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PubMed2002

Molecular cloning and characterization of mouse calcitonin gene-related peptide receptor.

The calcitonin gene-related peptide (CGRP) plays important roles as a neurotransmitter/neuromodulator in the central nervous system, and as a potent vasodilator when secreted from peripheral, perivascular nerves through its specific receptors. In this study, we cloned mouse cDNA counterparts of the human CGRP receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) and examined the signal transduction mechanism through the CGRP receptor. Mouse CRLR (mCRLR) is a 462-amino acid G protein-coupled heptahelical receptor, and mouse RAMP1 (mRAMP1) is a 148-amino acid single membrane-spanning protein with a short cytoplasmic portion. Specific binding of (125)I-CGRP was detected only when both mCRLR and mRAMP1 cDNAs were cotransfected to COS-7 cells, and the Kd value of the receptor was 2.2 x 10(-10) M. CGRP induced a marked elevation of the intracellular cAMP levels in COS-7 cells cotransfected with mCRLR and mRAMP1. CGRP signaling through the mCRLR/mRAMP1 receptor complex was found to increase the promoter activities of cyclic AMP responsive element and serum responsive element in the co-transfected HeLa cells. These results indicate that mCRLR and mRAMP1 constitute a functional mouse CGRP receptor for the transduction of CGRP signaling by PKA and extracellular signal-regulated kinase signal transduction pathways.

Amino Acid Sequence↗
PubMed2001

Multiprotein complex signaling and the plasticity problem.

Synaptic transmission of distinct patterns of spikes, or 'neural code', leads to plastic changes in synapses and other parts of the neuron, as well as learning in animals. Recent findings indicate that specialized multiprotein structures associated with neurotransmitter receptors and cell-adhesion proteins function as molecular devices that both read the neural code and initiate long-term changes in synaptic structure and function.

Adaptor Proteins, Signal Transducing↗
PubMed1977

A model for macromolecular selection in complementary instructing systems.

In this paper we consider a model for the selection and evolution of biological macromolecules when their reproduction is based on complementary instruction. The model is an extension of one of Eigen's models for selection and takes in account explicitly the formation of both single stranded and double stranded molecular complexes. We construct exact solutions to the rate equations for the case of constant rate parameters and error distributions. Criteria for selection are discussed.

Biological Evolution↗
PubMed1997

Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.

Binding Sites↗
PubMed1989

Evidence that the interferon-induced Daudi cell human lupus inclusions are de novo synthesized complexes of ribonucleoprotein and membrane.

Quantitative electron microscope autoradiography has been used to define the macromolecular composition of the interferon-induced human lupus-type inclusions (LI) in the human B lymphoblastoid cell line, Daudi. LI were first apparent in Daudi cell cultures 12 h after the addition of 100 units/ml of the purified recombinant human leukocyte interferon, IFLrA. Radiolabels were added at this time and allowed to incorporate over the following 12 h during which an estimated greater than 99% of the LI material present at 24 h was formed. The LI-incorporated radiolabels were present only during this discrete 12-h period after the interferon activation of LI cell pathways in order to detect LIs de novo synthesized macromolecular components. The estimate relative specific activities of the LI-incorporated radiolabels were: choline at 4.042, mannose at 2.631, uridine at 0.664, glucosamine at 0.578, and amino acids at 0.477. With thymidine the estimated LI specific activity was 0.000. LI isolated from whole cells retained the tubular elements and the interwoven membrane network. These results provide direct evidence that the interferon-induced Daudi cell LI are de novo synthesized complexes of ribonucleoprotein and membrane.

Amino Acids↗
PubMedDate not supplied

Differential gene expression profile between PC-14 cells treated with free cisplatin and cisplatin-incorporated polymeric micelles.

Cisplatin (CDDP)-incorporated polymeric micelles (CDDP/m) are a macromolecular carrier system possessing a time-modulated decaying property accompanied by sustained release of free drug. The gene expression profiles in nonsmall cell lung cancer PC-14 cells treated with free CDDP and CDDP/m were evaluated by a cDNA expression array for 807 genes. Although the total gene expression profile of the cells treated with CDDP/m approximated that of free CDDP in the hierarchical clustering analysis, a number of genes showed differential expression according to whether the cells had been treated with CDDP or CDDP/m. Ultimately, 50 genes with significant differential expression between cells treated with CDDP and CDDP/m were selected by principal component (PC) analysis and the unpaired t-test. The genes selected, including genes related to cell cycle regulation, apoptosis-related proteins, detoxification, and DNA repair enzymes, were considered to be related to CDDP-induced cytotoxicity. Interestingly, CDDP/m down-regulated the genes encoding integrins and matrix metalloproteinases (MMPs), which play an integral role in tumor invasion, metastasis, and angiogenesis, whereas free CDDP up-regulated them. The results suggest that use of the macromolecular carriers may yield additional therapeutic effects over free drug.

