bluuebunny/crossref_metadata_2025 huggingface dataset:
Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia
Authors: Namura Shobu , Iihara Koji , Takami Shinya , Nagata Izumi , Kikuchi Haruhiko , Matsushita Koji , Moskowitz Michael A. , Bonventre Joseph V. , Alessandrini Alessandro
Published: July 2002

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100–200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.
Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment
Authors: Hastrup Hanne , Karlin Arthur , Javitch Jonathan A.
Published: July 2002

There is evidence both for and against Na
+
- and Cl
−
-dependent neurotransmitter transporters forming oligomers. We found that cross-linking the human dopamine transporter (DAT), which is heterologously expressed in human embryonic kidney 293 cells, either with copper phenanthroline (CuP) or the bifunctional reagent bis-(2-methanethiosulfonatoethyl)amine hydrochloride (bis-EA) increased the apparent molecular mass determined with nonreducing SDS/PAGE from ≈85 to ≈195 kDa. After cross-linking, but not before, coexpressed, differentially epitope-tagged DAT molecules, solubilized in Triton X-100, were coimmunoprecipitated. Thus, the 195-kDa complex was a homodimer. Cross-linking of DAT did not affect tyramine uptake. Replacement of Cys-306 with Ala prevented cross-linking. Replacement of all of the non-disulfide-bonded cysteines in the extracellular and membrane domains, except for Cys-306, did not prevent cross-linking. We conclude that the cross-link is between Cys-306 at the extracellular end of TM6 in each of the two DATs. The motif GVXXGVXXA occurs at the intracellular end of TM6 in DAT and is found in a number of other neurotransmitter transporters. This sequence was originally found at the dimerization interface in glycophorin A, and it promotes dimerization in model systems. Mutation of either glycine disrupted DAT expression and function. The intracellular end of TM6, like the extracellular end, is likely to be part of the dimerization interface.
Male mate choice selects for female coloration in a fish
Authors: Amundsen Trond , Forsgren Elisabet
Published: July 2002

Although sexual selection theory has proved successful in explaining a wide array of male ornaments, the function of ornaments occurring in females is largely unknown. Traditionally, female ornaments have been considered nonfunctional, being merely a genetically correlated response to selection for male ornamentation. However, this hypothesis is only relevant to species in which the ornament is basically the same in the two sexes. Alternatively, female ornaments may be influenced by selection acting directly on the females, either through female–female competition or male choice. We tested the latter hypothesis in mate-choice experiments with two-spotted gobies (
Gobiusculus flavescens
). In this small marine fish, females have bright yellow-orange bellies during the breeding season, a conspicuous trait that is not present in males. We conducted two aquarium experiments to test whether males preferred to mate with more colorful females. In the first experiment, males had a choice between two females that varied in natural coloration (and belly roundness). In the second experiment, we manipulated belly coloration and kept roundness constant. Males spent more time with colorful than with drab females in both experiments and also performed far more courtship displays toward colorful females. Our study provides experimental evidence that males prefer ornamented females in a fish that is not sex-role reversed, supporting the hypothesis that female ornamentation is sexually selected.
Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia
Authors: Taylor Cormac T. , Furuta Glenn T. , Synnestvedt Kristin , Colgan Sean P.
Published: July 2002

Hypoxia activates a number of gene products through degradation of
the transcriptional coactivator cAMP response element binding protein
(CREB). Other transcriptional regulators (e.g., β-catenin and
NF-κB) are controlled through phosphorylation-targeted proteasomal
degradation, and thus, we hypothesized a similar degradative pathway
for CREB. Differential display analysis of mRNA derived from hypoxic
epithelia revealed a specific and time-dependent repression of
protein phosphatase 1 (PP1), a serine phosphatase important in CREB
dephosphorylation. Subsequent studies identified a previously
unappreciated proteasomal-targeting motif within the primary structure
of CREB (DSVTDS), which functions as a substrate for PP1. Ambient
hypoxia resulted in temporally sequential CREB serine phosphorylation,
ubiquitination, and degradation (
in vitro
and
in
vivo
). HIV-tat peptide-facilitated loading of intact epithelia
with phosphopeptides corresponding to this proteasome targeting motif
resulted in inhibition of CREB ubiquitination. Further studies revealed
that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas
proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia
establishes conditions that target CREB to proteasomal degradation.
These studies may provide unique insight into a general mechanism of
transcriptional regulation by hypoxia.
RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences
Authors: Dunn Walter , Trang Phong , Khan Umair , Zhu Jiaming , Liu Fenyong
Published: July 2002

