bluuebunny/crossref_metadata_2025 huggingface dataset:
BEN/SC1/DM-GRASP, a homophilic adhesion molecule, is required for in vitro myeloid colony formation by avian hemopoietic progenitors.
Authors: Corbel C , Pourquié O , Cormier F , Vaigot P , Le Douarin N M
Published: July 2002

BEN/SC1/DM-GRASP is a membrane glycoprotein of the immunoglobulin superfamily isolated in the chick by several groups, including ours. Its expression is strictly developmentally regulated in several cell types of the nervous and hemopoietic systems and in certain epithelia. Each of these cell types expresses isoforms of BEN which differ by their level of N-glycosylation and by the presence or absence of the HNK-1 carbohydrate epitope. In the present work, the influence of glycosylation on BEN homophilic binding properties was investigated by two in vitro assays. First, each BEN isoform was covalently coupled to microspheres carrying different fluorescent dyes and an aggregation test was performed. We found that homophilic aggregates form indifferently between the same or different BEN isoforms, showing that glycosylation does not affect BEN homophilic binding properties. This was confirmed in the second test, where the BEN-coated microspheres bound to the neurites of BEN- expressing neurons, irrespective of the isoform considered. The transient expression of the BEN antigen on hemopoietic progenitors prompted us to see whether it might play a role in their proliferation and differentiation. When added to hemopoietic progenitor cells in an in vitro colony formation assay anti-BEN immunoglobulin strongly inhibited myeloid, but not erythroid, colony formation although both types of precursors express the molecule.
Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity.
Authors: Mizuno T M , Bergen H , Funabashi T , Kleopoulos S P , Zhong Y G , Bauman W A , Mobbs C V
Published: July 2002

Mutations in the obese (ob) gene lead to obesity. This gene has been recently cloned, but the factors regulating its expression have not been elucidated. To address the regulation of the ob gene with regard to body weight and nutritional factors, Northern blot analysis was used to assess ob mRNA in adipose tissue from mice [lean, obese due to diet, or genetically (yellow agouti) obese] under different nutritional conditions. ob mRNA was elevated in both forms of obesity, compared to lean controls, correlated with elevations in plasma insulin and body weight, but not plasma glucose. In lean C57BL/6J mice, but not in mice with diet-induced obesity, ob mRNA decreased after a 48-hr fast. Similarly, in lean C57BL/6J controls, but not in obese yellow mice, i.p. glucose injection significantly increased ob mRNA. For up to 30 min after glucose injection, ob mRNA in lean mice significantly correlated with plasma glucose, but not with plasma insulin. In a separate study with only lean mice, ob mRNA was inhibited >90% by fasting, and elevated approximately 2-fold 30 min after i.p. injection of either glucose or insulin. These results suggest that in lean animals glucose and insulin enhance ob gene expression. In contrast to our results in lean mice, in obese animals ob mRNA is elevated and relatively insensitive to nutritional state, possibly due to chronic exposure to elevated plasma insulin and/or glucose.
Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.
Authors: Malmström V , Michaëlsson E , Burkhardt H , Mattsson R , Vuorio E , Holmdahl R
Published: July 2002

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.
In vitro
motility of AtKCBP, a calmodulin-binding kinesin protein of
Arabidopsis
Authors: Song Hebok , Golovkin Maxim , Reddy A. S. N. , Endow Sharyn A.
Published: July 2002

