bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of
quaking
in myelination: Regulation of alternative splicing
Authors: Wu Jiang I. , Reed Robyn B. , Grabowski Paula J. , Artzt Karen
Published: July 2002

Proteomic diversity is frequently achieved by alternative RNA-splicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system.
quaking
(
qk
), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The
qk
locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized
qk
isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the
qk
mouse is demonstrated.
Negative regulation of central nervous system myelination by polysialylated-neural cell adhesion molecule
Authors: Charles P. , Hernandez M. P. , Stankoff B. , Aigrot M. S. , Colin C. , Rougon G. , Zalc B. , Lubetzki C.
Published: July 2002

Many factors have been shown to promote myelination, but few have been shown to be inhibitory. Here, we show that polysialylated-neural cell adhesion molecule (PSA-NCAM) can negatively regulate myelin formation. During development, PSA-NCAM is first expressed on all growing fibers; then, axonal expression is down-regulated and myelin deposition occurs only on PSA-NCAM-negative axons. Similarly, in cocultures of oligodendrocytes and neurons, PSA-NCAM expression on axons is initially high, but decreases as myelination proceeds. Importantly, if expression of PSA-NCAM is prematurely decreased in cultures, by either antibody-mediated internalization or enzymatic removal of the PSA moieties with endoneuraminidase N (endo-N), myelination increases 4- to 5-fold. In the optic nerve, premature cleavage of PSA moieties by intravitreous injection of endo-N also induces a transient increase in the number of myelinated internodes, but does not interfere with the onset of myelination. Previously, we showed that axonal electrical activity strongly induced myelination, which could be prevented by tetrodotoxin (TTX), an action potential blocker. Interestingly, removal of PSA moieties does not reverse the inhibition of myelination by TTX. Together, this suggests that myelination is tightly controlled by both positive (electrical activity) and negative (PSA-NCAM expression) regulatory signals.
Anticonvulsant activity of a nonpeptide galanin receptor agonist
Authors: Saar Külliki , Mazarati Andrey M. , Mahlapuu Riina , Hallnemo Gerd , Soomets Ursel , Kilk Kalle , Hellberg Sven , Pooga Margus , Tolf Bo-Ragnar , Shi Tiejun S. , Hökfelt Tomas , Wasterlain Claude , Bartfai Tamas , Langel Ülo
Published: July 2002

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace
125
I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace
125
I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties
in vivo
. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.
A flagellar K
+
-dependent Na
+
/Ca
2+
exchanger keeps Ca
2+
low in sea urchin spermatozoa
Authors: Su Yi-Hsien , Vacquier Victor D.
Published: July 2002

The metabolism, flagellar beating, and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. As is true of most cells, swimming sperm maintain intracellular Ca
2+
concentrations at submicromolar levels. Here we describe a K
+
-dependent Na
+
/Ca
2+
exchanger (suNCKX) from sea urchin sperm. The suNCKX is phylogenetically related to other NCKXs, which use high relative intracellular K
+
, and high relative extracellular Na
+
, to couple the efflux of 1 Ca
2+
and 1 K
+
to the influx of 4 Na
+
. The 652-aa suNCKX shares structural topology with other NCKX proteins, and has two protein kinase A sites and a His-rich region in its cytoplasmic loop. The suNCKX is encoded by a single gene, which is highly expressed in testes. The suNCKX activity of whole sperm shows Na
+
and K
+
dependence, and like other NCKXs can run in reverse exchange mode. An inhibitor blocks the suNCKX activity and sperm motility. suNCKX localizes to the plasma membrane over the sperm flagellum. The suNCKX may play a major role in keeping Ca
2+
low in swimming sperm.
Activation of NF-κB is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes
Authors: Purcell Nicole H. , Tang Guilin , Yu Chenfei , Mercurio Frank , DiDonato Joseph A. , Lin Anning
Published: July 2002

