bluuebunny/crossref_metadata_2025 huggingface dataset:
p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway
Authors: Nickel Walter , Sohn Kai , Bünning Carsten , Wieland Felix T.
Published: July 2002

A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8–p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8–p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8–p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8–p23 fusion protein within the ER–Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi.
A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors
Authors: Naranda Tatjana , Goldstein Avram , Olsson Lennart
Published: July 2002

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.
Mosquito transferrin, an acute-phase protein that is up-regulated upon infection
Authors: Yoshiga Toyoshi , Hernandez Vida P. , Fallon Ann M. , Law John H.
Published: July 2002

When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both
Aedes aegypti
and
Aedes albopictus
. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an
A. aegypti
transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3′ end exactly matched a cDNA encoding a polypeptide that is up-regulated when
A. aegypti
encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994)
Exp. Parasitol.
79, 312–321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.
The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments
Authors: Fan Huizhou , Huang Aiping , Villegas Cristy , Wright Jim A.
Published: July 2002

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in
ras
-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential
in vivo
. In three
ras
-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic
ras
,
myc
, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic
ras
showed significantly increased anchorage independence as compared with control
ras
-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.
Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?
Authors: Bharathan Geeta , Janssen Bart-Jan , Kellogg Elizabeth A. , Sinha Neelima
Published: July 2002

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.
Ammonia acquisition in enteric bacteria: Physiological role of the ammonium/methylammonium transport B (AmtB) protein
Authors: Soupene Eric , He Luhong , Yan Dalai , Kustu Sydney
Published: July 2002

Homologues of the
amtB
gene of enteric bacteria
exist in all three domains of life. Although their products are
required for transport of the ammonium analogue methylammonium in
washed cells, only in
Saccharomyces cerevisiae
have they
been shown to be necessary for growth at low NH
4
+
concentrations. We now demonstrate that an
amtB
strain
of
Escherichia coli
also grows slowly at low
NH
4
+
concentrations in batch culture, but only at pH
values below 7. In addition, we find that the growth defect of an
S. cerevisiae
triple-mutant strain lacking the function
of three homologues of the ammonium/methylammonium transport B (AmtB)
protein [called methylammonium/ammonium permeases (MEP)] that was
observed at pH 6.1 is relieved at pH 7.1. These results provide direct
evidence that AmtB participates in acquisition of
NH
4
+
/NH
3
in bacteria as well as eucarya.
Because NH
3
is the species limiting at low pH for a given
total concentration of NH
4
+
+ NH
3
, results
with both organisms indicate that AmtB/MEP proteins function in
acquisition of the uncharged form. We confirmed that accumulation of
[
14
C]methylammonium depends on its conversion to
γ-
N
-methylglutamine, an energy-requiring reaction
catalyzed by glutamine synthetase, and found that at pH 7, constitutive
expression of AmtB did not relieve the growth defects of a mutant
strain of
Salmonella typhimurium
that appears to require
a high internal concentration of
NH
4
+
/NH
3
. Hence, contrary to previous
views, we propose that AmtB/MEP proteins increase the rate of
equilibration of the uncharged species, NH
3
, across the
cytoplasmic membrane rather than actively transporting—that is,
concentrating—the charged species, NH
4
+
.
Analysis of xylem formation in pine by cDNA sequencing
Authors: Allona Isabel , Quinn Michelle , Shoop Elizabeth , Swope Kristi , Cyr Sheila St. , Carlis John , Riedl John , Retzel Ernest , Campbell Malcolm M. , Sederoff Ronald , Whetten Ross W.
Published: July 2002

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (
Pinus taeda
L.). A total of 1,097 single-pass sequences were obtained from 5′ ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.
Neomycin inhibits angiogenin-induced angiogenesis
Authors: Hu Guo-fu
Published: July 2002