External guide sequences (EGSs) are oligonucleotides that consist
of a sequence complementary to a target mRNA and recruit intracellular
RNase P for specific degradation of the target RNA. In this
study, DNA-based EGS molecules were chemically synthesized to target
the mRNA coding for the protease of human cytomegalovirus (HCMV). The
EGS molecules efficiently directed human RNase P to cleave the target
mRNA sequence
in vitro
. When EGSs were exogenously
administered into HCMV-infected human foreskin fibroblasts, a reduction
of about 80–90% in the expression level of the protease and a
reduction of about 300-fold in HCMV growth were observed in the cells
that were treated with a functional EGS, but not in cells that were not
treated with the EGS or with a “disabled” EGS carrying nucleotide
mutations that precluded RNase P recognition. Moreover, packaging of
the viral DNA genome into the capsid was blocked in the cells treated
with the functional EGS. These results indicate that HCMV protease is
essential for viral DNA encapsidation. Moreover, our study provides
direct evidence that exogenous administration of a DNA-based EGS
can be used as a therapeutic approach for inhibiting gene expression
and replication of a human virus.
Granzyme B can cause mitochondrial depolarization and cell death in the absence of BID, BAX, and BAK
Authors: Thomas Dori A. , Scorrano Luca , Putcha Girish V. , Korsmeyer Stanley J. , Ley Timothy J.
Published: July 2002

Granzyme B (GzmB) is a serine protease that is used by activated cytotoxic T lymphocytes to induce target cell apoptosis. Although GzmB directly cleaves the Bcl2 family member BID on target cell entry,Bid-deficient (andBax, Bakdoubly deficient) cells are susceptible to GzmB-induced death, even though they fail to release cytochromecfrom mitochondria. GzmB still induces mitochondrial depolarization inBax, Bakdouble knockout cells without cytochromecrelease or opening of the permeability transition pore. Because GzmB cannot directly cause depolarization of isolated mitochondria, novel intracellular factor(s) may be required for GzmB to depolarize mitochondriain situ. GzmB therefore utilizes two distinct mitochondrial pathways to amplify the proapoptotic signal that it delivers to target cells.
Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.
Authors: Huang L C , Clarkin K C , Wahl G M
Published: July 2002

The tumor suppressor p53 contributes to maintaining genome stability by inducing a cell cycle arrest or apoptosis in response to conditions that generate DNA damage. Nuclear injection of linearized plasmid DNA, circular DNA with a large gap, or single-stranded circular phagemid is sufficient to induce a p53-dependent arrest. Supercoiled and nicked plasmid DNA, and circular DNA with a small gap were ineffective. Titration experiments indicate that the arrest mechanism in normal human fibroblasts can be activated by very few double strand breaks, and only one may be sufficient. Polymerase chain reaction assays showed that end-joining activity is low in serum-arrested human fibroblasts, and that higher joining activity occurs as cells proceed through G1 or into S phase. We propose that the exquisite sensitivity of the p53-dependent G1 arrest is partly due to inefficient repair of certain types of DNA damage in early G1.
Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.
Authors: Steele D F , Butler C A , Fox T D
Published: July 2002

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.
Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.
Authors: Rasty S , Thatikunta P , Gordon J , Khalili K , Amini S , Glorioso J C
Published: July 2002

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.
Three quaternary structures for a single protein.
Authors: Huang D B , Ainsworth C F , Stevens F J , Schiffer M
Published: July 2002

