bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron accumulation in Alzheimer disease is a source of redox-generated free radicals
Authors: Smith Mark A. , Harris Peggy L. R. , Sayre Lawrence M. , Perry George
Published: July 2002

Damage from free radicals has been demonstrated in susceptible neuronal populations in cases of Alzheimer disease. In this study, we investigated whether iron, a potent source of the highly reactive hydroxyl radical that is generated by the Fenton reaction with H
2
O
2
, might contribute to the source of radicals in Alzheimer disease. We found, using a modified histochemical technique that relies on the formation of mixed valence iron complexes, that redox-active iron is associated with the senile plaques and neurofibrillary tangles—the pathological hallmark lesions of this disease. This lesion-associated iron is able to participate in
in situ
oxidation and readily catalyzes an H
2
O
2
-dependent oxidation. Furthermore, removal of iron was completely effected using deferoxamine, after which iron could be rebound to the lesions. Characterization of the iron-binding site suggests that binding is dependent on available histidine residues and on protein conformation. Taken together, these findings indicate that iron accumulation could be an important contributor toward the oxidative damage of Alzheimer disease.
Modulation of potassium channel function by methionine oxidation and reduction
Authors: Ciorba Matthew A. , Heinemann Stefan H. , Weissbach Herbert , Brot Nathan , Hoshi Toshinori
Published: July 2002

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such
in vivo
activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.
Activities of human recombination protein Rad51
Authors: Gupta Ravindra C. , Bazemore L. Rochelle , Golub Efim I. , Radding Charles M.
Published: July 2002

Homologous pairing and strand exchange, which are catalyzed by
Escherichia coli
RecA protein, are central to homologous recombination. Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51. For the studies described here, we purified HsRad51 from
E. coli
. Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences. The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than (null)/1;10 those of RecA. In addition, HsRad51 bound γ-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog. By contrast, RecA promoted both of the latter reactions in control experiments. These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.
Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking
Authors: Kanamori Takashi , Nishikawa Shuh-Ichi , Shin Injae , Schultz Peter G. , Endo Toshiya
Published: July 2002

Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [
dl
-2-amino-3-(
p
-benzoylphenyl)propanoic acid (
1
) and
dl
-2-amino-5-(
p
-benzoylphenyl)pentanoic acid (
2
)] were incorporated at specific sites throughout the cytochrome
b
2
-dihydrofolate reductase fusion proteins, p
b
2
(220)-DHFR and p
b
2
Δ19(220)-DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively.
In vitro
import of p
b
2
(220)-DHFR and p
b
2
Δ19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted p
b
2
Δ19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted p
b
2
(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of p
b
2
(220)-DHFR, but not p
b
2
Δ19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.
Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells
Authors: Weigmann Anja , Corbeil Denis , Hellwig Andrea , Huttner Wieland B.
Published: July 2002

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from
Caenorhabditis elegans
predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.
Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin
Authors: Murphy John E. , Zhou Shangzhen , Giese Klaus , Williams Lewis T. , Escobedo Jaime A. , Dwarki Varavani J.
Published: July 2002

The
ob/ob
mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in
ob/ob
mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.
Anchoring of protein kinase A facilitates hormone-mediated insulin secretion
Authors: Lester Linda B. , Langeberg Lorene K. , Scott John D.
Published: July 2002

Impaired insulin secretion is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). One possible therapeutic agent for NIDDM is the insulinotropic hormone glucagon-like peptide 1 (GLP-1). GLP-1 stimulates insulin secretion through several mechanisms including activation of protein kinase A (PKA). We now demonstrate that the subcellular targeting of PKA through association with
A
-
k
inase-
a
nchoring
p
roteins (AKAPs) facilitates GLP-1-mediated insulin secretion. Disruption of PKA anchoring by the introduction of anchoring inhibitor peptides or expression of soluble AKAP fragments blocks GLP-1 action in primary islets and cAMP-responsive insulin secretion in clonal beta cells (RINm5F). Displacement of PKA also prevented cAMP-mediated elevation of intracellular calcium suggesting that localized PKA phosphorylation events augment calcium flux.
Cryptic single-stranded-DNA binding activities of the phage λ P and
Escherichia coli
DnaC replication initiation proteins facilitate the transfer of
E. coli
DnaB helicase onto DNA
Authors: Learn Brian A. , Um Soo-Jong , Huang Li , McMacken Roger
Published: July 2002