A class of angiogenesis inhibitor has emerged from our mechanistic study of the action of angiogenin, a potent angiogenic factor. Neomycin, an aminoglycoside antibiotic, inhibits nuclear translocation of human angiogenin in human endothelial cells, an essential step for angiogenin-induced angiogenesis. The phospholipase C-inhibiting activity of neomycin appears to be involved, because U-73122, another phospholipase C inhibitor, has a similar effect. In contrast, genistein, oxophenylarsine, and staurosporine, inhibitors of tyrosine kinase, phosphotyrosine phosphatase, and protein kinase C, respectively, do not inhibit nuclear translocation of angiogenin. Neomycin inhibits angiogenin-induced proliferation of human endothelial cells in a dose-dependent manner. At 50 μM, neomycin abolishes angiogenin-induced proliferation but does not affect the basal level of proliferation and cell viability. Other aminoglycoside antibiotics, including gentamicin, streptomycin, kanamycin, amikacin, and paromomycin, have no effect on angiogenin-induced cell proliferation. Most importantly, neomycin completely inhibits angiogenin-induced angiogenesis in the chicken chorioallantoic membrane at a dose as low as 20 ng per egg. These results suggest that neomycin and its analogs are a class of agents that may be developed for anti-angiogenin therapy.
Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes
Authors: Kordis Dusan , Gubensek Franc
Published: July 2002

We have shown previously by Southern blot analysis that Bov-B long interspersed nuclear elements (LINEs) are present in different Viperidae snake species. To address the question as to whether Bov-B LINEs really have been transmitted horizontally between vertebrate classes, the analysis has been extended to a larger number of vertebrate, invertebrate, and plant species. In this paper, the evolutionary origin of Bov-B LINEs is shown unequivocally to be in Squamata. The previously proposed horizontal transfer of Bov-B LINEs in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The horizontal transfer of Bov-B LINEs from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution. The ancestor of Colubroidea snakes is a possible donor of Bov-B LINEs to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINEs in Ruminantia and the fossil data of Ruminantia to be 40–50 My ago. The phylogenetic relationships of Bov-B LINEs from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINEs have been maintained stably by vertical transmission since the origin of Squamata in the Mesozoic era.
Temperature dependence of amyloid β-protein fibrillization
Authors: Kusumoto Yoko , Lomakin Aleksey , Teplow David B. , Benedek George B.
Published: July 2002

Fibrillogenesis of the amyloid β-protein (Aβ) is believed to play a central role in the pathogenesis of Alzheimer’s disease. Previous studies of the kinetics of Aβ fibrillogenesis showed that the rate of fibril elongation is proportional to the concentration of monomers. We report here the study of the temperature dependence of the Aβ fibril elongation rate constant,
k
e
, in 0.1 M HCl. The rate of fibril elongation was measured at Aβ monomer concentrations ranging from 50 to 400 μM and at temperatures from 4°C to 40°C. Over this temperature range,
k
e
increases by two orders of magnitude. The temperature dependence of
k
e
follows the Arrhenius law,
k
e
=
A
exp (−
E
A
/
kT
). The preexponential factor
A
and the activation energy
E
A
are ≈6 × 10
18
liter/(mol·sec) and 23 kcal/mol, respectively. Such a high value of
E
A
suggests that significant conformational changes are associated with the binding of Aβ monomers to fibril ends.
Spatial scales of desert locust gregarization
Authors: Collett Matthew , Despland Emma , Simpson Stephen J. , Krakauer David C.
Published: July 2002

Central to swarm formation in migratory locusts is a crowding-induced change from a “solitarious” to a “gregarious” phenotype. This change can occur within the lifetime of a single locust and accrues across generations. It represents an extreme example of phenotypic plasticity. We present computer simulations and a laboratory experiment that show how differences in resource distributions, conspicuous only at small spatial scales, can have significant effects on phase change at the population level; local spatial concentration of resource induces gregarization. Simulations also show that populations inhabiting a locally concentrated resource tend to change phase rapidly and synchronously in response to altered population densities. Our results show why information about the structure of resource at small spatial scales should become key components in monitoring and control strategies.
Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells
Authors: Ye Keqiang , Ke Yong , Keshava Nagalakshmi , Shanks John , Kapp Judith A. , Tekmal Rajeshwar R. , Petros John , Joshi Harish C.
Published: July 2002

