bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant thyroid hormone receptor and retinoid Xreceptor stimulate ligand-dependent transcription in vitro.
Authors: Lee I J , Driggers P H , Medin J A , Nikodem V M , Ozato K
Published: May 2006

The thyroid hormone and retinoid X receptors form
a heterodimer with each other and mediate thyroid hormone (T3)-dependent
transcription. Retinoid X receptor, in addition, forms a homodimer and mediates
9-cis-retinoic acid-dependent transcription. Here, recombinant thyroid hormone
receptor and recombinant retinoid X receptor beta expressed from baculovirus
vectors have been studied for ligand-mediated activation of transcription in
vitro. We show that the two recombinant receptors, most likely as a heterodimer,
cooperatively enhance transcription in vitro from a template containing
functional T3 responsive elements. The enhancement was specific for the T3
responsive element and was greatest when T3 was added to the reaction
(approximately 14-fold increase). Albeit to a lesser degree, the two receptors
also directed transcription in the absence of T3. Template competition
experiments suggest that the two receptors enhance formation of the
preinitiation complex and that activation by T3 occurs when the ligand binds the
receptor prior to (or during), but not after, the formation of the preinitiation
complex. Although 9-cis-retinoic acid had no effect on the T3-dependent
transcription, this ligand activated transcription in vitro directed by
recombinant retinoic X receptor beta, most likely as a homodimer. This
activation was observed when using nuclear extracts from embryonal carcinoma
cells as a source of basal transcription factors, but not those from B
lymphocytes. These results demonstrate that transcriptional activation mediated
by T3 and 9-cis-retinoic acid can be reconstituted in vitro with the respective
recombinant receptors.
Nuclear translocation of angiogenin in proliferatingendothelial cells is essential to its angiogenic activity.
Authors: Moroianu J , Riordan J F
Published: May 2006

The intracellular pathway of human angiogenin in
calf pulmonary artery endothelial (CPAE) cells has been studied by
immunofluorescence microscopy. Proliferating CPAE cells specifically endocytose
native angiogenin and translocate it to the nucleus, where it accumulates in the
nucleoli. Nuclear translocation of angiogenin does not occur in
nonproliferative, confluent CPAE cells. These cells were previously found to
express an angiogenin-binding protein (AngBP) that was identified as smooth
muscle alpha-actin. Exogenous actin, an anti-actin antibody, heparin, and
heparinase treatment all inhibit the internalization of angiogenin, suggesting
the involvement of cell surface AngBP/actin and heparan sulfate proteoglycans in
this process. It has been established that two regions of angiogenin are
essential for its angiogenic activity, one is its endothelial cell binding site
and the other its catalytic site capable of cleaving RNA. CPAE cells do not
internalize four enzymatically active angiogenin derivatives whose cell binding
site is modified, but they do internalize two enzymatically inactive mutants
whose cell binding site is intact. Thus, the putative cell binding site of
angiogenin is necessary for both endocytosis and nuclear translocation, but the
catalytic site is not. Three other angiogenic molecules are also translocated to
the nucleus of growing CPAE cells. Overall, the results suggest that nuclear
translocation of angiogenin and other angiogenic molecules is a critical step in
the process of angiogenesis.
Structure of the Escherichia coli Fis-DNA complexprobed by protein conjugated with 1,10-phenanthroline copper(I)complex.
Authors: Pan C Q , Feng J A , Finkel S E , Landgraf R , Sigman D , Johnson R C
Published: May 2006

The Escherichia coli Fis (factor for inversion
stimulation) protein functions in many diverse biological systems including
recombination, transcription, and DNA replication. Although Fis is a
site-specific DNA-binding protein, it lacks a well-defined consensus recognition
sequence. The electrophoretic mobility of Fis-DNA complexes, along with
considerations of the Fis crystal structure, indicates that significant
deformation of DNA occurs upon Fis binding. To investigate the structure of
Fis-DNA complexes, the chemical nuclease 1,10-phenanthroline-copper complex
(OP-Cu) has been linked to four specific sites within the Fis DNA-binding
domain. Two of these Fis-OP derivatives were active in cleaving DNA. The
scission patterns obtained on four different Fis binding sites indicate that Fis
positions itself on these highly divergent DNA sequences in a very similar
fashion. The patterns of cleavage of a derivative at Asn-98 generally support a
model of a Fis-DNA complex that contains specific bends within the
core-recognition sequence. Data from a second Fis-OP derivative at Asn-73
provides evidence for greater wrapping of flanking DNA around the sides of the
Fis protein than was previously postulated. The cleavage efficiency of flanking
segments varies, suggesting that the extent of DNA wrapping is sequence
dependent. Specific amino acids on Fis are implicated in promoting this DNA
wrapping.
Peroxisome proliferator and retinoid signalingpathways co-regulate preadipocyte phenotype and survival.
Authors: Chawla A , Lazar M A
Published: May 2006

