bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic action of thrombin is required for electrical activity-dependent synapse reduction.
Authors: Liu Y , Fields R D , Festoff B W , Nelson P G
Published: May 2006

Molecular mechanisms of activity-dependent synapse reduction were studied in an in vitro mammalian neuromuscular preparation. Synapse reduction in this model is activity-dependent and is substantially reduced by the broad-spectrum protease inhibitor, leupeptin, suggesting the role of activity-dependent proteolytic action in the process. Our present experiments show that a potent and specific thrombin inhibitor, hirudin, at nanomolar concentration completely blocked the activity-dependent synapse reduction. Furthermore, a naturally occurring serine protease inhibitor, protease nexin I (PNI), which closely colocalizes with acetylcholine receptors at the neuromuscular junction, inhibited the synapse reduction at the same low concentration. In contrast, neither cystatin, a cysteine protease inhibitor, nor aprotinin, a serine protease inhibitor that does not inhibit thrombin, blocked the synapse reduction. Similarly, neither of the inhibitors of the calcium-activated proteases calpain I and II prevented the reduction of synapses. These results strongly suggest that serine proteolytic action by thrombin or thrombin-like molecules is required for synapse reduction in our in vitro model of the mammalian neuromuscular junction.
Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.
Authors: Beck D L , Van Dolleweerd C J , Lough T J , Balmori E , Voot D M , Andersen M T , O'Brien I E , Forster R L
Published: May 2006

White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.
Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.
Authors: Maiti S , Walker G C , Cowen B R , Pippenger R , Moser C C , Dutton P L , Hochstrasser R M
Published: May 2006

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.
Conformational isomerism and the diversity of antibodies.
Authors: Foote J , Milstein C
Published: May 2006

The fact that one cell encodes a single antibody sequence does not necessarily mean that the resulting antibody folds into a single structure, although this is a common assumption. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site. The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex hapten-binding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that ligand binding involved isomerization, as well as associative steps. The existence of an equilibrium between at least two antibody conformations, with ligands binding preferentially to one form, was deduced from the variation with hapten concentration of the apparent rate of each phase.
Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.
Authors: Otzen D E , Itzhaki L S , elMasry N F , Jackson S E , Fersht A R
Published: May 2006

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure. The structure of the transition state appears to be an expanded form of the native state in which secondary and tertiary elements have been partly formed concurrently. This is consistent with a "global collapse" model of folding rather than a framework model in which folding is initiated from fully preformed local secondary structural elements. This may be a general feature for the folding of proteins lacking a folding intermediate and is perhaps representative of the early stages of folding for multidomain or multimodule proteins. The major transition state for the folding of barnase, for example, has some fully formed secondary and tertiary structural elements in the major transition state, and barnase appears to form by a framework process. However, the fully formed framework may be preceded by a global collapse, and a unified folding scheme is presented.
Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.
Authors: Li A , Daggett V
Published: May 2006

Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics simulations. The major transition state of unfolding was identified on the basis of structural and conformational changes in the protein during the unfolding reaction. The native tertiary contacts in the hydrophobic core were considerably disrupted in the transition state, whereas the secondary structure was partially intact. The extent of structural change of the protein around a particular residue was represented quantitatively by the ratio of the number of contacts the residue makes in the transition state relative to the native state, phi MD, which allows quantitative comparison with the experimentally determined phi F values. For the region of the unfolding trajectory that is identified as the transition state, the phi MD and phi F values are in good agreement, suggesting that the transition state identified in the unfolding simulation corresponds to that probed with protein engineering methods. Although speculative, the transition state identified in the simulation is consistent with available experimental data and provides an in-depth view of what the transition state of unfolding may look like.
A single-base substitution in the proximal Sp1 site of the human low density lipoprotein receptor promoter as a cause of heterozygous familial hypercholesterolemia.
Authors: Koivisto U M , Palvimo J J , Jänne O A , Kontula K
Published: May 2006