An alkaloid from opium, noscapine, is used as an antitussive drug and has low toxicity in humans and mice. We show that noscapine binds stoichiometrically to tubulin, alters its conformation, affects microtubule assembly, and arrests mammalian cells in mitosis. Furthermore, noscapine causes apoptosis in many cell types and has potent antitumor activity against solid murine lymphoid tumors (even when the drug was administered orally) and against human breast and bladder tumors implanted in nude mice. Because noscapine is water-soluble and absorbed after oral administration, its chemotherapeutic potential in human cancer merits thorough evaluation.
Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation
Authors: Lehman Chris W. , Botchan Michael R.
Published: July 2002

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.
Agrobacterium
transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene
ipt
Authors: Chou Alan Y. , Archdeacon John , Kado Clarence I.
Published: July 2002

Virulence genes of
Agrobacterium tumefaciens
are under the control of positive and negative transcriptional regulators. We found that the transcriptional regulator Ros controls expression of the plant oncogene
ipt
, which encodes isopentenyl transferase, in
A. tumefaciens
. This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant. An
ipt
promoter∷
cat
reporter gene fusion showed a 10-fold increase in
ipt
promoter activity in
A. tumefaciens ros
mutant strains when compared with wild type. Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in
ros
mutant strains.
In vitro
studies using purified Ros showed it binds directly to the
ipt
promoter. Analysis of the deduced Ros amino acid sequence identified a novel type of C
2
H
2
zinc finger. In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C
2
H
2
motif. Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn
2+
and DNA binding activities of Ros. The existence of such a regulator in
Agrobacterium
may be due to horizontal interkingdom retrotransfer of the
ros
gene from plant to bacteria.
Poly(ADP-ribose) polymerase activation mediates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism
Authors: Mandir Allen S. , Przedborski Serge , Jackson-Lewis Vernice , Wang Zhao-Qi , Simbulan-Rosenthal Cynthia M. , Smulson Mark E. , Hoffman Brian E. , Guastella Daniel B. , Dawson Valina L. , Dawson Ted M.
Published: July 2002

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes parkinsonism in humans and nonhuman animals, and its use has led to greater understanding of the pathogenesis of Parkinson’s disease. However, its molecular targets have not been defined. We show that mice lacking the gene for poly(ADP-ribose) polymerase (PARP), which catalyzes the attachment of ADP ribose units from NAD to nuclear proteins after DNA damage, are dramatically spared from MPTP neurotoxicity. MPTP potently activates PARP exclusively in vulnerable dopamine containing neurons of the substantia nigra. MPTP elicits a novel pattern of poly(ADP-ribosyl)ation of nuclear proteins that completely depends on neuronally derived nitric oxide. Thus, NO, DNA damage, and PARP activation play a critical role in MPTP-induced parkinsonism and suggest that inhibitors of PARP may have protective benefit in the treatment of Parkinson’s disease.
An equilibrium statistical model for the spreading phase of open-ocean convection
Authors: DiBattista Mark T. , Majda Andrew J.
Published: July 2002

A “most probable state” equilibrium statistical theory for random distributions of hetons in a closed basin is developed here in the context of two-layer quasigeostrophic models for the spreading phase of open-ocean convection. The theory depends only on bulk conserved quantities such as energy, circulation, and the range of values of potential vorticity in each layer. The simplest theory is formulated for a uniform cooling event over the entire basin that triggers a homogeneous random distribution of convective towers. For a small Rossby deformation radius typical for open-ocean convection sites, the most probable states that arise from this theory strongly resemble the saturated baroclinic states of the spreading phase of convection, with a stabilizing barotropic rim current and localized temperature anomaly.
Laser-mediated, site-specific inactivation of RNA transcripts
Authors: Grate Dilara , Wilson Charles
Published: July 2002

The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and
in vitro
characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.
Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: A full
in vitro
analysis
Authors: Warner Timothy D. , Giuliano Francesco , Vojnovic Ivana , Bukasa Antoaneta , Mitchell Jane A. , Vane John R.
Published: July 2002