The structure of a multisubunit protein (immunoglobulin light chain) was solved in three crystal forms, differing only in the solvent of crystallization. The three structures were obtained at high ionic strength and low pH, high ionic strength and high pH, and low ionic strength and neutral pH. The three resulting "snapshots" of possible structures show that their variable-domain interactions differ, reflecting their stabilities under specific solvent conditions. In the three crystal forms, the variable domains had different rotational and translational relationships, whereas no alteration of the constant domains was found. The critical residues involved in the observed effect of the solvent are tryptophans and histidines located between the two variable domains in the dimeric structure. Tryptophan residues are commonly found in interfaces between proteins and their subunits, and histidines have been implicated in pH-dependent conformation changes. The quaternary structure observed for a multisubunit protein or protein complex in a crystal may be influenced by the interactions of the constituents within the molecule or complex and/or by crystal packing interactions. The comparison of buried surface areas and hydrogen bonds between the domains forming the molecule and between the molecules forming the crystals suggest that, for this system, the interactions within the molecule are most likely the determining factors.
Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.
Authors: Sharma A , Okabe J , Birch P , McClellan S B , Martin M J , Platt J L , Logan J S
Published: July 2002

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.
Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.
Authors: Madrenas J , Schwartz R H , Germain R N
Published: July 2002

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
Authors: Petzer A L , Hogge D E , Landsdorp P M , Reid D S , Eaves C J
Published: July 2002

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.
The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.
Authors: Sanchez A , Trappier S G , Mahy B W , Peters C J , Nichol S T
Published: July 2002

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.
Mixed synapses discovered and mapped throughout mammalian spinal cord.
Authors: Rash J E , Dillman R K , Bilhartz B L , Duffy H S , Whalen L R , Yasumura T
Published: July 2002

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.
The B
12
-dependent ribonucleotide reductase from the archaebacterium
Thermoplasma acidophila
: An evolutionary solution to the ribonucleotide reductase conundrum
Authors: Tauer Andreas , Benner Steven A.
Published: July 2002

A coenzyme B
12
-dependent ribonucleotide reductase was purified from the archaebacterium
Thermoplasma acidophila
and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in
Escherichia coli
. The deduced amino acid sequence shows that the catalytic domain of the B
12
-dependent enzyme from
T. acidophila
, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from
E. coli
, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the
T. acidophila
sequence and that of a B
12
-dependent reductase from
Mycobacterium tuberculosis
establish homology between the Fe-dependent enzymes and the catalytic domain of the
Lactobacillus leichmannii
B
12
-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the
Lactobacillus
enzyme. Sequence similarities show that the N-terminal domain of the
T. acidophila
ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from
E. coli
, which uses neither B
12
nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic
E. coli
enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B
12
ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in “post-genomic” biochemistry.
Four primordial modes of tRNA-synthetase recognition, determined by the (G,C) operational code
Authors: Rodin Sergei N. , Ohno Susumu
Published: July 2002

In distinction to single-stranded anticodons built of G, C, A, and U bases, their presumable double-stranded precursors at the first three positions of the acceptor stem are composed almost invariably of G-C and C-G base pairs. Thus, the “second” operational RNA code responsible for correct aminoacylation seems to be a (G,C) code preceding the classic genetic code. Although historically rooted, the two codes were destined to diverge quite early. However, closer inspection revealed that two complementary catalytic domains of class I and class II aminoacyl-tRNA synthetases (aaRSs) multiplied by two, also complementary, G2-C71 and C2-G71 targets in tRNA acceptors, yield four (2 × 2) different modes of recognition. It appears therefore that the core four-column organization of the genetic code, associated with the most conservative central base of anticodons and codons, was in essence predetermined by these four recognition modes of the (G,C) operational code. The general conclusion follows that the genetic code
per se
looks like a “frozen accident” but only beyond the “2 × 2 = 4” scope. The four primordial modes of tRNA–aaRS recognition are amenable to direct experimental verification.
Cholinergic stimulation alters performance and task-specific regional cerebral blood flow during working memory
Authors: Furey M. L. , Pietrini P. , Haxby J. V. , Alexander G. E. , Lee H. C. , VanMeter J. , Grady C. L. , Shetty U. , Rapoport S. I. , Schapiro M. B. , Freo U.
Published: July 2002