We have identified a Finnish family with a typical phenotype of heterozygous familial hypercholesterolemia (FH) due to a single-base substitution in the proximal Sp1 binding site of the low density lipoprotein (LDL) receptor gene promoter. The mutation, a C-->T substitution at nucleotide -43, cosegregated with the FH phenotype in six available family members and abolished binding of Sp1 transcription factor to this site. As a consequence, transcriptional activity of the mutated LDL receptor promoter was only about 1/20th of that of the wild-type promoter, as judged by transfection studies in HeLa cells. Studies of primary fibroblast cultures established from a family member revealed a markedly reduced LDL receptor mRNA concentration as well as reduction of binding, internalization, and degradation of 125I-labeled LDL to values < 50% of those in normal fibroblasts. This DNA alteration is thus a naturally occurring promoter mutation causing a severe disorder of human lipoprotein metabolism.
Rolling circle DNA replication in vitro by a complex of herpes simplex virus type 1-encoded enzymes.
Authors: Skaliter R , Lehman I R
Published: May 2006

Extracts of insect cells infected with baculoviruses recombinant for the herpes simplex virus 1 (HSV-1)-encoded enzymes that are required for its replication can promote the rolling circle replication of circular plasmid templates. Replication is independent of a HSV-1 origin of replication (oris) or the HSV-1 origin binding protein and is inhibited by the origin binding protein when the plasmid contains oris. Replication is dependent on a complex composed of the HSV-1-encoded DNA polymerase and its processivity enhancing factor (the UL42 protein), ICP8 (the HSV-1-encoded single-strand DNA binding protein), and the HSV-1-encoded helicase-primase. The complex can be purified by size-exclusion and anion-exchange chromatography.
Bcl-2 expression during T-cell development: early loss and late return occur at specific stages of commitment to differentiation and survival.
Authors: Gratiot-Deans J , Merino R , Nuñez G , Turka L A
Published: May 2006

During T-cell development CD3-CD4-CD8- (double-negative) thymocytes proliferative and produce an enormous number of CD3loCD4+CD8+ (double-positive) thymocytes which are destined to die intrathymically unless rescued by positive selection. Those which survive become mature CD3hiCD4/8+ (single-positive) cells and are the precursor of peripheral blood lymphocytes. The product of the bcl-2 protooncogene has been implicated in preventing programmed cell death and is required for prolonged lymphocyte survival following maturation. Previously we and others have reported that Bcl-2 protein expression is biphasic, being high in proliferating double-negative stem cells, low in all double-positive thymocytes except for 1-5% of these cells, and restored in mature, single-positive thymocytes. However, it remained unclear which signaling and selection events regulate Bcl-2 during T-cell maturation. Now we have utilized four-color flow cytometry in normal and genetically altered mice for a detailed analysis of Bcl-2 expression as it relates to T-cell receptor (TCR) expression and positive selection. These studies show that (i) expression of a transgenic TCR in double-negative thymocytes does not lead to premature loss of Bcl-2; thus, Bcl-2 downregulation is not solely due to TCR expression; (ii) Bcl-2 expression is lost at the early transitional CD3-/loCD4-CD8+ stage, prior to expression of CD4; (iii) the Bcl-2+ double-positive thymocytes are those which have undergone positive selection; and (iv) upregulation of Bcl-2 during positive selection requires participation of the CD4 or CD8 co-receptor. These results demonstrate that Bcl-2 and TCR expression are regulated independently during T-cell development, and suggest a role for the CD4 or CD8 co-receptor in Bcl-2 induction during positive selection.
Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.
Authors: Labahn J , Granzin J , Schluckebier G , Robinson D P , Jack W E , Schildkraut I , Saenger W
Published: May 2006

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.
Mutations and altered expression of p16INK4 in human cancer.
Authors: Okamoto A , Demetrick D J , Spillare E A , Hagiwara K , Hussain S P , Bennett W P , Forrester K , Gerwin B , Serrano M , Beach D H
Published: May 2006

Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. Regulators of the G1 checkpoint include an inhibitor of cyclin-dependent kinase, p16INK4; two tumor-suppressor proteins, p53 and RB (the product of the retinoblastoma-susceptibility gene); and cyclin D1. Neither p16INK4 nor the RB protein was detected in 28 of 29 tumor cell lines from human lung, esophagus, liver, colon, and pancreas. The presence of p16INK4 protein is inversely correlated with detectable RB or cyclin D1 proteins and is not correlated with p53 mutations. Homozygous deletions of p16INK4 were detected in several cell lines, but intragenic mutations of this gene were unusual in either cell lines or primary tumors. Transfection of the p16INK4 cDNA expression vector into carcinoma cells inhibits their colony-forming efficiency and the p16INK4 expressing cells are selected against with continued passage in vitro. These results are consistent with the hypothesis that p16INK4 is a tumor-suppressor protein and that genetic and epigenetic abnormalities in genes controlling the G1 checkpoint can lead to both escape from senescence and cancer formation.
Abnormal class I assembly and peptide presentation in the nonobese diabetic mouse.
Authors: Li F , Guo J , Fu Y , Yan G , Faustman D
Published: May 2006