AtKCBP is a calcium-dependent calmodulin-binding protein from
Arabidopsis
that contains a conserved kinesin microtubule motor domain. Calmodulin has been shown previously to bind to heavy chains of the unconventional myosins, where it is required for
in vitro
motility of brush border myosin I, but AtKCBP is the first kinesin-related heavy chain reported to be capable of binding specifically to calmodulin. Other kinesin proteins have been identified in
Arabidopsis
, but none of these binds to calmodulin, and none has been demonstrated to be a microtubule motor. We have tested bacterially expressed AtKCBP for the ability to bind microtubules to a glass surface and induce gliding of microtubules across the glass surface. We find that AtKCBP is a microtubule motor protein that moves on microtubules toward the minus ends, with the opposite polarity as kinesin. In the presence of calcium and calmodulin, AtKCBP no longer binds microtubules to the coverslip surface. This contrasts strikingly with the requirement of calmodulin for
in vitro
motility of brush border myosin I. Calmodulin could regulate AtKCBP binding to microtubules in the cell by inhibiting the binding of the motor to microtubules. The ability to bind to calmodulin provides an evolutionary link between the kinesin and myosin motor proteins, but our results indicate that the mechanisms of interaction and regulation of kinesin and myosin heavy chains by calmodulin are likely to differ significantly.
Developmental parameters of cell death in the wing disc of
Drosophila
Authors: Milán M. , Campuzano S. , García-Bellido A.
Published: July 2002

Apoptotic cell death in wing imaginal discs takes place in single cells or small clusters of neighboring cells. These cells are distributed throughout the anlage at early stages and in recognizable territories at late larval and pupal stages. Apoptotic cells remain in the epithelium 2–4 h, prior to being engulfed in place by hemolymph cells. Experimentally induced apoptosis in single cells or territories is accompanied by nonautonomous death of adjacent cells and of cells further away in adjacent territories. These effects are followed by changes in cell proliferation in both territories. Apogenetic mosaics in mutant discs show cell death throughout the anlage. Apoptosis provides a mechanism, in addition to cell proliferation control, for matching territories with different positional values or different genetic specifications.
Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells
Authors: Moser Tobias , Neher Erwin
Published: July 2002

Whole-cell membrane capacitance measurements are frequently used to monitor neuronal and nonneuronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization-induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles between 0.6 and 2 fF. For comparison, measurements of membrane capacitance were combined with amperometric recordings of catecholamine release during intracellular perfusion of chromaffin cells with high [Ca
2+
]. Crosscorrelation of both signals yielded a mean capacitance contribution of individual catecholaminergic vesicles of 1.3 fF. We suggest that depolarization-induced capacitance increases in mouse adrenal chromaffin cells mainly represent fusion of chromaffin granules.
Ligand binding was acquired during evolution of nuclear receptors
Authors: Escriva Hector , Safi Rachid , Hänni Catherine , Langlois Marie-Claire , Saumitou-Laprade Pierre , Stehelin Dominique , Capron André , Pierce Raymond , Laudet Vincent
Published: July 2002

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution.
Extracellular calcium sensed by a novel cation channel in hippocampal neurons
Authors: Xiong Z.-G. , Lu W.-Y. , MacDonald J. F.
Published: July 2002

Extracellular concentrations of Ca
2+
change rapidly and transiently in the brain during excitatory synaptic activity. To test whether such changes in Ca
2+
can play a signaling role we examined the effects of rapidly lowering Ca
2+
on the excitability of acutely isolated CA1 and cultured hippocampal neurons. Reducing Ca
2+
excited and depolarized neurons by activating a previously undescribed nonselective cation channel. This channel had a single-channel conductance of 36 pS, and its frequency of opening was inversely proportional to the concentration of Ca
2+
. The inhibition of gating of this channel was sensitive to ionic strength but independent of membrane potential. The ability of this channel to sense Ca
2+
provides a novel mechanism whereby neurons can respond to alterations in the extracellular concentration of this key signaling ion.
Identification and characterization of
Saccharomyces cerevisiae EXO1
, a gene encoding an exonuclease that interacts with MSH2
Authors: Tishkoff Daniel X. , Boerger Adrienne L. , Bertrand Pascale , Filosi Nicole , Gaida Gretchen M. , Kane Michael F. , Kolodner Richard D.
Published: July 2002