The bacteriophage λ P and
Escherichia coli
DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P·DnaB or a DnaB·DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the λ O replication initiator enhances interaction of the P·DnaB complex with ssDNA. Partial disassembly of a ssDNA:O·P·DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.
Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway
Authors: Dugan Laura L. , Creedon Douglas J. , Johnson Eugene M. , Holtzman David M.
Published: July 2002

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.
A proline-rich motif in p53 is required for transactivation- independent growth arrest as induced by Gas1
Authors: Ruaro Elisabetta M. , Collavin Licio , Del Sal Giannino , Haffner Rebecca , Oren Moshe , Levine Arnold J. , Schneider Claudio
Published: July 2002

The involvement of p53 in regulating diverse cellular processes dictates that it must respond to multiple signaling mechanisms, thus coordinating the response to various “stress conditions.” Genotoxic stress has served as a paradigm to dissect the transactivation-dependent branch of the pathway by which p53 can induce growth arrest. Alternate mechanisms have been invoked to explain transactivation-independent effects of p53, especially in the context of apoptosis. We have identified a p53-dependent pathway initiated by the gas1 product, a plasma membrane protein highly expressed during G0, which activates a transactivation-independent p53 growth arrest function. Through a detailed deletional analysis and site-specific mutagenesis of p53 we show that the Gas1-dependent signal transduction relies on a proline-rich region (amino acids 63–85) of murine p53.
In vivo
competition experiments using combinations of such mutants implicate this functional domain of p53 as a docking site in the transmission of antiproliferative signals.
Rescuing impairment of long-term potentiation in fyn-deficient mice by introducing Fyn transgene
Authors: Kojima Nobuhiko , Wang Jian , Mansuy Isabelle M. , Grant Seth G. N. , Mayford Mark , Kandel Eric R.
Published: July 2002

To examine the physiological role of the Fyn tyrosine kinase in neurons, we generated transgenic mice that expressed a fyn cDNA under the control of the calcium/calmodulin-dependent protein kinase IIα promoter. With this promoter, we detected only low expression of Fyn in the neonatal brain. In contrast, there was strong expression of the fyn-transgene in neurons of the adult forebrain. To determine whether the impairment of long-term potentiation (LTP) observed in adult fyn-deficient mice was caused directly by the lack of Fyn in adult hippocampal neurons or indirectly by an impairment in neuronal development, we generated fyn-rescue mice by introducing the wild-type fyn-transgene into mice carrying a targeted deletion in the endogenous fyn gene. In fyn-rescue mice, Schaffer collateral LTP was restored, even though the morphological abnormalities characteristic of fyn-deficient mice were still present. These results suggest that Fyn contributes, at least in part, to the molecular mechanisms of LTP induction.
A transcription activation system for regulated gene expression in transgenic plants
Authors: Moore Ian , Gälweiler Leo , Grosskopf Debora , Schell Jeff , Palme Klaus
Published: July 2002

A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines. The promoter, pOp, consists oflacoperators cloned upstream of a minimal promoter. No expression was detected from this promoter when placed upstream of a β-glucuronidase (GUS) reporter gene in transgenic plants. Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4. This factor comprised transcription-activation domain-II from Gal4 ofSaccharomyces cerevisiaefused to a mutantlac-repressor that binds its operator with increased affinity. When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter. The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed. The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention. It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.
Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes
Authors: Ren Mindong , Xu Genxing , Zeng Jianbo , De Lemos-Chiarandini Carmen , Adesnik Milton , Sabatini David D.
Published: July 2002

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996)
J. Cell Biol.
135, 913–924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37°C of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37°C or 16°C. We show that incubation at 16°C blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16°C. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16°C is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.
Conversion of a human low-density lipoprotein receptor ligandbinding repeat to a virus receptor: Identification of residues important for ligand specificity
Authors: Rong Lijun , Gendron Kristin , Bates Paul
Published: July 2002