Presentation of self-antigens by major histocompatibility complex (MHC) class I molecules requires the function of the MHC class II-linked genes Tap-1 and Tap-2. Evidence suggests that interruption of self-peptide presentation results in reduced cell surface expression of MHC class I molecules and the interruption correlates with progression to diabetic autoimmunity in nonobese diabetic (NOD) mice and humans. NOD mice possess a rare Tap-1 allele (Tap-1b); this is associated with reduced Tap-1 mRNA abundance in lymphocytes from diabetes-prone females and decreased conformationally correct class I molecules on the cell surface. In this study, we demonstrate that, similar to lymphoma cell lines with mutations in Tap-1 or Tap-2, the reduced expression of class I molecules on the surface of lymphocytes from diabetes-prone female NOD mice was normalized by incubation at low temperatures or by exposure to class I allele-specific peptides. As would be expected for cells that express surface class I molecules not associated with peptide, female NOD lymphocytes were resistant to lysis by class I-restricted, peptide-specific cytotoxic T lymphocytes. Furthermore, the rate of class I exit from the endoplasmic reticulum of lymphocytes from female NOD mice was delayed as demonstrated by delayed glycosylation. Male NOD mice, which are not prone to diabetes, lacked these functional defects in class I assembly and had near-normal levels of Tap-1 mRNA and exhibited normal density of class I epitopes that were peptide filled. These results are consistent with the possibility that the rare Tap-1b allele is associated with a quantitative defect in Tap-1 expression that influences disease course.
The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation.
Authors: Eason M G , Jacinto M T , Theiss C T , Liggett S B
Published: May 2006

Most guanine nucleotide binding protein (G protein)-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spanning region. This cysteine is known to be palmitoylated in rhodopsin, the beta 2-adrenergic receptor (beta 2AR) and the alpha 2A-adrenergic receptor (alpha 2AAR). For the beta 2AR, this cysteine has been shown to be important for stimulatory G protein (Gs) coupling and agonist-promoted desensitization. For the alpha 2AAR (human alpha 2 C10) palmitoylation occurs at Cys-442, but it is not known what function such fatty acid acylation subserves. The closely related alpha 2CAR subtype denoted alpha 2C4 lacks a cysteine in this region and has different G-protein-coupling characteristics and agonist regulatory properties as compared to alpha 2C10. To assess the role of the palmitoylcysteine in alpha 2AR function, we constructed a mutated alpha 2C10 having a phenylalanine (the analogous amino acid in the alpha 2C4 in this position) substituted for Cys-442, denoted alpha 2C10(Phe-442), and expressed this along with wild-type alpha 2C10 and alpha 2C4 in CHO cells. Functional coupling to inhibitory G protein (Gi) and to Gs was identical between wild-type alpha 2C10 and alpha 2C10(Phe-442). Agonist-promoted desensitization of both the Gi and Gs-mediated pathways was also found to be unaffected by this mutation. Cellular trafficking induced by agonist exposure was evaluated by delineation of intracellular (sequestered) versus cell surface receptors and by determination of net receptor loss. Mutation of Cys-442 did not alter the extent or rate of agonist-promoted sequestration induced by agonists or the recovery from sequestration. However, the downregulation of receptor number after prolonged agonist exposure was completely abolished by this mutation and converted alpha 2C10 to an alpha 2C4 phenotype in regard to this adaptive response. Another mutated alpha 2C10, in which Cys-442 was replaced by alanine, also failed to downregulate. Thus, the function of this cytoplasmic palmitoylcysteine is distinctly different between the alpha 2AR and other G-protein-coupled receptors such as the beta 2AR and rhodopsin, and this suggests that this molecular attribute may subserve diverse roles among members of this family of receptors. For the alpha 2ARs, this may represent an evolved feature that provides for differing needs for regulation of the alpha 2C10 and alpha 2C4 subtypes by agonist.
Identification of genes involved in DNA replication of the Autographa californica baculovirus.
Authors: Kool M , Ahrens C H , Goldbach R W , Rohrmann G F , Vlak J M
Published: May 2006