The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full
in vitro
analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.
A more unified picture for the thermodynamics of nucleic acid duplex melting: A characterization by calorimetric and volumetric techniques
Authors: Chalikian Tigran V. , Völker Jens , Plum G. Eric , Breslauer Kenneth J.
Published: July 2002

We use a combination of calorimetric and volumetric techniques to detect and to characterize the thermodynamic changes that accompany helix-to-coil transitions for five polymeric nucleic acid duplexes. Our calorimetric measurements reveal that melting of the duplexes is accompanied by positive changes in heat capacity (Δ
C
P
) of similar magnitude, with an average Δ
C
P
value of 64.6 ± 21.4 cal deg
−1
mol
−1
. When this heat capacity value is used to compare significantly different transition enthalpies (Δ
H
o
) at a common reference temperature,
T
ref
, we find Δ
H

T
ref

for duplex melting to be far less dependent on duplex type, base composition, or base sequence than previously believed on the basis of the conventional assumption of a near-zero value for Δ
C
P
. Similarly, our densimetric and acoustic measurements reveal that, at a given temperature, all the AT- and AU-containing duplexes studied here melt with nearly the same volume and compressibility changes. In the aggregate, our results, in conjunction with literature data, suggest a more unified picture for the thermodynamics of nucleic acid duplex melting. Specifically, when compared at a common temperature, the apparent large differences present in the literature for the transition enthalpies of different duplexes become much more compressed, and the melting of all-AT- and all-AU-containing duplexes exhibits similar volume and compressibility changes despite differences in sequence and conformation. Thus, insofar as thermodynamic properties are concerned, when comparing duplexes, the temperature under consideration is as important as, if not more important than, the duplex type, the base composition, or the base sequence. This general behavior has significant implications for our basic understanding of the forces that stabilize nucleic acid duplexes. This behavior also is of practical significance in connection with the use of thermodynamic databases for designing probes and for assessing the affinity and specificity associated with hybridization-based protocols used in a wide range of sequencing, diagnostic, and therapeutic applications.
Bcl-2 family members do not inhibit apoptosis by binding the caspase activator Apaf-1
Authors: Moriishi Kohji , Huang David C. S. , Cory Suzanne , Adams Jerry M.
Published: July 2002

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, thein vivoassociation studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 could be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain was necessary and sufficient for their interaction. Apaf-1 did not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-xL, Bcl-w, A1, Mcl-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein–Barr virus BHRF-1. Endogenous Apaf-1 also failed to coimmunoprecipitate with endogenous Bcl-2 or Bcl-xL, or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli did not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly.
Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands
Authors: Morita Kazumasa , Furuse Mikio , Fujimoto Kazushi , Tsukita Shoichiro
Published: July 2002

Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands. Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family. Three of these were reported previously as RVP1,Clostridium perfringensenterotoxin receptor, and TMVCF, but their physiological functions were not determined. Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1,Clostridium perfringensenterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively. All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule. Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species. Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin–Darby canine kidney cells, all showed a tendency to concentrate at TJs. Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney. These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.
Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor
Authors: Agnello Vincent , Ábel György , Elfahal Mutasim , Knight Glenn B. , Zhang Qing-Xiu
Published: July 2002

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.
Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands
Authors: Kieper William C. , Jameson Stephen C.
Published: July 2002

Recent data suggest that survival of resting, naïve T cells requires an interaction with self MHC molecules. From analysis of the class I MHC-restricted T cell receptor transgenic strain OT-I, we report a different response. Rather than merely surviving, these T cells proliferated slowly after transfer into T-depleted syngeneic hosts. This expansion required both T cell “space” and expression of normal levels of self class I MHC molecules. Furthermore, we demonstrate that during homeostatic expansion in a suitable environment, naïve phenotype (CD44low) OT-I T cells converted to memory phenotype (CD44med/high), despite the absence of foreign antigenic stimulation. On the other hand, cells undergoing homeostatic expansion did not acquire cytolytic effector function. The significance of these data for reactivity of T cells with self peptide/MHC ligands and the implications for normal and abnormal T cell homeostasis are discussed.
Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage
Authors: Chehab Nabil H. , Malikzay Asra , Stavridi Elena S. , Halazonetis Thanos D.
Published: July 2002