Culture of mouse 3T3-L1 preadipocytes in medium
containing delipidated bovine calf serum caused the cells to elongate and divide
after reaching confluency. The continued proliferation correlated with sustained
expression of the c-myc gene, which was repressed in control cells. Exposure of
the cells to activators of peroxisome proliferator-activated receptor (PPAR),
including clofibrate, WY-14,643, and 5,8,11,14-eicosatetraynoic acid, reversed
and prevented the effects of culturing preadipocytes in delipidated serum.
Continued exposure to PPAR activators led to adipose conversion, during which
PPAR and its heterodimerization partner (retinoid X receptor) were induced.
Retinoic acid (RA) had no effect on the growth or survival of preadipocytes
grown in the presence of normal bovine serum. However, treatment of cells
cultured in delipidated serum with RA caused death of the cells by apoptosis.
Thus, preadipocyte phenotype and survival are regulated by activators of nuclear
hormone receptors.
Involvement of cardiovirus leader in hostcell-restricted virus expression.
Authors: Kong W P , Ghadge G D , Roos R P
Published: May 2006

Theiler murine encephalomyelitis virus designates
a number of picornavirus strains that are classified into two subgroups on the
basis of their different biological activities. DA strain and other members of
the TO subgroup produce a chronic demyelinating disease in which the virus
persists and manifests a restricted expression. Mutagenesis studies of the DA
strain leader (L) coding region, which is located at the 5' end of the
polyprotein coding region, demonstrate that L is completely dispensable for
infection of some cells; in addition, nucleotides can be inserted into the L
coding region with no loss in infectivity, indicating that Theiler murine
encephalomyelitis virus may be used as a vector for delivering foreign
sequences. In other cells, L is critical for plaque formation and efficient
viral multiplication. These findings raise the possibility that L may play a
role in the DA-induced demyelinating restricted infection. The functions of L,
and even its presence within the genome, vary among picornaviruses, reflecting
the various requirements for viral growth among different host
cells.
Molecular and population genetic analysis of allelicsequence diversity at the human beta-globin locus.
Authors: Fullerton S M , Harding R M , Boyce A J , Clegg J B
Published: May 2006

Allelic sequence polymorphism at the beta-globin
locus was investigated in a group of 36 Melanesians. A 3-kilobase fragment
containing the gene and its flanking regions was sequenced in 60 normal (beta A)
and 12 thalassemic (intron 1, position 5, G-->C) chromosomes. Haplotype
relationships between linked polymorphisms were derived by allele-specific PCR
amplification and sequencing. Seventeen nucleotide polymorphisms and 2 length
variants were identified, and these sites segregated as 17 sequence haplotypes
in the normal chromosomes. This haplotype diversity is higher than that expected
on the basis of the nucleotide polymorphism observed and is probably due to
recombination and gene conversion. Nucleotide diversity at synonymous sites in
the sample is 0.14%, suggesting an average age of sequence divergence of
approximately 450,000 years, consistent with that expected for a neutrally
evolving human nuclear locus.
Hyperinsulinemia induces a reversible impairment ininsulin receptor function leading to diabetes in the sand rat model ofnon-insulin-dependent diabetes mellitus.
Authors: Kanety H , Moshe S , Shafrir E , Lunenfeld B , Karasik A
Published: May 2006

The insulin receptor was evaluated at different
disease stages in the sand rat (Psammomys obesus), a model for nutrition-induced
diabetes. Nondiabetic sand rats showed markedly low receptor number in liver
compared with albino rats. Their receptor had an intact tyrosine kinase activity
but a higher Km for ATP in the phosphorylation reaction of exogenous substrates.
The initial effects of overeating (i.e., development of hyperinsulinemia without
hyperglycemia) were associated in the sand rat with a dramatic decrease in in
vitro and in vivo insulin-induced receptor tyrosine kinase activity in both
liver and muscle. In muscle, this coincided with a decrease in receptor number
and an increase in basal tyrosine kinase activity. Similar changes were observed
upon development of hyperinsulinemia with hyperglycemia. Upon recovery from the
diabetic state by diet restriction, the impaired receptor kinase activation was
corrected. Complete restoration occurred only in animals that fully recovered
from the diabetic state and became normoinsulinemic. These observations indicate
that loss and gain of receptor tyrosine kinase activity were dependent on
insulin levels. Thus, overeating may lead to the development of hyperinsulinemia
through ineffective extraction of excess insulin by the scarce liver receptors.
Hyperinsulinemia, in turn, causes a reversible reduction in receptor kinase
activity, leading to insulin resistance. This sequence of events may be relevant
to diet-related changes in human non-insulin-dependent diabetes
mellitus.
Location of Golgi membranes with reference to dividingnuclei in syncytial Drosophila embryos.
Authors: Ripoche J , Link B , Yucel J K , Tokuyasu K , Malhotra V
Published: May 2006