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.
High-affinity reactions between antigen-specific T-cell receptors and peptides associated with allogeneic and syngeneic major histocompatibility complex class I proteins.
Authors: Sykulev Y , Brunmark A , Tsomides T J , Kageyama S , Jackson M , Peterson P A , Eisen H N
Published: May 2006

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (Ld) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL, termed QL9) with an intrinsic affinity (intrinsic equilibrium association constant) of 1-2 x 10(7) M-1. The TCR of the other clone (4G3) binds a syngeneic class I MHC protein (Kb) in association with an ovalbumin octapeptide (SIINFEKL, termed pOV8) with an intrinsic affinity of 1.5 x 10(6) M-1. A comparison of the two clones, combined with current views of T-cell repertoire selection in the thymus, leads us to propose that TCR affinities are generally likely to be higher for allogeneic MHC-peptide complexes than for syngeneic MHC-peptide complexes.
Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin.
Authors: Chun M , Liyanage U K , Lisanti M P , Lodish H F
Published: May 2006

Caveolae are small invaginations of the plasma membrane 50-100 nm in diameter. Since calcium channels, inositol 1,4,5-trisphosphate receptors, and heterotrimeric GTP-binding proteins (G proteins) are localized in caveolae, they may participate in signal transduction by G protein-coupled receptors. Here we show that the G protein-coupled endothelin receptor subtype A (ETA) and its bound endothelin ligand are found in plasma membrane caveolae. ETA and its bound ligand coimmunoprecipitate with caveolin, a structural component of caveolae, in extracts of cells expressing transfected ETA receptors. Confocal fluorescence microscopy shows colocalization of ETA receptors and caveolin in micropatches at or near the plasma membrane, in the absence of endothelin ligands. These observations demonstrate a functional role for plasma membrane caveolae in signal transduction by this G protein-coupled receptor.
Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties.
Authors: Schaller A , Ryan C A
Published: May 2006

A protein of 50-kDa (SBP50) was identified in plasma membranes of tomato leaves which resembles proteases of the family of Kex2p-like prohormone convertases. To our knowledge, proteases of this class have not been reported in plants previously. A biotinylated derivative of systemin, the 18-aa polypeptide inducer of proteinase inhibitors in tomato and potato leaves, was bound by SBP50 with high specificity. When a systemin derivative was labeled with biotin at residue 8 and with [35S]methionine at position 15, the biotin moiety but not the radioactive label was bound by SBP50. At least 4 aa from the C terminus that included [35S]methionine were missing, indicating that proteolytic cleavage had occurred. Whereas residues in systemin most important for binding SBP50 appear to be located in the N-terminal half of the molecule, amino acids crucial for proteinase inhibitor induction are located within the C terminus. The residues important for binding include a cleavage site for furin, a member of the family of Kex2p-like prohormone-processing enzymes. Processing of systemin at the predicted furin cleavage site was confirmed in vitro. An antiserum against a Kex2p-like protease from Drosophila inhibited binding of biotinylsystemin to SBP50 and recognized a protein of about 60 kDa in Western blot analyses of tomato plasma membrane proteins. The data suggest a possible role for a membrane bound, furin-like protease in the mechanism of defense gene signaling by systemin.
Four hundred-million-year-old vesicular arbuscular mycorrhizae.
Authors: Remy W , Taylor T N , Hass H , Kerp H
Published: May 2006

The discovery of arbuscules in Aglaophyton major, an Early Devonian land plant, provides unequivocal evidence that mycorrhizae were established >400 million years ago. Nonseptate hyphac and arbuscules occur in a specialized meristematic region of the cortex that continually provided new cells for fungal infection. Arbuscules are morphologically identical to those of living arbuscular mycorrhizae in consisting of a basal trunk and repeatedly branched bush-like tuft within the plant cell. Although interpretations of the evolution of mycorrhizal mutualisms continue to be speculative, the existence of arbuscules in the Early Devonian indicates that nutrient transfer mutualism may have been in existence when plants invaded the land.
Casein kinase II specifically nucleotidylylates in vitro the amino acid sequence of the protein encoded by the alpha 22 gene of herpes simplex virus 1.
Authors: Mitchell C , Blaho J A , Roizman B
Published: May 2006