A two-hybrid screen was used to identify
Saccharomyces cerevisiae
genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of
Schizosaccharomyces pombe
EXO1, a double-stranded DNA-specific 5′–3′ exonuclease.
S. cerevisiae
EXO1 interacted with both
S. cerevisiae
and human MSH2 in two-hybrid and coimmunoprecipitation experiments.
exo1
mutants showed a mutator phenotype, and epistasis analysis was consistent with
EXO1
functioning in the
MSH2
-dependent mismatch repair pathway.
exo1
mutations were lethal in combination with
rad27
mutations, and overexpression of
EXO1
suppressed both the temperature sensitive and mutator phenotypes of
rad27
mutants.
Functional impact of cerebral connections
Authors: Vanduffel Wim , Payne Bertram R. , Lomber Stephen G. , Orban Guy A.
Published: July 2002

Cerebral networks are complex sets of connections that resemble a ladder-like web of multiple parallel feedforward, lateral, and feedback connections. This static anatomical description has been pivotal in guiding our understanding of signal processing within cerebral networks. However, measures on both magnitude and functional significance of connections are extremely limited. Here, we compare the anatomically defined strengths of a set of cerebral pathways emerging from the visual middle suprasylvian (MS) cortex of the cat with measures of the functional impact the same region has over distant sites. These functional measures were obtained by analyzing the local and distant effects of MS cooling deactivation on deoxyglucose uptake. Relative to major efferent projections from MS cortex that have a strong influence, projections to early visual processing stages have weaker functional influences than predicted from the anatomy. For higher processing stages, the converse holds: projections from MS cortex have stronger functional influence than predicted from the anatomy. We conclude that these and future functional measures, obtained using the same combination of techniques, will furnish fundamental, new information that complements and extends current models of static cerebral networks, and lead to more realistic models of cerebral network function and component interactions.
Interaction between amyloid precursor protein and presenilins in mammalian cells: Implications for the pathogenesis of Alzheimer disease
Authors: Xia Weiming , Zhang Jimin , Perez Ruth , Koo Edward H. , Selkoe Dennis J.
Published: July 2002

Mutations in the
presenilin 1
(
PS1
) and
presenilin 2
(
PS2
) genes increase the production of the highly amyloidogenic 42-residue form of amyloid β-protein (Aβ
42
) in a variety of cell lines and transgenic mice. To elucidate the molecular mechanism of this effect, wild-type (wt) or mutant
PS1
and
PS2
genes were stably transfected into Chinese hamster ovary cells expressing endogenous or transfected β-amyloid precursor protein (APP). By immunoprecipitation/Western blot analysis, APP was consistently found to coimmunoprecipitate with PS1 or PS2 proteins. Several distinct PS1, PS2, or APP antibodies precipitated PS–APP complexes that were detectable by blotting with either APP or PS antibodies. Importantly, complex formation could be detected at endogenous protein levels in nontransfected cells. In various Chinese hamster ovary cell lines, the amounts of APP coprecipitated by PS antibodies were proportional to the expression levels of both APP and PS. APP–PS complexes also were recovered from human 293 and HS683 cells. Full maturation of APP was not required for the interaction; most APP molecules complexed with PS were solely N-glycosylated. Treatment of cells with brefeldin A or incubation at 20°C did not block complex formation, suggesting that the association between APP and PS occurs in part in the endoplasmic reticulum. Complex formation was detected for both wt and mutant PS and APP proteins. Deletion of the APP C-terminal domain did not abrogate complex formation, suggesting that the interaction does not occur in the cytoplasmic domains of the proteins. Our results demonstrate that wt and mutant PS1 and PS2 proteins form complexes with APP in living cells, strongly supporting the hypothesis that mutant PS interacts with APP in a way that enhances the intramembranous proteolysis of the latter by a γ-secretase cleaving at Aβ
42
.
β-Catenin mutations in cell lines established from human colorectal cancers
Authors: Ilyas M. , Tomlinson I. P. M. , Rowan A. , Pignatelli M. , Bodmer W. F.
Published: July 2002