Animals↗
PubMedDate not supplied

In vitro reconstituted skin as a tool for biology, pharmacology and therapy: a review.

Various culture procedures, suitable to maintain the differentiated phenotype of various types of cells in vitro, have been devised during the past decade. These culture systems use macromolecular components extracted from extracellular matrixes or synthetic polymers which provide cells with a three-dimensional, spatially structured support. Substantial information has come from the use of collagen lattices. Many types of cells, of mesenchymal or other origin, are able to organize collagen fibrils in these models and to form a connective tissue-like structure. Prerequisites for this process are active function of the cytoskeleton and the expression of alpha(2)beta(1) integrin collagen receptors. Interaction of cells with such a matrix has profound effects on morphologic status, proliferation, cellular metabolism, and state of differentiation. The in vitro procedures reviewed and described in this article offer the possibility to combine different types of cells to approximate the in vivo environment and to investigate such physiologic processes as cell-cell and cell-matrix interactions which are otherwise not easily accessible. In pathologic conditions of the skin, these models have proven to be useful tools in investigating diseases relating to impaired recognition of extracellular matrix structures or alterations of cytoskeletal assembly. In pharmacologic and toxicologic studies, activity of drugs and potentially useful therapeutic substances has been evaluated. The use of cells and matrix components derived from skin has lead to refined systems which could be adapted and extended to other organs in an attempt to better understand pathophysiologic mechanisms.

Journal Article↗
PubMed2003

Dendrimer-based macromolecular MRI contrast agents: characteristics and application.

Numerous macromolecular MRI contrast agents prepared employing relatively simple chemistry may be readily available that can provide sufficient enhancement for multiple applications. These agents operate using a approximately 100-fold lower concentration of gadolinium ions in comparison to the necessary concentration of iodine employed in CT imaging. Herein, we describe some of the general potential directions of macromolecular MRI contrast agents using our recently reported families of dendrimer-based agents as examples. Changes in molecular size altered the route of excretion. Smaller-sized contrast agents less than 60 kDa molecular weight were excreted through the kidney resulting in these agents being potentially suitable as functional renal contrast agents. Hydrophilic and larger-sized contrast agents were found better suited for use as blood pool contrast agents. Hydrophobic variants formed with polypropylenimine diaminobutane dendrimer cores created liver contrast agents. Larger hydrophilic agents are useful for lymphatic imaging. Finally, contrast agents conjugated with either monoclonal antibodies or with avidin are able to function as tumor-specific contrast agents, which also might be employed as therapeutic drugs for either gadolinium neutron capture therapy or in conjunction with radioimmunotherapy.

Animals↗
PubMed2005

Destination-selective long-distance movement of phloem proteins.

The phloem macromolecular transport system plays a pivotal role in plant growth and development. However, little information is available regarding whether the long-distance trafficking of macromolecules is a controlled process or passive movement. Here, we demonstrate the destination-selective long-distance trafficking of phloem proteins. Direct introduction, into rice (Oryza sativa), of phloem proteins from pumpkin (Cucurbita maxima) was used to screen for the capacity of specific proteins to move long distance in rice sieve tubes. In our system, shoot-ward translocation appeared to be passively carried by bulk flow. By contrast, root-ward movement of the phloem RNA binding proteins 16-kD C. maxima phloem protein 1 (CmPP16-1) and CmPP16-2 was selectively controlled. When CmPP16 proteins were purified, the root-ward movement of CmPP16-1 became inefficient, suggesting the presence of pumpkin phloem factors that are responsible for determining protein destination. Gel-filtration chromatography and immunoprecipitation showed that CmPP16-1 formed a complex with other phloem sap proteins. These interacting proteins positively regulated the root-ward movement of CmPP16-1. The same proteins interacted with CmPP16-2 as well and did not positively regulate its root-ward movement. Our data demonstrate that, in addition to passive bulk flow transport, a destination-selective process is involved in long-distance movement control, and the selective movement is regulated by protein-protein interaction in the phloem sap.

