the other is used to decrease slightly the apparent binding free energy of the two substrates of phosphoribulokinase by about 1.5 kJ/mol. Whereas the previous association of the two enzymes does not significantly alter substrate binding to phosphoribulokinase, it greatly affects catalysis and decreases by about 16 kJ/mol the height of the energy barrier pertaining to this step. Therefore, within multienzyme complexes, information and energy can be transferred between proteins. Statistical thermodynamics offers the possibility of estimating how this energy is used to alter the various kinetic parameters of the reaction.

Animals↗
PubMed1998

Direct detection of Mycobacterium tuberculosis complex and M. avium complex in tissue specimens from cattle through identification of specific rRNA sequences.

Mycobacterium Tuberculosis Direct test and Accuprobe M. avium Complex test, rely on recognition of specific rRNA sequences. The present investigation included sequentially a pre-evaluation study and a study on non-visible lesion tuberculin reactor cattle. As regards the first study, tests were performed on tissue samples from 14 reactors with tuberculous-like lesions and from as many lesion-free non-reactors, using culture findings as a reference. MTD and Accuprobe M. Avium Complex tests proved positive in 12 and 2 reactors respectively. Both tests proved negative in all non-reactors. Data consisted with culture findings. The aim of the second study was to verify the presence of mycobacteria in lymph node and tonsil homogenates from 32 non-visible lesion cattle. MTD and Accuprobe M. avium Complex test proved positive in 10 and 14 subjects respectively; no overlap was found. Our research demonstrates that low to minimal amounts of the main pathogenic mycobacteria can be reliably identified from cattle tissues by commercially available tests, which give simple, more rapid results than conventional techniques.

Animals↗
PubMed1986

Statistical data base for the biomechanical properties of the human shoulder complex--I: Kinematics of the shoulder complex.

In the last two decades, several multi-segmented mathematical models of the total-human-body have appeared in the literature. While these models can handle very sophisticated load-motion situations, their effectiveness depends heavily on the proper biomechanical description and simulation of the major articulating joints of the human body. Among these joints, the most complicated and the least successfully modeled one has been the shoulder complex mainly due to the lack of an appropriate biomechanical data base as well as the anatomical complexity of the shoulder region. In 1984, the senior author and his associates proposed a new kinematic data collection methodology by means of sonic emitters and associated data analysis technique. Based on this data collection methodology, Part I of this paper establishes a statistical data base for the shoulder complex sinus of the male population of ages 18-32. Estimates for the population mean and standard deviation as well as their confidence intervals are presented. The results are expressed in functional expansion form relative to a locally defined joint axis system as well as relative to the torso-fixed coordinate system in the form of globographic representation.

Adolescent↗
PubMed1994

Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes.

Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.

CD8 Antigens↗
PubMed1991

A complex regulatory DNA element associated with a major histocompatibility complex class I gene consists of both a silencer and an enhancer.

A novel regulatory element which contributes to the regulation of quantitative, tissue-specific differences in gene expression has been found between -771 and -676 bp upstream of the major histocompatibility complex (MHC) class I gene, PD1. Molecular dissection of this element reveals the presence of two overlapping functional activities: an enhancer and a silencer. Distinct nuclear factors bind to the overlapping enhancer and silencer DNA sequence elements within the regulatory domain. The levels of factors binding the silencer DNA sequence in different cell types are inversely related to levels of class I expression; in contrast, factors binding the enhancer DNA sequence can be detected in all cells. In cultured cell lines, inhibition of protein synthesis leads to the rapid loss of silencer complexes, with a concomitant increase in both enhancer complexes and MHC class I RNA. From these data, we conclude that a labile silencer factor competes with a constitutively expressed, stable enhancer factor for overlapping DNA-binding sites; the relative abundance of the silencer factor contributes to establishing steady-state levels of MHC class I gene expression.

Animals↗
PubMed1996

Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes.

Animals↗
PubMed1997

Insensitivity of V1 complex cell responses to small shifts in the retinal image of complex patterns.