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (
CTNNB1
) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with
CTNNB1
mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of
CTNNB1
mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to
APC
mutations in the early stages of tumorigenesis.
A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak
Authors: Cheng Emily H.-Y. , Nicholas John , Bellows David S. , Hayward Gary S. , Guo Hong-Guang , Reitz Marvin S. , Hardwick J. Marie
Published: July 2002

The Bcl-2 protein family is characterized by the ability to modulate cell death, and members of this family share two highly conserved domains called Bcl-2 homology 1 (BH1) and 2 (BH2) which have been shown to be critical for the death-repressor activity of Bcl-2 and Bcl-x
L
. Through sequence analysis we identified a novel viral Bcl-2 homolog, designated KSbcl-2, from human herpesvirus 8 (HHV8) or Kaposi sarcoma-associated herpesvirus. The overall amino acid sequence identity between KSbcl-2 and other Bcl-2 homologs is low (15–20%) but concentrated within the BH1 and BH2 regions. Overexpression of KSbcl-2 blocked apoptosis as efficiently as Bcl-2, Bcl-x
L
, or another viral Bcl-2 homolog encoded by Epstein–Barr virus, BHRF1. Interestingly, KSbcl-2 neither homodimerizes nor heterodimerizes with other Bcl-2 family members, suggesting that KSbcl-2 may have evolved to escape any negative regulatory effects of the cellular Bax and Bak proteins. Furthermore, the herpesvirus Bcl-2 homologs including KSbcl-2, BHRF1, and ORF16 of herpesvirus saimiri contain poorly conserved Bcl-2 homology 3 (BH3) domains compared with other mammalian Bcl-2 homologs, implying that BH3 may not be essential for anti-apoptotic function. This is consistent with our observation that amino acid substitutions within the BH3 domain of Bcl-x
L
had no effect on its death-suppressor activity.
Design of compounds that increase the absorption of polar molecules
Authors: Bowe Caryn Lang , Mokhtarzadeh Leila , Venkatesan Pushpa , Babu Suresh , Axelrod Helena R. , Sofia Michael J. , Kakarla Ramesh , Chan Tin Yau , Kim Jae Seung , Lee Hye J. , Amidon Gordon L. , Choe Sally Y. , Walker Suzanne , Kahne Daniel
Published: July 2002

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.
Degeneration of neurons, synapses, and neuropil and glial activation in a murine
Atm
knockout model of ataxia–telangiectasia
Authors: Kuljis Rodrigo O. , Xu Yang , Aguila M. Cecilia , Baltimore David
Published: July 2002

Neural degeneration is one of the clinical manifestations of ataxia–telangiectasia, a disorder caused by mutations in the Atm protein kinase gene. However, neural degeneration was not detected with general purpose light microscopic methods in previous studies using several different lines of mice with disrupted
Atm
genes. Here, we show electron microscopic evidence of degeneration of several different types of neurons in the cerebellar cortex of 2-month-old
Atm
knockout mice, which is accompanied by glial activation, deterioration of neuropil structure, and both pre- and postsynaptic degeneration. These findings are similar to those in patients with ataxia–telangiectasia, indicating that
Atm
knockout mice are a useful model to elucidate the mechanisms underlying neurodegeneration in this condition and to develop and test strategies to palliate and prevent the disease.
Inactivation of the α C protein antigen gene,bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus
Authors: Li Jing , Kasper Dennis L. , Ausubel Frederick M. , Rosner Bernard , Michel James L.
Published: July 2002

The α C protein of group BStreptococcus(GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein,bca, has been cloned and sequenced. The protein encoded bybcais related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein,bcanull mutants were generated in which thebcagene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of thebcanull mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909,bcamutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in anin vitroopsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge withbcamutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from thebcagene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.
A homeodomain gene
Ptx
3 has highly restricted brain expression in mesencephalic dopaminergic neurons
Authors: Smidt Marten P. , van Schaick Hermien S. A. , Lanctôt Christian , Tremblay Jacques J. , Cox Joke J. , van der Kleij Arno A. M. , Wolterink Gerrit , Drouin Jacques , Burbach J. Peter H.
Published: July 2002