The transcription factor NF-κB regulates expression of genes that
are involved in inflammation, immune response, viral infection, cell
survival, and division. However, the role of NF-κB in hypertrophic
growth of terminally differentiated cardiomyocytes is unknown. Here we
report that NF-κB activation is required for hypertrophic growth of
cardiomyocytes. In cultured rat primary neonatal ventricular
cardiomyocytes, the nuclear translocation of NF-κB and its
transcriptional activity were stimulated by several hypertrophic
agonists, including phenylephrine, endothelin-1, and angiotensin II.
The activation of NF-κB was inhibited by expression of a
“supersuppressor” IκBα mutant that is resistant to
stimulation-induced degradation and a dominant negative IκB kinase
(IKKβ) mutant that can no longer be activated by phosphorylation.
Furthermore, treatment with phenylephrine induced IκBα degradation
in an IKK-dependent manner, suggesting that NF-κB is a downstream
target of the hypertrophic agonists. Importantly, expression of the
supersuppressor IκBα mutant or the dominant negative IKKβ mutant
blocked the hypertrophic agonist-induced expression of the embryonic
gene atrial natriuretic factor and enlargement of cardiomyocytes.
Conversely, overexpression of NF-κB itself induced atrial natriuretic
factor expression and cardiomyocyte enlargement. These findings suggest
that NF-κB plays a critical role in the hypertrophic growth of
cardiomyocytes and may serve as a potential target for the intervention
of heart disease.
ADP ribosylation of human neutrophil peptide-1 regulates its biological properties
Authors: Paone Gregorino , Wada Akihiro , Stevens Linda A. , Matin Abul , Hirayama Toshiya , Levine Rodney L. , Moss Joel
Published: July 2002

In human airways, epithelial cells lining the lumen and intraluminal cells (e.g., polymorphonuclear cells) participate in the innate immune response. These cells secrete or express on their surfaces arginine-specific ADP ribosyltransferases. Defensins, antimicrobial proteins secreted by immune cells, are arginine-rich, leading us to hypothesize that ADP ribosylation could modify their biological activities. We found that an arginine-specific ADP ribosyltransferase-1 present on airway epithelial cells modifies Arg-14 of α defensin-1. ADP-ribosylated defensin-1 had decreased antimicrobial and cytotoxic activities but still stimulated T cell chemotaxis and IL-8 release from A549 cells. Further, ADP-ribosylated defensin-1 inhibited cytotoxic and antimicrobial activities of unmodified defensin-1. We identified ADP-ribosylated defensin-1 in bronchoalveolar lavage fluid from smokers but not from nonsmokers, confirming its existencein vivo. Thus, airway mono-ADP-ribosyltransferases could have an important regulatory role in the innate immune response through modification of α defensin-1 and perhaps other basic molecules, with alteration of their biological properties.
Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis
Authors: Bowman Tammy , Broome Martin A. , Sinibaldi Dominic , Wharton Walker , Pledger W. J. , Sedivy John M. , Irby Rosalyn , Yeatman Timothy , Courtneidge Sara A. , Jove Richard
Published: July 2002

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c
-myc
gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.
Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding
Authors: Chen Julian C.-H. , Krucinski Jolanta , Miercke Larry J. W. , Finer-Moore Janet S. , Tang Ann H. , Leavitt Andrew D. , Stroud Robert M.
Published: July 2002

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa α-helix, α6, links the C-terminal domain to the catalytic core. A kink in one of the two α6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein–DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52–210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52–288 structure.
Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II
Authors: Park Chan Bae , Yi Kwan-Su , Matsuzaki Katsumi , Kim Mi Sun , Kim Sun Chang
Published: July 2002

Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1–4), an extended helical region (residues 5–10), a hinge (residue 11), and a C-terminal regular α-helical region (residues 12–21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity ≈2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the α-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.
Identification and characterization of a mammalian protein interacting with 20S proteasome precursors
Authors: Burri Lena , Höckendorff Jörg , Boehm Ulrich , Klamp Thorsten , Dohmen R. Jürgen , Lévy Frédéric
Published: July 2002

The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalianUMP1is increased after IFN-γ treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.
Evidence from sequence-tagged-site markers of a recent progenitor-derivative species pair in conifers
Authors: Perron Martin , Perry Daniel J. , Andalo Christophe , Bousquet Jean
Published: July 2002