The role of cytoskeletal elements in the
cellularization of syncytial Drosophila embryos is becoming evident; however,
the distribution and role of organelles such as the Golgi complex, essential for
membrane biogenesis, remain unknown. We have cloned a Golgi-membrane-associated
polypeptide, beta-COP, from Drosophila. Immunocytochemical studies of syncytial
Drosophila embryos with anti-Drosophila beta-COP antibody reveal that Golgi
membranes are spatially segregated from the rapidly dividing nuclei. In early
embryos, the Golgi membranes are located in the embryonic cortex and nuclei are
confined to the core. This distribution of Golgi membranes may serve in
preparation of the embryonic cortex for the accommodation of nuclei upon their
eventual migration to the cortex and in biogenesis of the excessive plasma
membrane needed for cellularization of syncytial embryos.
Lack of telomere shortening during senescence inParamecium.
Authors: Gilley D , Blackburn E H
Published: May 2006

Paramecium tetraurelia cells have a limited clonal
life span and die after approximately 200 fissions if they do not undergo the
process of autogamy or conjugation. To test the possibility that cellular
senescence of this species is caused by telomere shortening, we analyzed the
genomic DNA of the macronucleus during the clonal life span of P. tetraurelia.
We found that telomeric DNA sequences were not shortened during the interval of
decreased fission rate and cellular death, defined as senescence in these cells.
However, the mean size of the macronuclear DNA was markedly decreased during the
clonal life span. We present a model that expands upon previous proposals that
accumulated DNA damage causes cellular senescence in P.
tetraurelia.
Delayed emergence of effects of memory-enhancing drugs: implications for the dynamics of long-term memory.
Authors: Mondadori C , Hengerer B , Ducret T , Borkowski J
Published: May 2006

Many theories of memory postulate that processing of information outlasts the learning situation and involves several different physiological substrates. If such physiologically distinct mechanisms or stages of memory do in fact exist, they should be differentially affected by particular experimental manipulations. Accordingly, a selective improvement of the processes underlying short-term memory should be detectable only while the information is encoded in the short-term mode, and a selective influence on long-term memory should be detectable only from the moment when memory is based on the long-term trace. Our comparative study of the time course of the effects of the cholinergic agonist arecoline, the gamma-aminobutyric acid type B receptor antagonist CGP 36742, the angiotensin-converting enzyme inhibitor captopril, and the nootropic oxiracetam, four substances with completely different primary sites of action, show that the memory-enhancing effects consistently come into evidence no sooner than 16-24 h after the learning trial. On the one hand, this finding suggests that all these substances act by way of the same type of mechanism; on the other hand, it demonstrates that the substrate modulated by the compounds forms the basis of memory only after 16-24 h. From the observation that animals also show clear signs of retention during the first 16 h--i.e., before the effects of the substances are measurable--it can be inferred that retention during this time is mediated by other mechanisms that are not influenced by any of the substances.
Suppression of calcium-dependent membrane currents in human fibroblasts by replicative senescence and forced expression of a gene sequence encoding a putative calcium-binding protein.
Authors: Liu S , Thweatt R , Lumpkin C K , Goldstein S
Published: May 2006

Human diploid fibroblasts (HDFs) possess Ca(2+)-dependent membrane currents. These currents were suppressed in late-passage normal (senescent) HDFs and prematurely senescent HDFs derived from a subject with Werner syndrome (WS), compared with early-passage normal (young) HDFs. When young HDFs were microinjected with mRNA transcribed in vitro from a cDNA (WS3-10) which encodes a protein bearing a putative Ca(2+)-binding site and whose endogenous gene is overexpressed in senescent and WS HDFs, membrane currents fell to levels present in senescent and WS HDFs. Thus, both replicative senescence and forced expression of the WS3-10 gene sequence lead to suppression of Ca(2+)-dependent membrane currents, which suggests that a causal connection exists between these two processes.
Solution structure of a trefoil-motif-containing cell growth factor, porcine spasmolytic protein.
Authors: Carr M D , Bauer C J , Gradwell M J , Feeney J
Published: May 2006

The porcine spasmolytic protein (pSP) is a 106-residue cell growth factor that typifies a family of eukaryotic proteins that contain at least one copy of an approximately 40-amino acid protein domain known as the trefoil motif. In fact, pSP contains two highly homologous trefoil domains. We have determined the complete three-dimensional solution structure of pSP by using a combination of two- and three-dimensional 1H NMR spectroscopy and distance geometry calculations. pSP is a relatively elongated molecule, consisting of two compact globular domains joined via a small interface. The protein's two trefoil domains adopt the same tertiary structure and contain a core C-terminal two-stranded antiparallel beta-sheet, preceded by a 6-residue helix that packs against the N-terminal beta-strand. The remainder of the protein backbone is taken up by two short loops that lie on either side of the beta-hairpin and are linked by an extended region that wraps around the C-terminal beta-strand. The topology of the protein backbone observed for the trefoil domains in pSP represents an unusual polypeptide fold. A striking feature of both trefoil domains is a surface patch formed from five conserved residues that have no obvious structural role. The two patches are located at the far ends of the protein molecule, and we propose that these residues form at least part of the receptor binding site, or sites, on pSP.
Cloning and characterization of two members of the vertebrate Dlx gene family.
Authors: Simeone A , Acampora D , Pannese M , D'Esposito M , Stornaiuolo A , Gulisano M , Mallamaci A , Kastury K , Druck T , Huebner K
Published: May 2006