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene,
Ptx3
, a member of the
Ptx
-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of
Ptx3
-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxy-dopamine-lesioned rats, an animal model for this disease. Thus,
Ptx3
is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.
Bidirectional binding of the TATA box binding protein to the TATA box
Authors: Cox Julia M. , Hayward Matthew M. , Sanchez Jennifer F. , Gegnas Laura D. , van der Zee Sarina , Dennis Jonathan H. , Sigler Paul B. , Schepartz Alanna
Published: July 2002

By selective attachment of a DNA cleavage agent to specific
residues in the yeast TATA box binding protein (yTBP), we demonstrate
that, in solution, yTBP binds to the TATA boxes of both the adenovirus
major late promoter and the yeast
CYC1
promoter with
only a modest preference in orientation and binds well to several
overlapping binding sites. The general factors TFIIA and TFIIB each
increase the rotational and translational selectivity of yTBP but are
not sufficient, at least individually, to confer a unique polarity to
the preinitiation complex. We conclude that TBP alone cannot define the
productive orientation of general factor assembly on a promoter.
Large conductance voltage- and calcium-dependent K
+
channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus
Authors: Meera Pratap , Wallner Martin , Song Min , Toro Ligia
Published: July 2002

Large conductance voltage- and Ca
2+
-dependent K
+
(MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels,
in vitro
translation, functional, and “
in vivo
” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.
Ca
2+
/calmodulin-binding peptides block phototransduction in
Limulus
ventral photoreceptors: Evidence for direct inhibition of phospholipase C
Authors: Richard Edwin A. , Ghosh Smita , Lowenstein John M. , Lisman John E.
Published: July 2002

Phototransduction in
Limulus
photoreceptors involves a G protein-mediated activation of phospholipase C (PLC) and subsequent steps involving InsP
3
-mediated release of intracellular Ca
2+
. While exploring the role of calmodulin in this cascade, we found that intracellular injection of Ca
2+
/calmodulin-binding peptides (CCBPs) strongly inhibited the light response. By chemically exciting the cascade at various stages, we found the primary target of this effect was not in late stages of the cascade but rather at the level of G protein and PLC. That PLCδ
1
contains a calmodulin-like structure raised the possibility that PLC might be directly affected by CCBPs. To test this possibility,
in vitro
experiments were conducted on purified PLC. The activity of this enzyme was strongly inhibited by CCBPs and also inhibited by calmodulin itself. Our results suggest that the calmodulin-like region of PLC has an important role in regulating this enzyme.
Ca
2+
-dependent and -independent interactions of the isoforms of the α
1A
subunit of brain Ca
2+
channels with presynaptic SNARE proteins
Authors: Kim D. Kyle , Catterall William A.
Published: July 2002

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α
1A
subunit of P/Q-type Ca
2+
channels have different patterns of interactions with synaptic proteins. The BI isoform of α
1A
specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca
2+
concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α
1A
interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α
1A
is Ca
2+
-dependent, with maximum affinity at 10–20 μM Ca
2+
. Although the rbA isoform of α
1A
binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca
2+
-dependent. These differential, Ca
2+
-dependent interactions of Ca
2+
channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca
2+
influx through these channels.
Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: Evidence for a novel oxidative pathway
Authors: Sugiyama Katsumi , Highet Robert J. , Woods Amina , Cotter Robert J. , Osawa Yoichi
Published: July 2002