Black spruce (
Picea mariana
[B.S.P.] Mill.) and
red spruce (
Picea
rubens
Sarg.) are two
conifer species known to hybridize naturally in northeastern North
America. We hypothesized that there is a progenitor-derivative
relationship between these two taxa and conducted a genetic
investigation by using sequence-tagged-site markers of expressed genes.
Based on the 26 sequence-tagged-site loci assayed in this study, the
unbiased genetic identity between the two taxa was quite high with a
value of 0.920. The mean number of polymorphic loci, the mean number of
alleles per polymorphic locus, and the average observed heterozygosity
were lower in red spruce (
P
= 35%,
A
P
= 2.1,
H
o
= 0.069) than in black spruce (
P
= 54%,
A
P
= 2.9,
H
o
= 0.103). No unique alleles were found in red spruce, and the observed
patterns of allele distribution indicated that the genetic diversity of
red spruce was essentially a subset of that found in black spruce. When
considered in combination with ecological evidence and simulation
results, these observations clearly support the existence of a
progenitor-derivative relationship and suggest that the reduced level
of genetic diversity in red spruce may result from allopatric
speciation through glaciation-induced isolation of a preexisting black
spruce population during the Pleistocene era. Our observations signal a
need for a thorough reexamination of several conifer species complexes
in which natural hybridization is known to occur.
Mammalian Bax-induced plant cell death can be down-regulated by overexpression of
Arabidopsis Bax Inhibitor-1
(
AtBI-1
)
Authors: Kawai-Yamada Maki , Jin Lihua , Yoshinaga Keiko , Hirata Aiko , Uchimiya Hirofumi
Published: July 2002

We recently isolated the
AtBI-1
(
Arabidopsis Bax Inhibitor-1
) gene, the expression of which suppressed Bax-induced cell death in yeast. To determine whether the same is true in the plant system, transgenic
Arabidopsis
plants overexpressing Bax protein under a dexamethasone (DEX)-inducible promoter were generated. On DEX treatment, such transgenic plants exhibited marked cell death at the whole-plant level, cell shrinkage, membranous destruction, and other apoptotic phenotypes. Transgenic Bax plants were retransformed with a vector containing the
AtBI-1
gene (tagged with green fluorescent protein) under the control of the cauliflower mosaic virus 35S promoter. Plants expressing both
Bax
and
AtBI-1
were able to maintain growth on DEX-treatment by sustaining intracellular integrity. Thus, we present here direct genetic evidence that the plant antiapoptotic protein AtBI-1 is biologically active in suppressing the mammalian Bax action
in planta
.
Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers
Authors: Jäger Elke , Gnjatic Sacha , Nagata Yasuhiro , Stockert Elisabeth , Jäger Dirk , Karbach Julia , Neumann Antje , Rieckenberg Julia , Chen Yao-Tseng , Ritter Gerd , Hoffman Eric , Arand Michael , Old Lloyd J. , Knuth Alexander
Published: July 2002

Cancer–testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens defined to date. Spontaneous humoral and CD8+ T-cell responses to NY-ESO-1 are detected in 40–50% of patients with advanced NY-ESO-1-expressing tumors. A clinical trial was initiated to study the immunological effects of intradermal vaccination with 3 HLA-A2-binding NY-ESO-1 peptides in 12 patients with metastatic NY-ESO-1-expressing cancers. Seven patients were NY-ESO-1 serum antibody negative, and five patients were NY-ESO-1 serum antibody positive at the outset of the study. Primary peptide-specific CD8+ T-cell reactions and delayed-type hypersensitivity responses were generated in four of seven NY-ESO-1 antibody-negative patients. Induction of a specific CD8+ T-cell response to NY-ESO-1 in immunized antibody-negative patients was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive patients did not develop significant changes in baseline NY-ESO-1-specific T-cell reactivity. However, stabilization of disease and regression of individual metastases were observed in three of five immunized patients. These results demonstrate that primary NY-ESO-1-specific CD8+ T-cell responses can be induced by intradermal immunization with NY-ESO-1 peptides, and that immunization with NY-ESO-1 may have the potential to alter the natural course of NY-ESO-1-expressing tumors.
Enterocolitis induced by autoimmune targeting of enteric glial cells: A possible mechanism in Crohn's disease?
Authors: Cornet Anne , Savidge Tor C. , Cabarrocas Julie , Deng Wen-Lin , Colombel Jean-Frederic , Lassmann Hans , Desreumaux Pierre , Liblau Roland S.
Published: July 2002