A number of vertebrate genes of the Dlx gene family have been cloned in mouse, frog, and zebrafish. These genes contain a homeobox related to that of Distalless, a gene expressed in the developing head and limbs of Drosophila embryos. We cloned and studied the expression of two members of this family, which we named Dlx5 and Dlx6, in human and mouse. The two human genes, DLX5 and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLX1 and DLX2 are closely linked in a convergent configuration at 2q32, near the HOXD (previously HOX4) locus. In situ hybridization experiments in mouse embryos revealed expression of Dlx5 and Dlx6 mRNA in restricted regions of ventral diencephalon and basal telencephalon, with a distribution very similar to that reported for Dlx1 and Dlx2 mRNA. A surprising feature of Dlx5 and Dlx6 is that they are also expressed in all skeletal structures of midgestation embryos after the first cartilage formation. The expression pattern of these genes, together with their chromosome localization, may provide useful cues for the study of congenital disorders in which there is a combination of craniofacial and limb defects.
Immunization with purified N1 and N2 influenza virus neuraminidases demonstrates cross-reactivity without antigenic competition.
Authors: Johansson B E , Kilbourne E D
Published: May 2006

Based on the absence of serologic cross-reactivity, the neuraminidases (NAs) of influenza A viruses are divided into antigenically discrete subtypes, analogous to the hemagglutinin (HA) major antigens with which they share the virion surface. An innovative approach to influenza vaccination takes advantage of the infection-permissive nature of immunization with NA as the minor surface antigen. However, evidence that HA dominates immune response when HA and NA are presented together in the intact virion prompted investigation of possible competing effects during immunization of NA subtype mixtures ultimately required for human vaccination. Immunization of BALB/c mice with purified N1- and N2-subtype NAs demonstrated no antigenic competition in primary or secondary response. However, when homotypic or heterotypic infection followed immunization, cross-reactive antibodies between N1 and N2 were found and "reverse antigen competition" occurred with initial NA priming suppressing response to HA following infection with virus containing homologous NA. These studies of antigen mixtures have implications for the use of combined and chimeric vaccines for diseases other than influenza.
Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold.
Authors: Bazan J F , Weaver L H , Roderick S L , Huber R , Matthews B W
Published: May 2006

Amino acid sequence comparison suggests that the structure of Escherichia coli methionine aminopeptidase (EC 3.4.11.18) and the C-terminal domain of Pseudomonas putida creatinase (EC 3.5.3.3) are related. A detailed comparison of the three-dimensional folds of the two enzymes confirms this homology: with an approximately 260-residue chain segment, 218 C alpha atoms of the structures superimpose within 2.5 A; only 41 of these overlapping positions (i.e., 19%) feature identical amino acids in the two protein chains. Notwithstanding this striking correspondence in structure, methionine aminopeptidase binds and is stimulated by Co2+, while creatinase is not a metal-dependent enzyme. Searches of protein data banks using sequence and structure-based profiles reveal other enzymes, including aminopeptidase P (EC 3.4.11.9), prolidase (EC 3.4.13.9), and agropine synthase, that likely share the same "pita-bread" fold common to creatinase and methionine aminopeptidase.
The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.
Authors: Ding D X , Rivas C I , Heaney M L , Raines M A , Vera J C , Golde D W
Published: May 2006

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an alpha and beta subunit, which together form the high-affinity receptor. The alpha subunit by itself binds ligand at low affinity, whereas the isolated beta subunit does not bind GM-CSF. It is generally believed that the high-affinity receptor is responsible for the multiple functions of GM-CSF and that the isolated alpha subunit (GMR alpha) does not transduce a signal. Xenopus laevis oocytes injected with RNA encoding human GMR alpha expressed up to 10(10) low-affinity sites for GM-CSF (Kd = 6 nM). GM-CSF binding to the alpha subunit expressed in Xenopus oocytes caused activation of 2-deoxyglucose transport through endogenous glucose transporters. 2-Deoxyglucose transport was stimulated by similar low concentrations of GM-CSF in HL-60 leukemia cells as well as normal human neutrophils and Xenopus oocytes expressing GMR alpha. Engagement of the isolated alpha subunit in oocytes did not lead to protein phosphorylation or tyrosine phosphorylation of mitogen-activated protein kinase (MAP kinase). Staurosporin and genistein inhibited GM-CSF-induced tyrosine phosphorylation of MAP kinase in human neutrophils and HL-60 cells without affecting GM-CSF-stimulated uptake of 2-deoxyglucose. These results provide direct evidence that the isolated alpha subunit signals for hexose transport and can do so without engagement of the kinase cascade. Our data also indicate that signaling for hexose uptake may occur in a phosphorylation-independent manner in cells expressing the high-affinity GM-CSF receptor.
The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase.
Authors: Geremia R A , Mergaert P , Geelen D , Van Montagu M , Holsters M
Published: May 2006