Modulation of the cholinergic neurotransmitter system results in changes in memory performance, including working memory (WM), in animals and in patients with Alzheimer disease. To identify associated changes in the functional brain response, we studied performance measures and regional cerebral blood flow (rCBF) using positron emission tomography (PET) in healthy subjects during performance of a WM task. Eight control subjects received an infusion of saline throughout the study and 13 experimental subjects received a saline infusion for the first 2 scans followed by a continuous infusion of physostigmine, an acetylcholinesterase inhibitor, for the subsequent 8 scans. rCBF was measured using H
2
15
O and PET in a sequence of 10 PET scans that alternated between rest and task scans. During task scans, subjects performed the WM task for faces. Physostigmine both improved WM efficiency, as indicated by faster reaction times, and reduced WM task-related activity in anterior and posterior regions of right midfrontal gyrus, a region shown previously to be associated with WM. Furthermore, the magnitudes of physostigmine-induced change in reaction time and right midfrontal rCBF correlated. These results suggest that enhancement of cholinergic function can improve processing efficiency and thus reduce the effort required to perform a WM task, and that activation of right prefrontal cortex is associated with task effort.
An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish
Authors: Bakkers Jeroen , Semino Carlos E. , Stroband Henri , Kijne Jan W. , Robbins Phillips W. , Spaink Herman P.
Published: July 2002

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized
in vitro
by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled
N
-acetyl-
d
-[U
14
C]glucosamine and by HPLC in combination with enzymatic conversion using the
Bradyrhizobium
NodZ α-1,6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of
Catharanthus roseus
. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the
Xenopus
DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in
Xenopus
and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the
Bradyrhizobium
NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.
Intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction
Authors: Kawamata Takakazu , Dietrich W. Dalton , Schallert Timothy , Gotts Jeffrey E. , Cocke Robert R. , Benowitz Larry I. , Finklestein Seth P.
Published: July 2002

Focal cerebral infarction (stroke) due to unilateral occlusion of the middle cerebral artery in mature rats produces deficits in sensorimotor function of the contralateral limbs that recover partially over time. We found that biweekly intracisternal injection of basic fibroblast growth factor (bFGF; 0.5 μg/injection), a potent neurotrophic polypeptide, markedly enhanced recovery of sensorimotor function of the contralateral limbs during the first month after stroke without apparent adverse side effects. Immunostaining for growth-associated protein 43 (GAP-43), a molecular marker of axonal sprouting, showed a selective increase in GAP-43 immunoreactivity in the intact sensorimotor cortex contralateral to cerebral infarcts following bFGF treatment. These results show that bFGF treatment can enhance functional recovery after stroke, and that the mechanism may include stimulation of neuronal sprouting in the intact brain.
Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal
Authors: Benjamin Laura E. , Keshet Eli
Published: July 2002

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously “on,” tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.
Primary structure and tissue distribution of two novel proline-rich γ-carboxyglutamic acid proteins
Authors: Kulman John D. , Harris Jeff E. , Haldeman Betty A. , Davie Earl W.
Published: July 2002

Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal γ-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational γ-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called
p
roline-
r
ich
G
la
p
roteins (PRGP1 and PRGP2). Unlike the γ-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.
Phage display of a catalytic antibody to optimize affinity for transition-state analog binding
Authors: Baca Manuel , Scanlan Thomas S. , Stephenson Robert C. , Wells James A.
Published: July 2002

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH→ Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.
The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety
Authors: Aspberg Anders , Miura Ryu , Bourdoulous Sandrine , Shimonaka Motoyuki , Heinegård Dick , Schachner Melitta , Ruoslahti Erkki , Yamaguchi Yu
Published: July 2002

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes
in vivo
. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.
Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry
Authors: Neubauer Gitte , Gottschalk Alexander , Fabrizio Patrizia , Séraphin Bertrand , Lührmann Reinhard , Mann Matthias
Published: July 2002

Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast
Saccharomyces cerevisiae
by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m
3
G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B′ homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.
Conferring RNA polymerase Activity to a DNA polymerase: A single residue in reverse transcriptase controls substrate selection
Authors: Gao Guangxia , Orlova Marianna , Georgiadis Millie M. , Hendrickson Wayne A. , Goff Stephen P.
Published: July 2002