Cucurbita↗
PubMed2002

Ultrastructural studies define soluble macromolecular, particulate, and cellular transendothelial cell pathways in venules, lymphatic vessels, and tumor-associated microvessels in man and animals.

We present de novo studies and review published efforts from our laboratory, spanning 12 years (from 1988 to 2000), where we have used ultrastructural approaches to study the functional anatomy of the microvasculature in man and animals in health and disease. These efforts have defined a new endothelial cell organelle, termed the vesiculo-vacuolar organelle (VVO), which participates in the regulated transendothelial cell passage of soluble macromolecules. The studies defining this organelle utilized ultrathin serial sections, three-dimensional computer-assisted reconstructions, and ultrastructural electron-dense tracers to establish luminal to abluminal transendothelial cell continuity of VVOs. Commonality of VVOs and caveolae is suggested by the ultrastructural anatomy of individual units of VVOs and caveolae, the presence of caveolin in both structures, and a mathematical analysis of morphometric data, all of which suggest that VVOs form from fusions of individual size units equivalent to vesicles of caveolar size. Ultrastructural studies have localized potent permeability factors and their specific receptors to VVOs in in vivo tumor and allergic inflammation models. Regulation of permeability through VVOs has been quantified and shown to be increased in tumor microvessels and in control vessels exposed to potent permeability-inducing mediators. The transendothelial cell passage of particulate macromolecules occurs by vacuolar transport in tumor vessels; in permeability factor-exposed control vessels, colloidal carbon traversed endothelial cells via the development of pores that did not communicate with or disrupt intercellular junctions by gap formation. Serial section and computer-assisted reconstructions established these findings and suggested the possible development of transendothelial cell pores from VVOs. Serial sectioning and computer-assisted three-dimensional reconstructions of ultrastructural samples of an acute inflammation model revealed a transendothelial cell traffic route for motile neutrophils and platelets in the absence of classical ultrastructural criteria for regulated secretion from either cell.

Animals↗
PubMed2000

AutoDep: a web-based system for deposition and validation of macromolecular structural -information.

This paper describes the design and full implementation of a new concept in data deposition and validation: AutoDep (copyright Brookhaven Science Associates LLC). AutoDep changes the traditional procedure for data acceptance and validation of the primary databases into an interactive depositor-driven operation which almost eliminates the delay between the acceptance of the data and its public release. The system takes full advantage of the knowledge and expertise of the experimenters, rather than relying on the database curators for the complete and accurate description of the structural experiment and its results. AutoDep, developed by the Protein Data Bank at Brookhaven National Laboratory (BNL) as a flexible and portable system, has already been adopted by other primary databases and implemented on different platforms/operating systems. AutoDep was introduced at BNL in 1996 [see Manning (1996), Protein Data Bank Quart. Newslett. 77, 2 (ftp://ftp.rcsb. org/pub/pdb/doc/newsletters/bnl/newsletter96jul/newslttr+ ++.txt); Manning (1996), Protein Data Bank Quart. Newslett. 78, 2 (ftp://ftp. rcsb.org/pub/pdb/doc/newsletters/bnl/newsletter96oct/+ ++newslttr.txt)].

Computer Security↗
PubMed1996

Polymeric drugs derived from ibuprofen with improved antiinflammatory profile.

A new methacrylic monomeric compound bearing Ibuprofen (MIA) and the respective polymethacrylic macromolecular derivative (polyMIA) were evaluated for antiinflammatory properties in order to study their pharmacological activity profile. The macromolecular drug is constituted of Ibuprofen residues linked covalently to a polymethacrylic support, as a side substituent of the repeating unit, by means of a spacer group (p-aminophenoxy) that, in addition, is the most representative metabolite of Paracetamol. The Ibuprofen is slowly released from the polymeric support by hydrolysis of the ester bond in a biological medium. Different antiinflammatory testing methods were used; and the results obtained indicate that the monomeric and polymeric forms display higher potency to inhibit acute inflammatory processes than the traditional Ibuprofen form, even doubling the power of Ibuprofen. In addition, they present higher activity against chronic experimental processes, as well as an excellent antinociceptive behavior to inhibit the pain associated with arthritic diseases. According to previous reports and results presented in this article, we can conclude that the new polymethacrylic structures show higher analgesic, antipiretic, and antiinflammatory activities due to the possible activity of the macromolecular drug as well as to the controlled Ibuprofen release from the macromolecular delivery system. After releasing of the side active residue, a derivate of poly(sodium methacrylate) is formed, which is cleared from the body easily by the normal pathway of kidney metabolism.