The amino-terminal region of the low-density lipoprotein receptor (LDLR) contains seven imperfect repeats of a cysteine-rich, roughly 40-aa module (LDL-A module) that are critical for apolipoprotein binding. LDL-A modules are found in numerous cell-surface and secreted proteins and are believed to mediate extracellular protein–protein interactions. The cellular receptor for subgroup A Rous sarcoma virus (RSV) contains a single LDL-A module that binds the RSV envelope protein and allows viral infection. To define residues in an LDL-A module responsible for ligand recognition, we used a gain of function assay by using a chimeric protein in which the LDL-A module of Tva was replaced with a highly homologous module from human LDLR (LDL-A4) and determined whether this chimera or mutants produced in it could mediate RSV infection. LDL-A4 does not function as an RSV receptor; however, systematic replacement of the nonconserved residues of the LDL-A4 module in the chimeric protein with the corresponding residues from Tva identified three residues sufficient to alter ligand specificity and convert LDL-A4 to an efficient viral receptor. Mutations of the corresponding residues in the Tva LDL-A module decreased both envelope binding and viral receptor function, confirming the importance of these residues in ligand recognition by this module. Analysis of the hLDL-A5 structure demonstrates that these three residues are clustered at one end of the LDL-A module. These results demonstrate that using a single LDL-A module in a gain of function assay is a useful model to investigate ligand recognition by this module.
A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis
Authors: Denis Cécile , Methia Nassia , Frenette Paul S. , Rayburn Helen , Ullman-Culleré Mollie , Hynes Richard O. , Wagner Denisa D.
Published: July 2002

von Willebrand factor (vWf) deficiency causes severe von Willebrand disease in humans. We generated a mouse model for this disease by using gene targeting. vWf-deficient mice appeared normal at birth; they were viable and fertile. Neither vWf nor vWf propolypeptide (von Willebrand antigen II) were detectable in plasma, platelets, or endothelial cells of the homozygous mutant mice. The mutant mice exhibited defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events in ≈10% of neonates. As in the human disease, the factor VIII level in these mice was reduced strongly as a result of the lack of protection provided by vWf. Defective thrombosis in mutant mice was also evident in an
in vivo
model of vascular injury. In this model, the exteriorized mesentery was superfused with ferric chloride and the accumulation of fluorescently labeled platelets was observed by intravital microscopy. We conclude that these mice very closely mimic severe human von Willebrand disease and will be very useful for investigating the role of vWf in normal physiology and in disease models.
A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsensecontaining mRNAs in mammalian cells
Authors: Sun Xiaolei , Perlick Haley A. , Dietz Harry C. , Maquat Lynne E.
Published: July 2002

All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation. The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing. Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (
i
) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (
ii
) in the cytoplasm. Here, we provide evidence for a factor that functions to eliminate the production of nonsense-containing RNAs in mammalian cells. The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to
Saccharomyces cerevisiae
Upf1p, which is a group I RNA helicase that functions in the nonsense-mediated decay of mRNA in yeast. Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominant-negative fashion to abrogate the decay of nonsense-containing mRNA that takes place (
i
) in association with nuclei or (
ii
) in the cytoplasm. These findings provide evidence that nonsense-mediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.
Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia
Authors: Nogawa Shigeru , Forster Colleen , Zhang Fangyi , Nagayama Masao , Ross M. Elizabeth , Iadecola Costantino
Published: July 2002

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2(PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.
The effect of dynamic synapses on spatiotemporal receptive fields in visual cortex
Authors: Artun Ömer B. , Shouval Harel Z. , Cooper Leon N
Published: July 2002

Temporal dynamics are a general feature of synaptic transmission. Recently, novel aspects of temporal dynamics of synaptic transmission have been reported in the neocortex. Here, we examine the possible effects of these dynamics on the spatiotemporal receptive fields of simple cells in V1. We do this by examining a simple model of a cortical neuron that displays stimulus orientation selectivity as a consequence of the pattern of thalamocortical synaptic weights. In our model, the receptive field structure is encoded functionally in either presynaptic probability of release or postsynaptic efficacy. We show that these different assumptions about the origin of receptive field structure lead to very different spatiotemporal dynamics in the case of flashed-bar stimulus. In addition, the results of the reverse correlation study suggest a possible test for differentiating between models. We also show that the temporal code induced by dynamic synapses can be used to distinguish between different inputs that induce the same average firing rate.
Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding
Authors: Höök Fredrik , Rodahl Michael , Kasemo Bengt , Brzezinski Peter
Published: July 2002

We have studied the adsorption of two structurally similar forms of hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (
f
) (c.f. mass) and energy dissipation
(D)
(c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the Δ
D
vs. Δ
f
graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe
3+
of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Thus, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both
D
and
f
, demonstrating that the QCM technique provides unique information about the mechanisms of protein adsorption to solid surfaces.
Glutamyl-tRNA
Gln
amidotransferase in
Deinococcus radiodurans
may be confined to asparagine biosynthesis
Authors: Curnow Alan W. , Tumbula Debra L. , Pelaschier Joanne T. , Min Bokkee , Söll Dieter
Published: July 2002