Early pathological manifestations of Crohn's disease (CD) include vascular disruption, T cell infiltration of nerve plexi, neuronal degeneration, and induction of T helper 1 cytokine responses. This study demonstrates that disruption of the enteric glial cell network in CD patients represents another early pathological feature that may be modeled after CD8+T cell-mediated autoimmune targeting of enteric glia in double transgenic mice. Mice expressing a viral neoself antigen in astrocytes and enteric glia were crossed with specific T cell receptor transgenic mice, resulting in apoptotic depletion of enteric glia to levels comparable in CD patients. Intestinal and mesenteric T cell infiltration, vasculitis, T helper 1 cytokine production, and fulminant bowel inflammation were characteristic hallmarks of disease progression. Immune-mediated damage to enteric glia therefore may participate in the initiation and/or the progression of human inflammatory bowel disease.
Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1
Authors: Shen Yu , Luche Ralf , Wei Bo , Gordon Marcia L. , Diltz Curtis D. , Tonks Nicholas K.
Published: July 2002

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name,JnkStimulatoryPhosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.
Why fisheries collapse and what to do about it.
Authors: Roughgarden J , Smith F
Published: July 2002

With the collapse of fisheries in many parts of the world causing widespread economic harm, attention is focused on a possible cause and remedy of fishery collapse. Economic theory for managing a renewable resource, such as a fishery, leads to an ecologically unstable equilibrium as difficult to maintain as balancing a marble on top of a dome. A fishery should be managed for ecological stability instead--in the analogy, as easy to maintain as keeping a marble near the base of a bowl. The goal of ecological stability is achieved if the target stock is above that producing maximum sustainable yield and harvested at less than the maximum sustainable yield. The cost of managing for ecological stability, termed "natural insurance," is low if the fishery is sufficiently productive. This cost is shown to pay for itself over the long term in a variable and uncertain environment. An ecologically stable target stock may be attained either with annually variable quotas following current practice or, preferably, through a market mechanism whereby fish are taxed at dockside if caught when the stock was below target and are untaxed otherwise. In this regulatory environment, the goal of maximizing short-term revenue coincides with the goal of ecological stability, thereby also maximizing long-term revenue. This new approach to fishery management is illustrated with the recently collapsed Newfoundland fishing industry. The Newfoundland cod fishery is expected to rebuild to an ecologically stable level in about 9 years and thereafter support an annual harvest of about 75% of the 1981-1990 average.
The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.
Authors: Miller S G , De Vos P , Guerre-Millo M , Wong K , Hermann T , Staels B , Briggs M R , Auwerx J
Published: July 2002

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.
Formation of stable cationic lipid/DNA complexes for gene transfer.
Authors: Hofland H E , Shephard L , Sullivan S M
Published: July 2002

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.
Activation of muscarinic acetylcholine receptors via their allosteric binding sites.
Authors: Jakubík J , Bacáková L , Lisá V , el-Fakahany E E , Tucek S
Published: July 2002

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.
Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.
Authors: Kieran M W , Perkins A C , Orkin S H , Zon L I
Published: July 2002

The interaction of the hormone erythropoietin and its receptor (EpoR) is though to be required for normal hematopoiesis. To define the role of EpoR in this process, the murine EpoR was disrupted by homologous recombination. Mice lacking the EpoR died in utero at embryonic day 11-12.5 with severe anemia. Embryonic erythropoiesis was markedly diminished, while fetal liver hematopoiesis was blocked at the proerythroblast stage. Other cell types known to express EpoR, including megakaryocytes, mast, and neural cells were morphologically normal. Reverse transcription-coupled PCR analysis of RNA from embryonic yolk sac, peripheral blood, and fetal liver demonstrated near normal transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, GATA-1, GATA-2, and alpha- and embryonic beta H1-globin but non for adult beta maj-globin. While colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies were not present in cultures derived from EpoR-/- liver or yolk sac cells, hemoglobin-containing BFU-E colonies were detected in cultures treated with recombinant Tpo and Kit ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E colonies expressed adult beta-globin and c-MPL and appeared morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo receptor c-MPL stimulates proliferation and terminal differentiation of these progenitors in vitro.
Temperature dependence of the isotope chemistry of the heavy elements.
Authors: Bigeleisen J
Published: July 2002