An important role for neurons in the early visual system is to convey information about the structure of visual stimuli. However, neuronal responses show substantial variation across presentations of the same stimulus. In awake monkeys, it has been assumed that a great deal of this variation is related to the scatter in eye position (inducing scatter in the retinal position of the stimulus). Here we investigate the implied consequence of this assumption, i.e., that the scatter variation in eye position degrades the decodability of the neural response. We recorded from 50 complex cells in primary visual cortex of fixating monkeys while different complex stimuli were presented. Three types of retinal shifts were considered: natural scatter in the fixation, systematic fixation point shift, and systematic stimulus position shift. The stimulus pattern accounts for >50% of the response variance, always six times that accounted for by the scatter in eye position during fixation. The retinal location of a stimulus had to be shifted by 10-12 min of arc, an amount almost two times larger than the smallest picture element, before the responses changed systematically. Nonetheless, changes of the stimulus at the single pixel level often gave rise to discriminable responses. Thus complex cells convey information about the spatial structure of a stimulus, independent of rigid stimulus displacements on the order of the receptive field size or smaller.

Animals↗
PubMed2001

Prothrombin protects factor Xa in the prothrombinase complex from inhibition by the heparin-antithrombin complex.

Heparin is a commonly used anticoagulant drug. It functions primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the most important targets. There are conflicting results as to whether anticoagulant heparins can catalyze the antithrombin inhibition of factor Xa in the prothrombinase complex (factor Va, negatively charged membrane surfaces, and calcium ion), which is the physiologically relevant form of the proteinase responsible for the activation of prothrombin to thrombin during the blood coagulation process. In this study, a novel assay system was developed to compare the catalytic effect of different molecular-weight heparins in the antithrombin inhibition of factor Xa, either in free form or assembled into the prothrombinase complex during the process of prothrombin activation. This assay takes advantage of the unique property of a recombinant mutant antithrombin, which, similar to the wild-type antithrombin, rapidly inhibits factor Xa, but not thrombin, in the presence of heparin. A direct prothrombinase inhibition assay, monitoring thrombin generation under near physiological concentrations of prothrombin and antithrombin in the presence of therapeutic doses of low- and high-molecular-weight heparins, indicates that factor Xa in the prothrombinase complex is protected from inhibition by antithrombin more than 1000 times, independent of the molecular size of heparin.

Amino Acid Sequence↗
PubMed2002

Potential insulinominetic agents of zinc(II) complexes with picolinamide derivatives: preparations of complexes, in vitro and in vivo studies.

Following the finding of in vitro insulinominetic activities of new prepared Zn(II) complexes with amide ligands (2-picolinamide (pa-a) and 6-methyl-2-picolinmethylamide (6mpa-ma)) in isolated rat adipocytes treated with epinephrine in terms of inhibition of free fatty acid release, their blood glucose normalizing effects were observed on daily intraperitoneal injections for 14 d in a type 2 diabetes mellitus model animal, KK-Ay mice. The blood glucose levels of KK-Ay mice were maintained in a normal range during the administration of both complexes. After the administration of each complex for 14 d, the improvement of glucose metabolism was confirmed as judged by the glucose tolerance test.

Adipocytes↗
PubMed1977

In vitro binding of tritiated hormone-cytostatic complexes in the cytosol of various rat tissues and the incorporation of these complexes into nuclei.

Cytosols from rat ventral prostate, liver and muscle were incubated with tritiated estrogen-cytostatic complexes or tritiated estrogen alone. Considerably more radioactivity was recovered in the 4 S region when samples from ventral prostate cytosol were analysed by sucrose density gradient centrifugation compared with samples from liver or muscle cytosols. Similar results were found using Sephadex gel filtration. However, when cytosols were incubated with tritated estrogen alone only unspecific binding was registered. After in vitro tissue incubation with the tritiated hormone-cytostatic complexes, radioactivity was found in purified nuclei of both ventral prostate and liver tissue. The possible nature of the macromolecules which bind hormone-cytostatic complexes is discussed.

Animals↗