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53–Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2
in vitro
. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.
Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway
Authors: Kovarik Pavel , Stoiber Dagmar , Eyers Patrick A. , Menghini Rossella , Neininger Armin , Gaestel Matthias , Cohen Philip , Decker Thomas
Published: July 2002

STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of themapkap-k2gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylatedin vitroby p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.
Phylogeny of rice genomes with emphasis on origins of allotetraploid species
Authors: Ge Song , Sang Tao , Lu Bao-Rong , Hong De-Yuan
Published: July 2002

The rice genus,Oryza, which comprises 23 species and 9 recognized genome types, represents an enormous gene pool for genetic improvement of rice cultivars. Clarification of phylogenetic relationships of rice genomes is critical for effective utilization of the wild rice germ plasm. By generating and comparing two nuclear gene (Adh1andAdh2) trees and a chloroplast gene (matK) tree of all rice species, phylogenetic relationships among the rice genomes were inferred. Origins of the allotetraploid species, which constitute more than one-third of rice species diversity, were reconstructed based on theAdhgene phylogenies. Genome types of the maternal parents of allotetraploid species were determined based on thematK gene tree. The phylogenetic reconstruction largely supports the previous recognition of rice genomes. It further revealed that the EE genome species is most closely related to the DD genome progenitor that gave rise to the CCDD genome. Three species of the CCDD genome may have originated through a single hybridization event, and their maternal parent had the CC genome. The BBCC genome species had different origins, and their maternal parents had either a BB or CC genome. An additional genome type, HHKK, was recognized forOryza schlechteriandPorteresia coarctata, suggesting thatP. coarctatais anOryzaspecies. The AA genome lineage, which contains cultivated rice, is a recently diverged and rapidly radiated lineage within the rice genus.
Calcium channel activation and self-biting in mice
Authors: Jinnah H. A. , Yitta S. , Drew T. , Kim B. S. , Visser J. E. , Rothstein J. D.
Published: July 2002

The L type calcium channel agonist (±)Bay K 8644 has been reported to cause characteristic motor abnormalities in adult mice. The current study shows that administration of this drug can also cause the unusual phenomenon of self-injurious biting, particularly when given to young mice. Self-biting is provoked by injecting small quantities of (±)Bay K 8644 directly into the lateral ventricle of the brain, suggesting a central effect of the drug. Similar behaviors can be provoked by administration of another L type calcium channel agonist, FPL 64176. The self-biting provoked by (±)Bay K 8644 can be inhibited by pretreating the mice with dihydropyridine L type calcium channel antagonists such as nifedipine, nimodipine, or nitrendipine. However, self-biting is not inhibited by nondihydropyridine antagonists including diltiazem, flunarizine, or verapamil. The known actions of (±)Bay K 8644 as an L type calcium channel agonist, the reproduction of similar behavior with another L type calcium channel agonist, and the protection afforded by certain L type calcium channel antagonists implicate calcium channels in the mediation of the self-biting behavior. This phenomenon provides a model for studying the neurobiology of this unusual behavior.
An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers
Authors: Gómez-Cadenas Aurelio , Verhey Steven D. , Holappa Lynn D. , Shen Qingxi , Ho Tuan-Hua David , Walker-Simmons M. K.
Published: July 2002