An earlier report has shown that eight viral proteins with a common amino acid sequence (R/P)RA(P/S)R are nucleotidylyated in vitro by nuclear extracts from cells infected with herpes simplex virus 1. One, the product of the alpha 22 gene, is nucleotidylylated in the absence of viral proteins made late in infection. A chimeric protein (GST22P) consisting of amino acids 50-200 of the alpha 22 coding sequence fused to the C terminus of the glutathione S-transferase was nucleotidylylated by enzymes in nuclear extracts of infected or mock-infected cells and also by a casein kinase II enzyme purified from the sea star. The enzyme did not nucleotidylylate common casein kinase II substrates (casein, phosvitin) and the reaction was inhibited by heparin. The results are consistent with the hypothesis that nucleotidylylation of the eight viral proteins involves casein kinase II.
The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast.
Authors: Lithgow T , Junne T , Suda K , Gratzer S , Schatz G
Published: May 2006

We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors. Defects of Mas20p-deficient cells are explained by the reduced level of Mas22p in these mutants. Mas22p has one acidic domain in the cytosol and a second acidic domain in the mitochondrial intermembrane space. We suggest that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.
Fractal analysis of proton exchange kinetics in lysozyme.
Authors: Dewey T G
Published: May 2006

Experimental data for the exchange of protons in tritiated lysozyme is reexamined by using a fractal model. The fraction of protons unexchanged, f, is seen to follow a stretched exponential, f infinity exp[(-t/tau)alpha], in the long time limit. Data over a range of temperatures are considered, and accurate fits are obtained with a single, unadjusted scaling exponent, alpha. The time constant, tau, follows an Arrhenius law and gives an activation energy comparable to that obtained for free peptide exchange. A model is proposed where proton exchange occurs as a result of solvent reacting with protein side groups in a restricted volume surrounding the protein. Dynamic fluctuations of the protein allow the protonated groups to enter this volume. Solvent also penetrates this volume, allowing proton exchange to occur. The fluctuations of reactants in this restricted volume dominate the kinetics and result in anomalous behavior. The topology of this reaction volume can be characterized by its fractal dimension. The fractal dimension of the space excluded by the protein is equal to 3-ds, where ds is the fractal dimension of the protein surface. The dimensionality of this "reaction space" can be used to predict the value of the exponent alpha. When the problem is treated as a reaction of the type A + B-->C in a confined region, the exponent is given by alpha = (3-ds)/4. By using the value of 2.17 previously established for the surface dimension of lysozyme [Pfeifer, P., Welz, U. & Wippermann, H. (1985) Chem. Phys. Lett. 113, 535-540], a corresponding alpha of 0.21 is calculated. Data for lysozyme at six different temperatures could be accurately fit by using this unadjusted value for alpha. These results show how the surface morphology of a protein influences diffusional processes of small molecules associating with the protein.
Group A streptococci efficiently invade human respiratory epithelial cells.
Authors: LaPenta D , Rubens C , Chi E , Cleary P P
Published: May 2006

Although infection by group A streptococci is a model of extracellular mucosal pathogenesis, these organisms can be associated with highly invasive infections resulting in sepsis and shock. Over the last 6 yr this species has renewed its reputation as a significant cause of sepsis and has piqued interest in the mechanism by which some strains are better able to breach mucosal barriers to gain access to the bloodstream than are others. An internalization assay was developed on the basis of resistance of intracellular streptococci to penicillin and gentamicin. Experiments showed that stationary-phase, as opposed to logarithmic-phase, bacteria are efficiently internalized and can persist in cultured human cells. Electron microscopy confirmed that streptococci were contained within intracellular vacuoles. Various strains of streptococci revealed significant differences in their capacity to be internalized. Two type M1 streptococci isolated from blood infections were internalized at frequencies equal to those reported for Salmonella and Listeria monocytogenes and greater than the frequency of a clonal variant from a case of pharyngitis.
Protein domains connect cell cycle stimulation directly to initiation of DNA replication.
Authors: Gjørup O V , Rose P E , Holman P S , Bockus B J , Schaffhausen B S
Published: May 2006

Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation.
De novo synthesis of an intron by the maize transposable element Dissociation.
Authors: Giroux M J , Clancy M , Baier J , Ingham L , McCarty D , Hannah L C
Published: May 2006