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium
Deinococcus radiodurans
suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to
Bacillus subtilis gatCAB
. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating
D. radiodurans
GluRS to produce the required mischarged Glu-tRNA
Gln
substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the
D. radiodurans
genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in
D. radiodurans
extracts, suggests that the
gatCAB
genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.
Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition
Authors: Jun Susie , Wallen Robert V. , Goriely Anne , Kalionis Bill , Desplan Claude
Published: July 2002

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of theDrosophila eye gonegene (eyg). It is unique among Pax proteins, because it contains only the RED domain.eygseems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.
Stream biodiversity: The ghost of land use past
Authors: Harding J. S. , Benfield E. F. , Bolstad P. V. , Helfman G. S. , Jones E. B. D.
Published: July 2002

The influence of past land use on the present-day diversity of stream invertebrates and fish was investigated by comparing watersheds with different land-use history. Whole watershed land use in the 1950s was the best predictor of present-day diversity, whereas riparian land use and watershed land use in the 1990s were comparatively poor indicators. Our findings indicate that past land-use activity, particularly agriculture, may result in long-term modifications to and reductions in aquatic diversity, regardless of reforestation of riparian zones. Preservation of habitat fragments may not be sufficient to maintain natural diversity in streams, and maintenance of such biodiversity may require conservation of much or all of the watershed.
Susceptibility to the biological effects of polyaromatic hydrocarbons is influenced by genes of the major histocompatibility complex
Authors: Elmets Craig A. , Athar Mohammad , Tubesing Karen A. , Rothaupt Dinah , Xu Hui , Mukhtar Hasan
Published: July 2002

Polyaromatic hydrocarbons are ubiquitous environmental chemicals that are important mutagens and carcinogens. The purpose of this study was to determine whether genes within the major histocompatibility complex (MHC) influence their biological activities. Cell-mediated immunity to dimethylbenz(a)anthracene (DMBA) was investigated in congenic strains of mice. On three different backgrounds, H-2kand H-2ahaplotype mice developed significantly greater contact-hypersensitivity responses to DMBA than H-2b, H-2d, and H-2smice. In B10.A(R1) mice, which are Kkand Id, a vigorous contact-hypersensitivity response was present, indicating that the response was governed by class I, rather than class II, MHC genes. C3H/HeN (H-2k) and C3H.SW (H-2s) strains were also compared for the development of skin tumors and the persistence of DMBA–DNA adducts. When subjected to a DMBA initiation, phorbol 12-tetradecanoate 13-acetate (TPA)-promotion skin-tumorigenesis protocol, C3H/HeN mice, (which develop cell-mediated immunity to DMBA) were found to have significantly fewer tumors than C3H.SW mice (a strain that failed to develop a cell-mediated immune response to DMBA). DMBA–DNA adducts were removed more rapidly in C3H/HeN than in C3H.SW mice. The results indicate that genes within the MHC play an important role in several of the biological activities of carcinogenic polyaromatic hydrocarbons. The observations are consistent with the hypothesis that cell-mediated immunity to chemical carcinogens serves to protect individuals by removing mutant cells before they can evolve into clinically apparent neoplasms.
Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays
Authors: Der Sandy D. , Zhou Aimin , Williams Bryan R. G. , Silverman Robert H.
Published: July 2002

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-α, -β, or -γ treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (α, β) or Type II (γ) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1α). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.
A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics
Authors: SantaLucia John
Published: July 2002

A unified view of polymer, dumbbell, and oligonucleotide
nearest-neighbor (NN) thermodynamics is presented. DNA NN
ΔG°
37
parameters from seven laboratories are
presented in the same format so that careful comparisons can be made.
The seven studies used data from natural polymers, synthetic polymers,
oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN
parameters; used different methods of data analysis; used different
salt concentrations; and presented the NN thermodynamics in different
formats. As a result of these differences, there has been much
confusion regarding the NN thermodynamics of DNA polymers and
oligomers. Herein I show that six of the studies are actually in
remarkable agreement with one another and explanations are provided in
cases where discrepancies remain. Further, a single set of parameters,
derived from 108 oligonucleotide duplexes, adequately describes polymer
and oligomer thermodynamics. Empirical salt dependencies are also
derived for oligonucleotides and polymers.
Use of D gene segments with irregular spacers in terminal deoxynucleotidyltransferase (TdT)+/+and TdT−/−mice carrying a human Ig heavy chain transgenic minilocus
Authors: Tuaillon Nadine , Capra J. Donald
Published: July 2002