Nod factors are signal molecules produced by Azorhizobium, Bradyrhizobium, and Rhizobium species that trigger nodule formation in leguminous host plants. The backbone of Nod factors consists of a beta-1,4-N-acetylglucosamine oligosaccharide from which the N-acetyl group at the nonreducing end is replaced by a fatty acid. The nodABC gene products are necessary for backbone biosynthesis. By incubation of cell extracts from Azorhizobium caulinodans with radioactive uridine diphosphate-N-acetylglucosamine, Nod factor precursors were identified and characterized as beta-1,4-N-acetylglucosamine oligosaccharides. By analysis of different nod gene mutants and by expression of nodC in Escherichia coli, the N-acetylglucosaminyltransferase activity was ascribed to the NodC protein. The results suggest that the first step in biosynthesis of Nod factors is the assembly of the oligosaccharide chain.
Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.
Authors: Culiat C T , Stubbs L J , Montgomery C S , Russell L B , Rinchik E M
Published: May 2006

Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors (approximately 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 5 transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (gamma 3, alpha 5, and beta 3) subunits. These mice therefore provide a whole-organism type A gamma-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the gamma 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.
Neurotensin excites basal forebrain cholinergic neurons: ionic and signal-transduction mechanisms.
Authors: Farkas R H , Nakajima S , Nakajima Y
Published: May 2006

The whole-cell patch-clamp technique was used to investigate the effect of neurotensin on cholinergic neurons cultured from the rat nucleus basalis of Meynert. Neurotensin excited the neurons by inducing an initial inward current carried, at least in part, by Na+ and by reducing inwardly rectifying K+ conductance. Reduction of the inwardly rectifying K+ conductance was mediated by a pertussis toxin-insensitive G protein.
Site-specific incorporation of biophysical probes into proteins.
Authors: Cornish V W , Benson D R , Altenbach C A , Hideg K , Hubbell W L , Schultz P G
Published: May 2006

Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled amino acid and the fluorescent amino acid. The ability to incorporate these probes site-specifically allows for novel studies of protein structure and dynamics. Moreover, this work demonstrates that the Escherichia coli protein biosynthetic machinery can tolerate unnatural amino acids with little resemblance to the natural amino acids.
Hydrodynamic steering effects in protein association.
Authors: Brune D , Kim S
Published: May 2006

Protein-ligand reaction rates are often limited by the rate of diffusional encounter of the protein and ligand in solution. Reaction rates, however, can be much greater than expected, given the necessity for correct orientation before reaction. A number of forces can affect the orientation of the protein and ligand in solution, and thus increase the reaction rate. We have considered hydrodynamic forces, produced when water molecules between protein and ligand must be pushed out of the way to allow their encounter. We have used the cleft enzymes as a model system, as they could be expected to show strong hydrodynamic effects. One particular type of hydrodynamic interaction stands out: a steering torque which occurs when the enzyme and substrate move toward each other in solution. The magnitude of this steering torque is compared to the mutual torque experienced by interacting "protein-sized" dipoles in solution. A simple model is used to demonstrate that the hydrodynamic steering torque can be 2 orders of magnitude greater than the electrostatic torque.
Dantrolene ameliorates the metabolic hallmarks of sepsis in rats and improves survival in a mouse model of endotoxemia.
Authors: Hotchkiss R S , Karl I E
Published: May 2006

Sepsis is the systemic inflammatory response resulting from serious infection and is the most common cause of death in intensive care units. Intracellular free calcium concentration ([Ca2+]i) is an important regulator of numerous cellular processes and when increased excessively may act as a potent cellular toxin. To determine if [Ca2+]i is responsible for the major metabolic changes which are hallmarks of sepsis, we examined if sodium dantrolene, a drug which decreases release of calcium from sarcoplasmic reticulum, affected the metabolic abnormalities in plasma and epitrochlearis muscles of rats made septic by cecal ligation and perforation. Dantrolene when added in vitro or when given in vivo decreases many of the metabolic hallmarks of sepsis--i.e., muscle protein breakdown approximately 30%, muscle glucose transport approximately 38%, muscle lactate formation approximately 28%, and plasma lactate approximately 29% (P < 0.05). In addition, we examined the ability of dantrolene to improve survival in a mouse model of endotoxemia. Dantrolene caused > 2-fold improvement in survival when it was administered concurrently with endotoxin (54% vs. 20% survival in dantrolene-treated and control mice, respectively (P < 0.001). Our results are consistent with the hypothesis that an increase in [Ca2+]i plays an important role in the metabolic abnormalities which occur during sepsis and that dantrolene administration may be an effective therapeutic strategy.
Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function.
Authors: Meek R L , Urieli-Shoval S , Benditt E P
Published: May 2006