The temperature coefficient of equilibrium isotope fractionation in the heavy elements is shown to be larger at high temperatures than that expected from the well-studied vibrational isotope effects. The difference in the isotopic behavior of the heavy elements as compared with the light elements is due to the large nuclear isotope field shifts in the heavy elements. The field shifts introduce new mechanisms for maxima, minima, crossovers, and large mass-independent isotope effects in the isotope chemistry of the heavy elements. The generalizations are illustrated by the temperature dependence of the isotopic fractionation in the redox reaction between U(VI) and U(IV) ions.
Paternal and maternal DNA lineages reveal a bottleneck in the founding of the Finnish population.
Authors: Sajantila A , Salem A H , Savolainen P , Bauer K , Gierig C , Pääbo S
Published: July 2002

An analysis of Y-chromosomal haplotypes in several European populations reveals an almost monomorphic pattern in the Finns, whereas Y-chromosomal diversity is significantly higher in other populations. Furthermore, analyses of nucleotide positions in the mitochondrial control region that evolve slowly show a decrease in genetic diversity in Finns. Thus, relatively few men and women have contributed the genetic lineages that today survive in the Finnish population. This is likely to have caused the so-called "Finnish disease heritage"-i.e., the occurrence of several genetic diseases in the Finnish population that are rare elsewhere. A preliminary analysis of the mitochondrial mutations that have accumulated subsequent to the bottleneck suggests that it occurred about 4000 years ago, presumably when populations using agriculture and animal husbandry arrived in Finland.
An essential role for p300/CBP in the cellular response to hypoxia
Authors: Arany Zoltàn , Huang L. Eric , Eckner Richard , Bhattacharya Shoumo , Jiang Chian , Goldberg Mark A. , Bunn H. Franklin , Livingston David M.
Published: July 2002

p300 and CBP are homologous transcription adapters targeted by the
E1A oncoprotein. They participate in numerous biological processes,
including cell cycle arrest, differentiation, and transcription
activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How
they participate in these processes is not yet known. In a search for
specific p300 binding proteins, we have cloned the intact cDNA for
HIF-1α. This transcription factor mediates hypoxic induction of genes
encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular
endothelial growth factor. Hypoxic conditions lead to the formation of
a DNA binding complex containing both HIF-1α and p300/CBP.
Hypoxia-induced transcription from the Epo promoter was specifically
enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP.
Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by
E1A. Hence, p300/CBP–HIF complexes participate in the induction of
hypoxia-responsive genes, including one (vascular endothelial growth
factor) that plays a major role in tumor angiogenesis. Paradoxically,
these data, to our knowledge for the first time, suggest that
p300/CBP are active in both transformation suppression and tumor
development.
Constitutive expression of the cold-regulated
Arabidopsis thaliana COR15a
gene affects both chloroplast and protoplast freezing tolerance
Authors: Artus Nancy N. , Uemura Matsuo , Steponkus Peter L. , Gilmour Sarah J. , Lin Chentao , Thomashow Michael F.
Published: July 2002

Cold acclimation in plants is associated with the expression of
COR
(cold-regulated) genes that encode polypeptides of
unknown function. It has been widely speculated that products of these
genes might have roles in freezing tolerance. Here we provide direct
evidence in support of this hypothesis. We show that constitutive
expression of
COR15a
, a cold-regulated gene of
Arabidopsis thaliana
that encodes a chloroplast-targeted
polypeptide, enhances the
in vivo
freezing tolerance of
chloroplasts in nonacclimated plants by almost 2°C, nearly one-third
of the increase that occurs upon cold acclimation of wild-type plants.
Significantly, constitutive expression of
COR15a
also
affects the
in vitro
freezing tolerance of protoplasts.
At temperatures between −5 and −8°C, the survival of protoplasts
isolated from leaves of nonacclimated transgenic plants expressing
COR15a
was greater than that of protoplasts isolated
from leaves of nonacclimated wild-type plants. At temperatures between
−2 and −4°C, constitutive expression of
COR15a
had a
slight negative effect on survival. The implications of these data
regarding possible modes of
COR15a
action are discussed.
Yeast homologues of higher eukaryotic TFIID subunits
Authors: Moqtaderi Zarmik , Yale Jacqueline DePaulo , Struhl Kevin , Buratowski Stephen
Published: July 2002