Treatment of metmyoglobin with H2O2is known to lead to the crosslinking of an active site tyrosine residue to the heme [Catalano, C. E., Y. S. Choe, and P. R. Ortiz de Montellano (1989)J. Biol. Chem.264, 10534–10541]. We have found in this study that this reaction also leads to an altered heme product not covalently bound to the protein. This product was characterized by visible absorption, infrared absorption, and mass and NMR spectrometry as an iron chlorin product formed from the saturation of the double bond between carbon atoms at positions 17 and 18 of pyrrole ring D with concomitant addition of a hydroxyl group on the carbon atom at position 18 and lactonization of the propionic acid to the carbon atom at position 17. Studies with the use of18O-labeled H2O2, O2, and H2O clearly indicate that the source of the added oxygen on the heme is water. Evidently, water adds regiospecifically to a cationic site formed on a carbon atom at position 18 after oxidation of the ferric heme prosthetic group with peroxide. Prolonged incubation of the reaction mixture containing the iron hydroxychlorin product led to the formation of an iron dihydroxychlorin product, presumably from a slow addition of water to the initial iron hydroxychlorin. The iron chlorin products characterized in this study are distinct from the meso-oxyheme species, which is thought to be formed during peroxide-mediated degradation of metmyoglobin, cytochrome P450, ferric heme, and model ferric hemes, and give further insight into the mechanism of H2O2-induced heme alterations.
Conformation-dependent phosphorylation of p53
Authors: Adler Victor , Pincus Matthew R. , Minamoto Toshinari , Fuchs Serge Y. , Bluth Mark J. , Brandt-Rauf Paul W. , Friedman Fred K. , Robinson Richard C. , Chen James M. , Wang Xin Wei , Harris Curtis C. , Ronai Ze’ev
Published: July 2002

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione
S
-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 (
His
p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and β subunit of casein kinase II (CKIIβ) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length
His
p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53
wt
) undergoes better phosphorylation by CKIIβ and by WCE kinases than mutant forms of p53 at amino acid 249 (p53
249
) or 273 (p53
273
). Moreover, shifting the kinase reaction’s temperature from 37°C to 18°C reduces the phosphorylation of mutant p53 to a greater extent than of p53
wt
. Comparing truncated forms of p53 revealed that the ability of CKIIβ, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97–155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97–117 that increase p53 phosphorylation by CKIIβ while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53
249
exhibits significant changes to the conformation of p53 within amino acids 97–117. In summary, phosphorylation-related analysis of different p53 forms
in vitro
indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.
Anti-viral protection conferred by recombinant adenylate cyclase toxins fromBordetella pertussiscarrying a CD8+T cell epitope from lymphocytic choriomeningitis virus
Authors: Saron M. F. , Fayolle C. , Sebo P. , Ladant D. , Ullmann A. , Leclerc C.
Published: July 2002

The elucidation of the mechanisms of antigen presentation by major histocompatibility complex class I molecules has stimulated the search for nonreplicative vectors that could deliver CD8+T cell epitopes to the cytosol of antigen-presenting cells to trigger the activation of specific cytotoxic T lymphocytes (CTLs)in vivo. In the present study, we investigated the potential ability of an invasive adenylate cyclase toxin fromBordetella pertussis, carrying a CD8+T cell epitope from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), to stimulate protective anti-viral immunity. Mice immunized with this recombinant toxin developed strong CTL responses against LCMV-infected target cells. Moreover, these mice were protected against an intracerebral challenge with a virulent strain of LCMV that killed all nonimmunized mice within 7 days. This protection was abolished afterin vivoelimination of CD8+T cells. A mutant toxin devoid of adenylate cyclase activity (i.e., cAMP synthesizing activity) was constructed by insertion of a dipeptide into the catalytic site of the molecule. This genetically detoxified invasive toxin carrying the LCMV epitope stimulated a strong CTL response against both peptide-coated and virus-infected target cells, and mice immunized with this molecule were fully protected against a lethal intracerebral LCMV challenge. To our knowledge, this study represents the first demonstration that a genetically detoxified bacterial toxin carrying a viral CTL epitope can stimulate efficient protective immunity.
A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway
Authors: Juilfs Dawn M. , Fülle Hans-Jürgen , Zhao Allan Z. , Houslay Miles D. , Garbers David L. , Beavo Joseph A.
Published: July 2002