Altered lipoprotein metabolism and vascular injury are considered to be major parts of the pathogenesis of atherosclerotic lesions. Serum amyloid A (SAA) is a family of acute-phase reactants found residing mainly on high density lipoproteins (HDL) in the circulation. Several functions for the SAAs have been proposed that could be important in atherosclerosis. These include involvement in cholesterol metabolism, participation in detoxification, depression of immune responses, and interference with platelet functions. Like other acute-phase reactants, the liver is a major site of SAA synthesis. However, studies in the mouse have revealed that several cell types including macrophages express SAA. Furthermore, we recently found that SAA mRNA expression can be induced in the human monocyte/macrophage cell line, THP-1. In the present study, human atherosclerotic lesions of coronary and carotid arteries were examined for expression of SAA mRNA by in situ hybridization. Surprisingly, SAA mRNA was found in most endothelial cells and some smooth muscle cells as well as macrophage-derived "foam cells," adventitial macrophages, and adipocytes. In addition, cultured smooth muscle cells expressed SAA1, SAA2, and SAA4 mRNAs when treated with interleukin 1 or 6 (IL-1 or IL-6) in the presence of dexamethasone. These findings give further credence to the notion that the SAAs are involved in lipid metabolism or transport at sites of injury and in atherosclerosis or may play a role in defending against viruses or other injurious agents such as oxidized lipids. Furthermore, expression of SAAs by endothelial cells is compatible with the evidence that SAA modulates platelet aggregation and function and possibly adhesion at the endothelial cell surface.
Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning.
Authors: Meiri N , Masos T , Rosenblum K , Miskin R , Dudai Y
Published: May 2006

Transgenic mice designated alpha MUPA overproduce in the brain murine urokinase-type plasminogen activator (uPA), an extracellular protease implicated in tissue remodeling. We have now localized, by in situ hybridization, extensive signal of uPA mRNA in the alpha MUPA cortex, hippocampus, and amygdala, sites that were not labeled in counterpart wild-type mice. Furthermore, biochemical measurements reveal a remarkably high level of enzymatic activity of uPA in the cortex and hippocampus of alpha MUPA compared with wild-type mice. We have used the alpha MUPA mice to examine whether the abnormal level of uPA in the cortex and the limbic system affects learning ability. We report that alpha MUPA mice perform poorly in tasks of spatial, olfactory, and taste-aversion learning, while displaying normal sensory and motor capabilities. Our results suggest that uPA is involved in neural processes subserving a variety of learning types.
Electrically conducting polymers can noninvasively control the shape and growth of mammalian cells.
Authors: Wong J Y , Langer R , Ingber D E
Published: May 2006

Electrically conducting polymers are novel in that their surface properties, including charge density and wettability, can be reversibly changed with an applied electrical potential. Such properties might render conducting polymers unique for biological applications. However, the majority of research on conducting polymers has been carried out under nonbiological conditions. We synthesized optically transparent polypyrrole thin films and studied them in environments suitable for protein adsorption and mammalian cell culture. In vitro studies demonstrated that extracellular matrix molecules, such as fibronectin, adsorb efficiently onto polypyrrole thin films and support cell attachment under serum-free conditions. When aortic endothelial cells were cultured on fibronectin-coated polypyrrole (oxidized) in either chemically defined medium or the presence of serum, cells spread normally and synthesized DNA. In contrast, when the polymer was switched to its neutral state by applying an electrical potential, both cell extension and DNA synthesis were inhibited without affecting cell viability. Application of a similar electrical potential to cells cultured on indium tin oxide surfaces had no effect on cell shape or function. These data suggest that electrically conducting polymers may represent a type of culture substrate which could provide a noninvasive means to control the shape and function of adherent cells, independent of any medium alteration.
A discrete Fourier analysis for evolutionary trees.
Authors: Hendy M D , Penny D , Steel M A
Published: May 2006

Discrete Fourier transformations have recently been developed to model the evolution of two-state characters (the Cavender/Farris model). We report here the extension of these transformations to provide invertible relationships between a phylogenetic tree T (with three probability parameters of nucleotide substitution on each edge corresponding to Kimura's 3ST model) and the expected frequencies of the nucleotide patterns in the sequences. We refer to these relationships as spectral analysis. In either model with independent and identically distributed site substitutions, spectral analysis allows a global correction for all multiple substitutions (second- and higher-order interactions), independent of any particular tree. From these corrected data we use a least-squares selection procedure, the closest tree algorithm, to infer an evolutionary tree. Other selection criteria such as parsimony or compatibility analysis could also be used; each of these criteria will be statistically consistent for these models. The closest tree algorithm selects a unique best-fit phylogenetic tree together with independent edge length parameters for each edge. The method is illustrated with an analysis of some primate hemoglobin sequences.
Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.
Authors: Giese K , Pagel J , Grosschedl R
Published: May 2006