D gene segments with irregular spacers (DIR) are D gene segments that are specific to higher primates. Their use is controversial because of their G+C-rich long sequences. In the human, it has always been tempting to assume that a complementarity-determining region 3 sequence has been added by terminal deoxynucleotidyltransferase (TdT) activity and is not derived from DIR recombination. Herein, we examine the use of human DIR gene segments by cross-breeding the human Ig heavy chain minilocus pHC1 transgenic mice and TdT-deficient mice. In the absence of TdT and with a defined set of human D gene segments, it is relatively easy to demonstrate that DIR2 is used to form human Ig heavy chains, contributing to 7% of the human heavy chain rearrangements. VHDJHrearrangements (where H is heavy chain) in the minilocus TdT−/−mice use small portions of DIR2 located throughout the coding sequence. These results constitute the strongest evidence to date that DIR gene segments are used to form human antibodies. Additionally, we show that direct and inverted DIR2JHand VHDIR2 rearrangements occur in the minilocus transgenic mice. During these rearrangements, DM23′ signal sequence and a new DIR2 5′ signal sequence are used. These rearrangements generally follow the 12/23 recombination rule. Our results at the VHDJH, DJH, and VHD levels indicate that DIR2 is used to form human heavy chains in transgenic mice. The rearrangement of this gene segment likely involves, however, other mechanisms in addition to the classical VHDJHrecombination.
Negative regulation of granulocytic differentiation in the myeloid precursor cell line 32Dcl3 byear-2, a mammalian homolog ofDrosophila seven-up, and a chimeric leukemogenic gene,AML1/ETO(MTG8)
Authors: Ahn Mee-Young , Huang Gang , Bae Suk-Chul , Wee Hee-Jun , Kim Woo-Young , Ito Yoshiaki
Published: July 2002

The polyomavirus enhancer binding protein 2αB (AML1/PEBP2αB/Cbfa2) plays a pivotal role in granulocyte colony-stimulating factor (G-CSF)-mediated differentiation of a myeloid progenitor cell line, 32Dc13. In this article, we report the identification of a PEBP2αB interacting protein, Ear-2, an orphan member of the nuclear hormone receptor superfamily that directly binds to and can inhibit the function of PEBP2αB. Ear-2 is expressed in proliferating 32Dc13 cells in presence of interleukin 3 but is down-regulated during differentiation induced by G-CSF. Interestingly, AML1/ETO(MTG8), a leukemogenic chimeric protein can block the differentiation of 32Dc13 cells, which is accompanied by the sustained expression ofear-2. Overexpression of Ear-2 can prevent G-CSF-induced differentiation, strongly suggesting thatear-2is a key negative regulator of granulocytic differentiation. Our results indicate that a dynamic balance existing between PEBP2αB and Ear-2 appears to determine the choice between growth or differentiation for myeloid cells.
Deregulated expression of
TCL1
causes T cell leukemia in mice
Authors: Virgilio Laura , Lazzeri Cristina , Bichi Roberta , Nibu Ken-ichi , Narducci Maria Grazia , Russo Giandomenico , Rothstein Jay L. , Croce Carlo M.
Published: July 2002

The
TCL1
oncogene on human chromosome 14q32.1 is involved in the development of T cell leukemia in humans. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. The
TCL1
oncogene is activated in these leukemias by juxtaposition to the α or β locus of the T cell receptor, caused by chromosomal translocations t(14:14)(q11:q32), t(7:14)(q35:q32), or by inversions inv(14)(q11:q32). To show that transcriptional alteration of
TCL1
is causally involved in the generation of T cell neoplasia we have generated transgenic mice that carry the
TCL1
gene under the transcriptional control of the p56
lck
promoter element. The
lck
-
TCL1
transgenic mice developed mature T cell leukemias after a long latency period. Younger mice presented preleukemic T cell expansions expressing
TCL1
, and leukemias developed only at an older age. The phenotype of the murine leukemias is CD4
−
CD8
+
, in contrast to human leukemias, which are predominantly CD4
+
CD8
−
. These studies demonstrate that transcriptional activation of the
TCL1
protooncogene can cause malignant transformation of T lymphocytes, indicating the role of
TCL1
in the initiation of malignant transformation in T prolymphocytic leukemias and T chronic lymphocytic leukemias.
Profound neuronal plasticity in response to inactivation of the dopamine transporter
Authors: Jones Sara R. , Gainetdinov Raul R. , Jaber Mohamed , Giros Bruno , Wightman R. Mark , Caron Marc G.
Published: July 2002