The traditional classification of nucleic acid polymerases as either DNA or RNA polymerases is based, in large part, on their fundamental preference for the incorporation of either deoxyribonucleotides or ribonucleotides during chain elongation. The refined structure determination of Moloney murine leukemia virus reverse transcriptase, a strict DNA polymerase, recently allowed the prediction that a single amino acid residue at the active site might be responsible for the discrimination against the 2′OH group of an incoming ribonucleotide. Mutation of this residue resulted in a variant enzyme now capable of acting as an RNA polymerase. In marked contrast to the wild-type enzyme, the
K
m
of the mutant enzyme for ribonucleotides was comparable to that for deoxyribonucleotides. The results are consistent with proposals of a common evolutionary origin for both classes of enzymes and support models of a common mechanism of nucleic acid synthesis underlying catalysis by all such polymerases.
Molecular origin of cancer: Catechol estrogen-3,4-quinones as endogenous tumor initiators
Authors: Cavalieri E. L. , Stack D. E. , Devanesan P. D. , Todorovic R. , Dwivedy I. , Higginbotham S. , Johansson S. L. , Patil K. D. , Gross M. L. , Gooden J. K. , Ramanathan R. , Cerny R. L. , Rogan E. G.
Published: July 2002

Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon–DNA adducts generate oncogenic mutations found in mouse skin papillomas (
Proc. Natl. Acad. Sci. USA
92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E
1
) and estradiol (E
2
) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA
in vitro
to form the depurinating adduct 4-OHE
1
(E
2
)-1(α,β)-N7Gua at 59–213 μmol/mol DNA–phosphate whereas the level of stable adducts was 0.1 μmol/mol DNA–phosphate. In female Sprague–Dawley rats treated by intramammillary injection of E
2
-3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 μmol 4-OHE
2
-1(α,β)-N7Gua/molDNA–phosphate. When 4-OHE
1
(E
2
) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87–440 μmol of 4-OHE
1
(E
2
)-1(α, β)-N7Gua was formed. After treatment with 4-OHE
2
, rat mammary tissue contained 1.4 μmol of adduct/mol DNA–phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.
Endosperm balance number manipulation for direct
in vivo
germplasm introgression to potato from a sexually isolated relative (
Solanum commersonii
Dun.)
Authors: Carputo Domenico , Barone Amalia , Cardi Teodoro , Sebastiano Angela , Frusciante Luigi , Peloquin Stanley J.
Published: July 2002

Diploid (2
n
= 2
x
= 24)
Solanum
species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2
n
= 4
x
= 48, 4EBN) and haploid (2
n
= 2
x
= 24, 2EBN)
S. tuberosum
Group Tuberosum. To sexually overcome these crossing barriers in the diploid species
S. commersonii
(1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F
1
hybrids between an
in vitro
-doubled clone of
S. commersonii
(2
n
= 4
x
= 48, 2EBN) and diploid 2EBN clones were successfully used in 3
x
× 4
x
crosses with
S. tuberosum
Group Tuberosum, resulting in pentaploid/near pentaploid BC
1
progenies. This provided evidence of 2
n
(3
x
) egg formation in the triploid female parents. Two selected BC
1
pentaploid hybrids were successfully backcrossed both as male and as female parents with
S. tuberosum
Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC
2
progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of
S. commersonii
genomes was confirmed by the presence of
S. commersonii
-specific randomly amplified polymorphic DNA markers in the BC
2
population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4
x
(4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other
in vitro
or
in vivo
, direct or indirect, approaches previously reported.
Evidence for a role for DNA polymerase β in mammalian meiosis
Authors: Plug Annemieke W. , Clairmont Caroline A. , Sapi Eva , Ashley Terry , Sweasy Joann B.
Published: July 2002

DNA polymerase β (pol β) is an enzyme possessing both polymerase and deoxyribose phophatase activities. Although pol β is not believed to participate in the replication of genomic DNA, several studies have indicated a role for pol β in DNA repair. The high level of expression of pol β in mouse and rat testes raises the possibility that pol β participates in mammalian meiosis. Using antibody localization, we detect foci that stain with pol β antisera at discrete sites along homologous chromosomes as they synapse and progress through prophase of meiosis I. These data suggest that pol β participates in meiotic events associated with synapsis and recombination.