Analgesics↗
PubMed1999

T cell lines specific for polyomavirus T-antigen recognize T-antigen complexed with nucleosomes: a molecular basis for anti-DNA antibody production.

We have previously demonstrated that in vivo expression of the polyomavirus DNA-binding T-antigen initiated production of IgG antibodies to T-antigen and to DNA, but not to a panel of autoantigens not related to nucleosomes, indicating an antigen-selective T cell-dependent B cell response. In this study, we demonstrate that CD4-positive T cells from both normal and systemic lupus erythematosus (SLE) patients readily proliferate in response to pure T-antigen, and also to T-antigen in complex with nucleosomes. T-antigen-specific T cell lines from both normal individuals and SLE patients proliferate in response to nucleosome-T-antigen complexes, but not to nucleosomes or histones. B cells co-cultured with T-antigen-specific T cells and stimulated with nucleosome-T-antigen complexes produce anti-T-antigen and anti-DNA antibodies, indicating that such CD4-positive T cells have the potential to interact with B cells specific for individual components of nucleosome-T-antigen complexes. Thus, a non-self DNA-binding protein like polyomavirus T-antigen may initiate and maintain an antibody response to DNA when T-antigen is actively expressed.

Adult↗
PubMed2001

Signaling pathway of ciliary neurotrophic factor in neuroblastoma cell lines.

BACKGROUND: Ciliary neurotrophic factor (CNTF) is a member of the interleukin-6 (IL-6) cytokine family and affects the survival and differentiation of several classes of neurons. For signal transduction, CNTF requires a receptor complex, composed of the IL-6 signal transducing molecule gp130, leukemia inhibitory factor receptor (LIFR)-beta, and CNTFR-alpha. There are two major independent pathways (Jak-STAT and Ras-MAPK) in cell signaling, and some recent reports show interaction between these pathways. The signal of the IL-6 family is mainly transduced to the Jak-STAT pathway through gp130. However, it has not been examined in neuroblastoma in detail. PROCEDURE AND RESULTS: Here we examine the signaling pathway of CNTF in 11 neuroblastoma cell lines. Northern blot analysis revealed that 3 of the 11 cell lines expressed c-fos mRNA after CNTF stimulation. Cell lysates were immunoprecipitated with agarose-conjugated antiphosphotyrosine antibody and blotted with anti-gp130, anti-Jak1, or anti-STAT3 antibody. Tyrosine phosphorylation of gp130, Jak1, and STAT3 was observed after CNTF stimulation in these three cell lines. Furthermore, tyrosine phosphorylation of ERK1 (one of the MAPKs) was also observed in all of them. CONCLUSIONS: These results demonstrate that CNTF signaling is conserved in some of the neuroblastoma cell lines and suggest that not only a Jak-STAT pathway but a MAPK pathway is activated by CNTF through gp130 in neuroblastoma cell lines.

Antigens, CD↗
PubMed1991

Prediction of the rotational diffusion behavior of biopolymers on the basis of their solution or crystal structure.

Two low structure-resolution methods are proposed for prediction of rotational diffusion parameters. The indirect procedure is based on the structure of a molecule in solution or in crystal, and uses the structure parameters of radius of gyration, and low-resolution molecular surface and volume, determined from measured or theoretically calculated small-angle x-ray scattering intensities, to estimate a frictional equivalent ellipsoid of revolution. The direct method starts mainly from the crystallographic structure of a molecule and calculates the triaxial inertia equivalent ellipsoid, experimentally calibrated by translation diffusion data, to simulate the frictional behavior. The predicted harmonic mean of the rotational correlation times of compact globular macromolecules with molar masses of 14,000-65,000 g/mol agree with experimental results within the error limits. The prediction method is recommended for expert systems in structure research and for detection of internal protein flexibility or marker mobility by nmr and electron paramagnetic resonance experiments.

Crystallography↗
PubMed1992

Tubulin dimer formation via the release of alpha- and beta-tubulin monomers from multimolecular complexes.

The functional subunit of microtubules is a heterodimer consisting of alpha- and beta-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse alpha-tubulin isotypes involves a multimolecular complex. The synthesis of mouse beta-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized alpha- and beta-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers.

Cell-Free System↗