The mechanisms by which introns are gained or lost in the evolution of eukaryotic genes remain poorly understood. The discovery that transposable elements sometimes alter RNA splicing to allow partial or imperfect removal of the element from the primary transcripts suggests that transposons are a potential and continuing source of new introns. To date, splicing events that precisely restore the wild-type RNA sequence at the site of insertion have not been detected. Here we describe alternative RNA splicing patterns that result in precise removal of a Dissociation (Ds) insertion and one copy of its eight-nucleotide host site duplication from an exon sequence of the maize shrunken2-mutabe1 (sh2-m1) mutant. In one case, perfect splicing of Ds was associated with aberrant splicing of an intron located 32 bp upstream of the insertion site. The second transcript type was indistinguishable from wild-type mRNA, indicating that Ds was spliced like a normal intron in about 2% of the sh2-m1 transcripts. Our results suggest that the transposition of Ds into sh2 in 1968, in effect, marked the creation of a new intron in a modern eukaryotic gene. The possibility of precise intron formation by a transposable element demonstrated here may be a general phenomenon of intron formation, since consensus intron splice sites can be explained by insertions that duplicate host sequences upon integration. A model is presented.
Chemiluminescent activation of the antiviral activity of hypericin: a molecular flashlight.
Authors: Carpenter S , Fehr M J , Kraus G A , Petrich J W
Published: May 2006

Hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. The absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. The chemiluminescent oxidation of luciferin by the luciferase from the North American firefly Photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral activity of hypericin. Light-induced virucidal activity of hypericin was demonstrated against equine infectious anemia virus, a lentivirus structurally, genetically, and antigenically related to the human immunodeficiency virus. The implications for exploiting chemiluminescence as a "molecular flashlight" for effecting photodynamic therapy against virus-infected cells and tumor cells are discussed.
Lazaroids improve the survival of grafted rat embryonic dopamine neurons.
Authors: Nakao N , Frodl E M , Duan W M , Widner H , Brundin P
Published: May 2006

In rodent models of Parkinson disease in which transplants of dissociated rodent and human embryonic mesencephalic tissue, rich in dopamine neurons, have been studied, only 5-20% of the dopamine neurons survive the implantation procedure. We have investigated the effects of inhibiting free radical generation with two lazaroids, U-74389G and U-83836E, on the survival of embryonic rat dopamine neurons. U-74389G is a 21-aminosteroid, and U-83836E combines the piperazinyl pyrimidine portion of 21-aminosteroids with the antioxidant ring of alpha-tocopherol. In an initial study, we found that the lazaroids markedly prolonged the period after tissue dissociation that an embryonic mesencephalic cell suspension exhibits high cell viability in vitro, as assessed by using a dye exclusion method. In a second series of experiments, addition of lazaroids to dissociated mesencephalic graft tissue increased the yield of surviving rat dopamine neurons 2.6-fold after implantation in the dopamine-denervated rat striatum. The improved survival correlated with an earlier onset of graft-induced functional effects in the amphetamine-induced rotation test. Thus, inhibition of free radical generation can significantly increase the yield of grafted embryonic dopamine neurons. Addition of lazaroids to the graft preparation is a relatively simple modification of the transplantation protocol and could readily be applied in a clinical setting. Moreover, since iron-dependent lipid peroxidation has been suggested to play a role in the death of nigral dopamine neurons in Parkinson disease and lazaroids are particularly potent inhibitors of such processes, the findings may have implications for the pathogenesis of this disease.
Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase.
Authors: Szabó C , Southan G J , Thiemermann C
Published: May 2006