The mammalian sex-determining gene SRY (sex-determining region on Y chromosome) encodes a member of the high mobility group (HMG) family of regulatory proteins. The HMG domain of the SRY protein represents a DNA binding motif that displays rather unusually weak evolutionary conservation of amino acids between human and mouse sequences. Together with the previous finding that the human (h) SRY gene is unable to induce a male phenotype in genetically female transgenic mice, these observations raise questions concerning the DNA binding properties of SRY proteins. Here, we present data that indicate that the DNA binding and bending properties of the HMG domains of murine (m) SRY and hSRY differ from each other. In comparison, mSRY shows more-extensive major-groove contacts with DNA and a higher specificity of sequence recognition than hSRY. Moreover, the extent of protein-induced DNA bending differs from the HMG domains of hSRY and mSRY. These differences in DNA binding by hSRY and mSRY may, in part, account for the functional differences observed with these gene products.
Kinetics of an RNA conformational switch.
Authors: LeCuyer K A , Crothers D M
Published: May 2006

The spliced leader RNA from Leptomonas collosoma has two competing secondary structures of nearly equal free energy. Short, complementary oligonucleotides can drive the structure from one form of the other. We report stopped-flow rapid-mixing and temperature-jump measurements of the kinetics of the structural switch. At high concentrations of oligonucleotide, the rate of binding becomes limited by the rate of the structural switch, which occurs on a time scale of a fraction of a second. The low activation energy observed for the process implies a branch migration type of mechanism in which portions of the two competing helices transiently coexist.
Seroepidemiology of water-borne hepatitis in India and evidence for a third enterically-transmitted hepatitis agent.
Authors: Arankalle V A , Chadha M S , Tsarev S A , Emerson S U , Risbud A R , Banerjee K , Purcell R H
Published: May 2006

Many epidemics of water-borne hepatitis have occurred throughout India. These were thought to be epidemics of hepatitis A until 1980, when evidence for an enterically transmitted non-A, non-B hepatitis was first reported. Subsequently, hepatitis E virus was discovered and most recent epidemics of enterically transmitted non-A, non-B hepatitis have been attributed to hepatitis E virus infection. However, only a limited number of cases have been confirmed by immuno electron microscopy, polymerase chain reaction, or seroconversion. In the present study we have performed a retrospective seroepidemiologic study of 17 epidemics of water-borne hepatitis in India. We have confirmed that 16 of the 17 epidemics were caused at least in part by serologically closely related hepatitis E viruses. However, one epidemic, in the Andaman Islands, and possibly a significant minority of cases in other epidemics, appears to have been caused by a previously unrecognized hepatitis agent.
Compartmentalization of two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides.
Authors: Konishi T , Sasaki Y
Published: May 2006

Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-CoA, the first intermediate in fatty acid synthesis. We studied the localization of two forms, the prokaryote and the eukaryote forms, of ACCase in pea leaves by comparing the biotin polypeptides of the two ACCases in protein extract from leaves and plastids. We found that the two forms of ACCase were in different cell compartments of pea leaves; the prokaryote form was in the plastids, and the eukaryote form was elsewhere, probably in the cytosol. This result suggested the existence of two sites of malonyl-CoA synthesis. The Gramineae, such as rice and wheat, which lack the accD gene encoding one of the subunits of the prokaryote form of ACCase in their chloroplast genomes, did not have the prokaryote form of the enzyme but had the eukaryote form. The selective grass herbicides of the diphenoxypropionic acid type and the cyclohexanedione type, in vitro, inhibited plastidic ACCase of the eukaryote form from wheat but did not inhibit that of the prokaryote form from pea, suggesting that the origin of the tolerance of intact pea plant toward these herbicides is partly in the insensitivity of the prokaryote form of the enzyme. The origin of the susceptibility of the Gramineae plants toward these herbicides seems to lie in the presence of the herbicide-sensitive eukaryote form and the absence of the insensitive prokaryote form due to the lack of the accD gene in plastid.
Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary.
Authors: Clancy R M , Levartovsky D , Leszczynska-Piziak J , Yegudin J , Abramson S B
Published: May 2006