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. We have found a subset of olfactory neurons that express the cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D (GC-D), suggesting that cGMP in these neurons also can have an important regulatory function in olfactory signaling. PDE2 and GC-D are both expressed in olfactory cilia where odorant signaling is initiated; however, only PDE2 is expressed in axons. In contrast to most other olfactory neurons, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb that are similar to the subset that have been termed “necklace glomeruli.” Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulin-dependent PDE (PDE1C2), adenylyl cyclase III, and cAMP-specific PDE (PDE4A). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distribution is distinct. PDE1C2 and adenylyl cyclase III are expressed almost exclusively in the olfactory cilia whereas PDE4A is present only in the cell bodies and axons. These data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in olfactory neurons and likely in other neurons as well. In addition, the data implies that an olfactory signal transduction pathway specifically modulated by cGMP is present in some neurons of the olfactory neuroepithelium.
Genotype determining low catechol-O-methyltransferase activity as a risk factor for obsessive-compulsive disorder
Authors: Karayiorgou Maria , Altemus Margaret , Galke Brandi L. , Goldman David , Murphy Dennis L. , Ott Jurg , Gogos Joseph A.
Published: July 2002

In the present study, we address the role of the gene for catechol-O-methyltransferase (COMT), a key modulator of dopaminergic and noradrenergic neurotransmission, in the genetic predisposition to obsessive-compulsive disorder (OCD). We show that a common functional allele of this gene, which results in a 3- to 4-fold reduction in enzyme activity, is significantly associated in a recessive manner with susceptibility to OCD, particularly in males. This association is further supported by psychiatric evaluation of patients who carry microdeletions encompassing thecomtgene. The mechanism underlying this sex-selective association remains to be defined and may include a sexual dimorphism in COMT activity, although close linkage with a nearby disease susceptibility locus cannot be excluded at this point.
Fertilization antigen-1: cDNA cloning, testis-specific expression, and immunocontraceptive effects
Authors: Zhu Xiaolong , Naz Rajesh K.
Published: July 2002

cDNA encoding for a sperm antigen, designated fertilization antigen (FA-1), was cloned and sequenced from murine testis cDNA-λgt11 expression library using FA-1 mAb. Computer-generated translation analysis of 649-bp cDNA yielded an ORF of 164 amino acids with the first ATG Met start codon at nucleotide 81 and the stop codon TAA at nucleotide 577 and a polyadenylylation tail following the stop codon. The translated protein has a calculated molecular mass of 18.2 kDa and a potential N-linked glycosylation site at amino acids 158–160, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced amino acid sequence indicated it to be a membrane-anchored peptide. Extensive computer search in the GenBank, National Biomedical Research Foundation, and Swiss sequence banks did not identify any known nucleotide/amino acid sequence having homology with FA-1 cDNA or deduced amino acids, indicating it to be a novel protein. Northern blot analysis and reverse transcription–PCR indicated testis-specific expression of FA-1 antigen. The FA-1 cDNA was subcloned into pGEX-2T vector and expressed in glutathione
S
-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with ZP3 of oocyte zona pellucida and its affinity-purified antibodies completely blocked sperm–zona pellucida interaction in mice. These findings suggest that the sperm-specific recombinant FA-1 antigen is an attractive candidate for the development of a contraceptive vaccine.
The chromatin unfolding domain of chromosomal protein HMG-14 targets the N-terminal tail of histone H3 in nucleosomes
Authors: Trieschmann Lothar , Martin Brian , Bustin Michael
Published: July 2002