The dopamine transporter (DAT) plays an important role in calibrating the duration and intensity of dopamine neurotransmission in the central nervous system. We have used a strain of mice in which the gene for the DAT has been genetically deleted to identify the DAT’s homeostatic role. We find that removal of the DAT dramatically prolongs the lifetime (300 times) of extracellular dopamine. Within the time frame of neurotransmission, no other processes besides diffusion can compensate for the lack of the DAT, and the absence of the DAT produces extensive adaptive changes to control dopamine neurotransmission. Despite the absence of a clearance mechanism, dopamine extracellular levels were only 5 times greater than control animals due to a 95% reduction in content and a 75% reduction in release. Paradoxically, dopamine synthesis rates are doubled despite a decrease of 90% in the levels of tyrosine hydroxylase and degradation is markedly enhanced. Thus, the DAT not only controls the duration of extracellular dopamine signals but also plays a critical role in regulating presynaptic dopamine homeostasis. It is interesting to consider that the switch to a dopamine-deficient, but functionally hyperactive, mode of neurotransmission observed in mice lacking the DAT may represent an extreme example of neuronal plasticity resulting from long-term psychostimulant abuse.
The CCR5 receptor acts as an alloantigen in CCR5Δ32 homozygous individuals: Identification of chemokineand HIV-1-blocking human antibodies
Authors: Ditzel Henrik J. , Rosenkilde Mette M. , Garred Peter , Wang Meng , Koefoed Klaus , Pedersen Court , Burton Dennis R. , Schwartz Thue W.
Published: July 2002

The chemokine receptor CCR5 is the major coreceptor for infection by macrophage-tropic R5 HIV-1. A 32-bp deletion in the gene coding for CCR5 (CCR5Δ32) occurs with a frequency of 10% in the Caucasian population and results in a receptor protein that is truncated and not expressed at the cell surface. CCR5Δ32 homozygous individuals are apparently normal but resistant to infection with R5 HIV-1. In two individuals homozygous for CCR5Δ32, who had been repeatedly exposed to CCR5-expressing blood cells through sexual activity, we have identified antibodies to CCR5 that bound specifically to the surface of CCR5-expressing cell lines. Serum from these individuals, in contrast to serum from CCR5
+/+
individuals, competed with radiolabeled RANTES for binding to the CCR5 receptor and inhibited infection of peripheral blood mononuclear cells with R5, but not X4, primary isolates of HIV-1. The identified human antibodies to CCR5 define an alloantigen that may cause allograft rejection in a mismatch situation even in individuals with no history of blood transfusions or i.v. drug abuse.
Differential signaling via the same axon of neocortical pyramidal neurons
Authors: Markram Henry , Wang Yun , Tsodyks Misha
Published: July 2002

The nature of information stemming from a single neuron and conveyed simultaneously to several hundred target neurons is not known. Triple and quadruple neuron recordings revealed that each synaptic connection established by neocortical pyramidal neurons is potentially unique. Specifically, synaptic connections onto the same morphological class differed in the numbers and dendritic locations of synaptic contacts, their absolute synaptic strengths, as well as their rates of synaptic depression and recovery from depression. The same axon of a pyramidal neuron innervating another pyramidal neuron and an interneuron mediated frequency-dependent depression and facilitation, respectively, during high frequency discharges of presynaptic action potentials, suggesting that the different natures of the target neurons underlie qualitative differences in synaptic properties. Facilitating-type synaptic connections established by three pyramidal neurons of the same class onto a single interneuron, were all qualitatively similar with a combination of facilitation and depression mechanisms. The time courses of facilitation and depression, however, differed for these convergent connections, suggesting that different pre-postsynaptic interactions underlie quantitative differences in synaptic properties. Mathematical analysis of the transfer functions of frequency-dependent synapses revealed supra-linear, linear, and sub-linear signaling regimes in which mixtures of presynaptic rates, integrals of rates, and derivatives of rates are transferred to targets depending on the precise values of the synaptic parameters and the history of presynaptic action potential activity. Heterogeneity of synaptic transfer functions therefore allows multiple synaptic representations of the same presynaptic action potential train and suggests that these synaptic representations are regulated in a complex manner. It is therefore proposed that differential signaling is a key mechanism in neocortical information processing, which can be regulated by selective synaptic modifications.