We performed experiments to determine whether nitric oxide promoted the formation of intracellular S-nitrosothiol adducts in human neutrophils. At concentrations sufficient to inhibit chemoattractant-induced superoxide anion production, nitric oxide caused a depletion of measurable intracellular glutathione as determined by both the monobromobimane HPLC method and the glutathione reductase recycling assay. The depletion of glutathione could be shown to be due to the formation of intracellular S-nitrosoglutathione as indicated by the ability of sodium borohydride treatment of cytosol to result in the complete recovery of measurable glutathione. The formation of intracellular S-nitrosylated compounds was confirmed by the capacity of cytosol derived from nitric oxide-treated cells to ADP-ribosylate glyceraldehyde-3-phosphate dehydrogenase. Depletion of intracellular glutathione was accompanied by a rapid and concomitant activation of the hexose monophosphate shunt (HMPS) following exposure to nitric oxide. Kinetic studies demonstrated that nitric oxide-dependent activation of the HMPS was reversible and paralleled nitric oxide-induced glutathione depletion. Synthetic preparations of S-nitrosoglutathione shared with nitric oxide the capacity to inhibit superoxide anion production and activate the HMPS. These data suggest that nitric oxide may regulate cellular functions via the formation of intracellular S-nitrosothiol adducts and the activation of the HMPS.
UV photoelectron and ab initio quantum mechanical characterization of valence electrons in Na(+)-water-2'-deoxyguanosine 5'-phosphate clusters: electronic influences on DNA alkylation by methylating and ethylating carcinogens.
Authors: Kim H S , LeBreton P R
Published: May 2006

UV photoelectron data for 1,9-dimethylguanine, 3-hydroxytetrahydrofuran, and water, and results from ab initio self-consistent field (SCF) and post-SCF molecular orbital calculations were employed to describe valence electrons in clusters of 2'-deoxyguanosine 5'-phosphate (5'-dGMP-) with four water molecules and a phosphate-bound sodium ion. Two clusters (A and B) were examined. In A, Na+ is coordinated to 5'-dGMP-. In B, Na+ and 5'-dGMP- are separated by water. In 5'-dGMP- clusters, the nucleotide valence ionization potentials (IPs) are different from those previously reported for isolated 5'-dGMP-. The smallest IP in isolated 5'-dGMP- (4.6 eV) arises from the phosphate group; the smallest IPs in A (8.2 eV) and B (7.9 eV) arise from the base. In the clusters, the IPs associated with the seven upper occupied base orbitals differ from corresponding IPs in 1,9-dimethylguanine by less than 0.4 eV. In A and B, the smaller IP of the base, compared with the phosphate group, is consistent with reactivity data indicating that DNA and RNA are subject to electrophilic SN2 attack by carcinogens such as N-methyl-N-nitrosourea, dimethyl and diethyl sulfate, and methyl and ethyl methanesulfonate, in which more than 75% of nucleotide alkylation occurs at the bases.
Mast cells synthesize, store, and release nerve growth factor.
Authors: Leon A , Buriani A , Dal Toso R , Fabris M , Romanello S , Aloe L , Levi-Montalcini R
Published: May 2006

Mast cells and nerve growth factor (NGF) have both been reported to be involved in neuroimmune interactions and tissue inflammation. In many peripheral tissues, mast cells interact with the innervating fibers. Changes in the behaviors of both of these elements occur after tissue injury/inflammation. As such conditions are typically associated with rapid mast cell activation and NGF accumulation in inflammatory exudates, we hypothesized that mast cells may be capable of producing NGF. Here we report that (i) NGF mRNA is expressed in adult rat peritoneal mast cells; (ii) anti-NGF antibodies clearly stain vesicular compartments of purified mast cells and mast cells in histological sections of adult rodent mesenchymal tissues; and (iii) medium conditioned by peritoneal mast cells contains biologically active NGF. Mast cells thus represent a newly recognized source of NGF. The known actions of NGF on peripheral nerve fibers and immune cells suggest that mast cell-derived NGF may control adaptive/reactive responses of the nervous and immune systems toward noxious tissue perturbations. Conversely, alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature.
A model of dendritic spine Ca2+ concentration exploring possible bases for a sliding synaptic modification threshold.
Authors: Gold J I , Bear M F
Published: May 2006

We used a biophysical model of an isolated dendritic spine to assess quantitatively the impact of changes in spine geometry, Ca2+ buffer concentration, and channel kinetics on Ca2+ dynamics following high-frequency activation of N-methyl-D-aspartate receptors. We found that varying the buffer concentration in the postsynaptic density from 50 to 500 microM can result in an 8-fold difference in the peak Ca2+ concentration following three pulses at 100 Hz. Similarly, varying the spine neck diameter from 0.1 to 0.55 micron can result in a 15-fold difference in the peak Ca2+ concentration. The amplification of peak Ca2+ concentration also depended on temporal summation of N-methyl-D-aspartate-mediated excitatory postsynaptic currents. Variation of the current duration on the order of 100 msec can significantly affect summation at a given stimulation frequency, resulting in a 10-fold difference in the peak Ca2+ concentration at 100 Hz. It is suggested that activity-dependent modifications of these parameters may be important for the regulation of synaptic plasticity in the brain.