Nonhistone chromosomal protein HMG-14 is a nucleosomal binding protein that unfolds the higher-order chromatin structure and enhances the transcriptional potential of chromatin, but not that of DNA. Both the transcriptional enhancement and the chromatin unfolding activities of HMG-14 are mediated through the C-terminal region of the protein. Here we study the molecular interactions of both this region and the N-terminal region of HMG-14 with nucleosome cores. By protein photocrosslinking we demonstrate that the N-terminal domain of HMG-14 targets a restricted region in histone H2B, whereas the C-terminal chromatin unfolding domain of HMG-14 targets a restricted region in the N terminus of histone H3. The N-terminal regions of the core histones are involved in the folding of oligonucleosomes and are the target of various activities associated with chromatin unfolding and transcriptional activation. We suggest that specific interactions between the C-terminal domain of HMG-14 and the N-terminal tail of histone H3 reduce the compaction of chromatin. These findings provide insights into the molecular mechanism whereby HMG-14/-17 proteins reduce the repressive effect of chromatin, and they also broaden the scope of the molecular interactions involving the N termini of the core histones in nucleosomes.
Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells
Authors: Gong Jianlin , Chen Dongshu , Kashiwaba Masahiro , Li Yongqing , Chen Ling , Takeuchi Hideya , Qu Hui , Rowse Gerald J. , Gendler Sandra J. , Kufe Donald
Published: July 2002

Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumor-associated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinoma-associated antigen in MUC1 transgenic mice unresponsive to MUC1 antigen. Immunization of adult MUC1 transgenic mice with irradiated MUC1-positive tumor cells was unsuccessful in reversing unresponsiveness to MUC1. By contrast, fusions of dendritic cells with MUC1-positive tumor cells induced cellular and humoral immunity against MUC1. Immunization with the dendritic cell fusions that express MUC1 resulted in the rejection of established metastases and no apparent autoimmunity against normal tissues. These findings demonstrate that unresponsiveness to the MUC1 tumor-associated antigen is reversible by immunization with heterokaryons of dendritic cells and MUC1-positive carcinoma cells.
Water does not flow across the tight junctions of MDCK cell epithelium
Authors: Kovbasnjuk Olga , Leader John P. , Weinstein Alan M. , Spring Kenneth R.
Published: July 2002

Although it has been known for decades that the tight junctions of fluid-transporting epithelia are leaky to ions, it has not been possible to determine directly whether significant transjunctional water movement also occurs. An optical microscopic technique was developed for the direct visualization of the flow velocity profiles within the lateral intercellular spaces of a fluid-absorptive, cultured renal epithelium (MDCK) and used to determine the velocity of the fluid flow across the tight junction. The flow velocity within the lateral intercellular spaces fell to near zero adjacent to the tight junction, showing that significant transjunctional flow did not occur, even when transepithelial fluid movement was augmented by imposition of osmotic gradients.
Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia
Authors: Aronica Eleonora M. , Gorter Jan A. , Grooms Sonja , Kessler John A. , Bennett Michael V. L. , Zukin R. Suzanne , Rosenbaum Daniel M.
Published: July 2002

Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other protein–nucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca
2+
influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca
2+
permeable, leads to excessive Ca
2+
influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca
2+
influx initiates apoptosis. Maintaining formation of Ca
2+
impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection.
Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways
Authors: Jiang Yi Wei , Veschambre Philippe , Erdjument-Bromage Hediye , Tempst Paul , Conaway Joan W. , Conaway Ronald C. , Kornberg Roger D.
Published: July 2002

A multiprotein complex isolated from murine cells is identified as a counterpart of the yeast Mediator of transcriptional regulation on the basis of the following: homologs of two subunits of yeast Mediator, Srb7 and Med7, copurify with the complex; peptide sequencing reveals, in addition, homologs of the yeast Mediator subunits Rgr1 and Med6; as with yeast Mediator, the mouse complex binds to the RNA polymerase II C-terminal domain (CTD) and stimulates phosphorylation of the CTD by TFIIH. Peptide sequencing also identifies a component of mouse Mediator as a relative of Ring-3 protein, a mitogen-activated nuclear protein kinase, raising the possibility of Mediator as an end point of signal transduction pathways.
Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis
Authors: de la Rosa Enrique J. , Vega-Núñez Elena , Morales Aixa V. , Serna José , Rubio Eva , de Pablo Flora
Published: July 2002

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.