Enhanced formation of nitric oxide (NO) by both the constitutive and the inducible isoforms of NO synthase (NOS) has been implicated in the pathophysiology of a variety of diseases, including circulatory shock. Non-isoform-selective inhibition of NO formation, however, may lead to side effects by inhibiting the constitutive isoform of NOS and, thus, the various physiological actions of NO. S-Methylisothiourea sulfate (SMT) is at least 10- to 30-fold more potent as an inhibitor of inducible NOS (iNOS) in immunostimulated cultured macrophages (EC50, 6 microM) and vascular smooth muscle cells (EC50, 2 microM) than NG-methyl-L-arginine (MeArg) or any other NOS inhibitor yet known. The effect of SMT on iNOS activity can be reversed by excess L-arginine in a concentration-dependent manner. SMT (up to 1 mM) does not inhibit the activity of xanthine oxidase, diaphorase, lactate dehydrogenase, monoamine oxidase, catalase, cytochrome P450, or superoxide dismutase. SMT is equipotent with MeArg in inhibiting the endothelial, constitutive isoform of NOS in vitro and causes increases in blood pressure similar to those produced by MeArg in normal rats. SMT, however, dose-dependently reverses (0.01-3 mg/kg) the hypotension and the vascular hyporeactivity to vasoconstrictor agents caused by endotoxin [bacterial lipopolysaccharide (LPS), 10 mg/kg, i.v.] in anesthetized rats. Moreover, therapeutic administration of SMT (5 mg/kg, i.p., given 2 hr after LPS, 10 mg/kg, i.p.) attenuates the rises in plasma alanine and aspartate aminotransferases, bilirubin, and creatinine and also prevents hypocalcaemia when measured 6 hr after administration of LPS. SMT (1 mg/kg, i.p.) improves 24-hr survival of mice treated with a high dose of LPS (60 mg/kg, i.p.). Thus, SMT is a potent and selective inhibitor of iNOS and exerts beneficial effects in rodent models of septic shock. SMT, therefore, may have considerable value in the therapy of circulatory shock of various etiologies and other pathophysiological conditions associated with induction of iNOS.
Potent bombesin antagonists with C-terminal Leu-psi(CH2-N)-Tac-NH2 or its derivatives.
Authors: Cai R Z , Reile H , Armatis P , Schally A V
Published: May 2006

Various pseudononapeptide bombesin (BN)-(6-14) antagonists with a reduced peptide bond (CH2-NH) between positions 13 and 14 can suppress the mitogenic activity of BN or gastrin-releasing peptide in 3T3 fibroblast cells and small cell lung carcinoma. In the search for more potent BN antagonists, 10 modified nonapeptide BN antagonists containing N-terminal D-Phe, D-Cpa, and D- or L-Tpi and C-terminal Leu-psi(CH2-N)-Tac-NH2, Leu-psi(CH2-N)-MeTac-NH2, or Leu-psi(CH2-N)-Me2Tac-NH2 have been synthesized by incubating [13 psi 14,CH2-NH,Cys14]BN-(6-14) or [13 psi 14-CH2-NH,Pen14]BN-(6-14) with formaldehyde or acetaldehyde (Cpa = 4-chlorophenylalanine, Tac = thiazolidine-4-carboxylic acid, Tpi = 2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indol-3-carboxylic acid, and Pen = penicillamine). The biological activities of these compounds were then evaluated. [D-Phe6,13 psi 14,CH2-N,Tac14]BN-(6-14) (RC-3950-II) and [D-Phe6,13 psi 14,CH2-N,Me2Tac14]BN-(6-14) (RC-3985-II) exhibited greater potency in inhibition of 125I-labeled [Tyr4]BN binding to Swiss 3T3 cells than their parent compounds [D-Phe6,13 psi 14,CH2-NH,Cys14]BN-(6-14) (RC-3950-I) and [D-Phe6,13 psi 14,CH2-NH,Pen14]BN-(6-14) (RC-3985-I). The order of binding affinities of these compounds was as follows: [13 psi 14,CH2-N,Tac14]BN-(6-14) > [13 psi 14,CH2-N,Me2Tac14]BN-(6-14) > [13 psi 14,CH2-N,MeTac14]BN-(6-14). In most cases, the analogs with C-terminal Leu-psi(CH2-N)-Tac-NH2 were also more potent growth inhibitors of 3T3 cells than compounds containing C-terminal Leu-psi(CH2-N)-Me2Tac-NH2 or Leu-psi(CH2-N)-MeTac-NH2. The best BN antagonists of this series, RC-3950-II and [D-Cpa6,13 psi 14,CH2-N,Tac14]BN- (6-14) (RC-3925-II), inhibited gastrin-releasing peptide-stimulated growth of Swiss 3T3 cells with IC50 values of 1 nM and 6 nM, respectively. Since antagonists of this class inhibit growth of various tumors in animal cancer models, some of them may have clinical applications.
An increase in dendritic spine density on hippocampal CA1 pyramidal cells following spatial learning in adult rats suggests the formation of new synapses.
Authors: Moser M B , Trommald M , Andersen P
Published: May 2006