In eukaryotic cells the TATA-binding protein (TBP) associates with
other proteins known as TBP-associated factors (TAFs) to form
multisubunit transcription factors important for gene expression by all
three nuclear RNA polymerases. Computer searching of the complete
Saccharomyces cerevisiae
genome revealed five previously
unidentified yeast genes with significant sequence similarity to known
human and
Drosophila
RNA polymerase II TAFs. Each of
these genes is essential for viability. A sixth essential gene
(
FUN81
) has previously been noted to be similar to human
TAF
II
18. Coimmunoprecipitation experiments show that all
six proteins are associated with TBP, demonstrating that they are true
TAFs. Furthermore, these proteins are present in complexes containing
the TAF
II
130 subunit, indicating that they are components
of TFIID. Based on their predicted molecular weights, these genes have
been designated
TAF67
,
TAF61(68)
,
TAF40
,
TAF23(25)
,
TAF19
(
FUN81
), and
TAF17
.
Yeast TAF61 is significantly larger than its higher eukaryotic
homologues, and deletion analysis demonstrates that the evolutionarily
conserved, histone-like domain is sufficient and necessary to support
viability.
TransfectedDrosophilacells as a probe for defining the minimal requirements for stimulating unprimed CD8+T cells
Authors: Cai Zeling , Brunmark Anders , Jackson Michael R. , Loh Dennis , Peterson Per A. , Sprent Jonathan
Published: July 2002

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfectedDrosophilacells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfectedDrosophilaAPC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+Drosophilacells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. TransfectedDrosophilacells are thus a useful tool for examining the minimal APC requirements for naive T cells.
The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.
Authors: Beyer R E , Segura-Aguilar J , Di Bernardo S , Cavazzoni M , Fato R , Fiorentini D , Galli M C , Setti M , Landi L , Lenaz G
Published: July 2002

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.
Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.
Authors: Fakhrai H , Dorigo O , Shawler D L , Lin H , Mercola D , Black K L , Royston I , Sobol R E
Published: July 2002

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.
Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.
Authors: Missale C , Boroni F , Sigala S , Buriani A , Fabris M , Leon A , Dal Toso R , Spano P
Published: July 2002

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.
Place cells and place navigation
Authors: Bures J. , Fenton A. A. , Kaminsky Yu. , Zinyuk L.
Published: July 2002

The assumption that hippocampal place cells (PCs) form the neural substrate of cognitive maps can be experimentally tested by comparing the effect of experimental interventions on PC activity and place navigation. Conditions that interfere with place navigation (darkness, cholinergic blockade) but leave PC activity unaffected obviously disrupt spatial memory at a post-PC level. Situations creating a conflict between egocentric and allocentric orientation (place navigation in the Morris water maze filled with slowly rotating water) slow down spatial learning. PC recording in rats searching food pellets in a rotating arena makes it possible to determine which firing fields are stable relative to the room (allocentrically dependent on sighted extramaze landmarks), to the surface of the arena (dependent on egocentric path integration mechanisms and intra-arena cues), or disappear during rotation. Such comparison is made possible by the computerized tracking system simultaneously displaying a rat’s locomotion and the respective firing rate maps both in the room reference and arena reference frames. More severe conflict between allocentric and egocentric inputs is produced in the field clamp situation when the rat searching food in a ring-shaped arena is always returned by rotation of the arena to the same allocentric position. Ten-minute exposure to this condition caused subsequent disintegration or remapping of 70% PCs (
n
= 100). Simultaneous examination of PC activity and navigation is possible in the place avoidance task. A rat searching food in a stationary or rotating arena learns to avoid an allocentrically or egocentrically defined location where it receives mild electric footshock. In the place preference task the rat releases pellet delivery by entering an unmarked goal area and staying in it for a criterion time. Both tasks allow direct comparison of the spatial reference frames used by the PCs and by the behaving animal.