The search for cellular correlates of learning is a major challenge in neurobiology. The hippocampal formation is important for learning spatial relations. A possible long-lasting consequence of such spatial learning is alteration of the size, shape, or number of excitatory synapses. The dendritic spine density is a good index for the number of hippocampal excitatory synapses. By using laser-scanning confocal microscopy, we observed a significantly increased spine density in CA1 basal dendrites of spatially trained rats when compared to nontrained controls. With unchanged dendritic length, the higher spine density reflects an increased number of excitatory synapses per neuron associated with spatial learning.
Identification of calreticulin as a rubella virus RNA binding protein.
Authors: Singh N K , Atreya C D , Nakhasi H L
Published: May 2006

Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-loop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem-loop sequence. In the present study, we have purified the RNA binding protein and identified it as a simian homologue of human calreticulin. The purified calreticulin binds to the RV RNA with specificity similar to the protein present in cytosolic extracts. Human calreticulin antibodies recognize several forms of simian calreticulin, one of which is phosphorylated in vivo. A 2-fold increase in phosphorylation of this form of calreticulin is observed in RV-infected cells. Recombinant human calreticulin can bind RV 3' (+)-stem-loop RNA only after undergoing in vitro phosphorylation. This binding activity is abrogated by pretreatment of phosphorylated recombinant human calreticulin with alkaline phosphatase. The RV RNA was also immunoprecipitated from RV-infected UV-crosslinked Vero 76 cells by using calreticulin antibodies. Our results show that phosphorylated calreticulin is an RNA binding protein and phosphorylation is necessary for this activity. Specific binding of calreticulin to the cis-acting element of RV RNA in vivo suggests a possible role for this interaction in viral replication.
Nonrandomness in protein sequences: evidence for a physically driven stage of evolution?
Authors: Pande V S , Grosberg A Y , Tanaka T
Published: May 2006

The sequences, or primary structures, of existing biopolymers--in particular, proteins--are believed to be a product of evolution. Are the sequences random? If not, what is the character of this nonrandomness? To explore the statistics of protein sequences, we use the idea of mapping the sequence onto the trajectory of a random walk, originally proposed by Peng et al. [Peng, C.-K., Buldyrev, S. V., Goldberger, A. L., Havlin, S., Sciortino, F., Simons, M. & Stanley, H. E. (1992) Nature (London) 356, 168-170] in their analysis of DNA sequences. Using three different mappings, corresponding to three basic physical interactions between amino acids, we found pronounced deviations from pure randomness, and these deviations seem directed toward minimization of the energy of the three-dimensional structure. We consider this result as evidence for a physically driven stage of evolution.
Cellular epigenetics: control of the size, shape, and spatial distribution of transformed foci by interactions between the transformed and nontransformed cells.
Authors: Rubin H
Published: May 2006

NIH 3T3 cells that are passaged frequently at low density in high (10%) calf serum lose their original capacity to produce transformed foci on a monolayer of nontransformed cells. They can then be used to form a monolayered background for the assay of the number of focus-forming cells from a transformed population. Continuation of the low-density passages for many weeks gives rise to a population that can suppress the full development of foci by a transformed line. The suppression appears to occur only after the background cells have become confluent and contact inhibited. It can also cause the disappearance of light foci that had developed before suppression began. Another subline of cells that were passaged at cloning density only once a week lose their focus-forming capacity more slowly than those passaged thrice weekly. When used as a background for the assay of a transformed line, they permit continuous expansion of the foci, with no sign of suppression. Not only the number and size of foci but also their detailed morphology is influenced by the background on which they are formed. A suppressive background can also determine the spatial distribution of foci, presumably as a result of gradients in local cell density of the background. The permissiveness of a nontransformed cell population for focus formation by transformed cells appears to be related to the capacity of the nontransformed population itself to undergo transformation when exposed to the constraints used to induce transformation. These findings indicate there are many degrees of capacity to suppress focus formation and to overcome suppression. They have significance for tumor development and for the epigenetic interactions of normal development.
A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity.
Authors: Eismann E , Müller F , Heinemann S H , Kaupp U B
Published: May 2006

Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells from the external and internal face. We studied ion selectivity and blockage by Ca2+ of wild-type and mutant channels in a heterologous expression system. External Ca2+ blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of approximately 2. The blockage from the internal face requires approximately 1000-fold higher Ca2+ concentrations. Neutralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeabilities nearly unaffected. The current blockage at -80 mV requires approximately 2000-fold higher external Ca2+ concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.