The phytohormone abscisic acid (ABA) induces genes-encoding
proteins involved in desiccation tolerance and dormancy in seeds, but
ABA also suppresses gibberellin (GA)-responsive genes encoding
hydrolytic enzymes essential for postgermination growth. A unique
serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in
seeds, has been identified. In this report, the effect of PKABA1 on the
signal transduction pathway mediating ABA induction and suppression of
genes has been determined in aleurone layers of barley seeds. Two
groups of gene constructs were introduced to barley aleurone layers by
using particle bombardment: the reporter constructs containing the
coding sequence of β-glucuronidase gene linked to hormone-responsive
promoters and the effector constructs containing the coding region of
protein kinases linked to a constitutive promoter. Constitutive
expression of PKABA1 drastically suppressed expression of low- and
high-pI α-amylase and protease genes induced by GA. However, the
presence of PKABA1 had only a small effect on the ABA induction of a
gene encoding a late embryogenesis abundant protein, HVA1. Our results
indicate that PKABA1 acts as a key intermediate in the signal
transduction pathway leading to the suppression of GA-inducible gene
expression in cereal aleurone layers.
Retinotopic mapping of lateral geniculate nucleus in humans using functional magnetic resonance imaging
Authors: Chen Wei , Zhu Xiao-Hong , Thulborn Keith R. , Ugurbil Kamil
Published: July 2002

Subcortical nuclei in the thalamus, which play an important role in many functions of the human brain, provide challenging targets for functional mapping with neuroimaging techniques because of their small sizes and deep locations. In this study, we explore the capability of high-resolution functional magnetic resonance imaging at 4 Tesla for mapping the retinotopic organization in the lateral geniculate nucleus (LGN). Our results show that the hemifield visual stimulation only activates LGN in the contralateral hemisphere, and the lower-field and upper-field visual stimulations activate the superior and inferior portion of LGN, respectively. These results reveal a similar retinotopic organization between the human and nonhuman primate LGN and between LGN and the primary visual cortex. We conclude that high-resolution functional magnetic resonance imaging is capable of functional mapping of suborganizations in small nuclei together with cortical activation. This will have an impact for studying the thalamocortical networks in the human brain.
Declining survival probability threatens the North Atlantic right whale
Authors: Caswell Hal , Fujiwara Masami , Brault Solange
Published: July 2002

The North Atlantic northern right whale
(Eubalaena glacialis)
is considered the most endangered large whale species. Its population has recovered only slowly since the cessation of commercial whaling and numbers about 300 individuals. We applied mark-recapture statistics to a catalog of photographically identified individuals to obtain the first statistically rigorous estimates of survival probability for this population. Crude survival decreased from about 0.99 per year in 1980 to about 0.94 in 1994. We combined this survival trend with a reported decrease in reproductive rate into a branching process model to compute population growth rate and extinction probability. Population growth rate declined from about 1.053 in 1980 to about 0.976 in 1994. Under current conditions the population is doomed to extinction; an upper bound on the expected time to extinction is 191 years. The most effective way to improve the prospects of the population is to reduce mortality. The right whale is at risk from entanglement in fishing gear and from collisions with ships. Reducing this human-caused mortality is essential to the viability of this population.
GADD45 induction of a G
2
/M cell cycle checkpoint
Authors: Wang Xin Wei , Zhan Qimin , Coursen Jill D. , Khan Mohammed A. , Kontny H. Udo , Yu Lijia , Hollander M. Christine , O’Connor Patrick M. , Fornace Albert J. , Harris Curtis C.
Published: July 2002

G
1
/S and G
2
/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G
2
/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G
2
/M boundary with a phenotype of MPM2 immunopositivity, 4
n
DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G
2
/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li–Fraumeni fibroblasts or in normal fibroblasts coexpressed with
p53
mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G
2
/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G
2
/M checkpoint is at least in part through modulation of the activity of the G
2
-specific kinase, cyclin B1/p34
cdc2
. Genetic and physiological evidence of a Gadd45-mediated G
2
/M checkpoint was obtained by using
GADD45
-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense
GADD45
expression have an impaired G
2
/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G
2
arrest after ionizing radiation. Lymphocytes from
gadd45
-knockout mice (
gadd45
−/−) also retained a G
2
/M checkpoint initiated by ionizing radiation and failed to arrest at G
2
/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G
2
/M checkpoints in response to specific types of DNA damage.
The 60-kDa heat shock protein modulates allograft rejection
Authors: Birk Ohad S. , Gur Sara L. , Elias Dana , Margalit Raanan , Mor Felix , Carmi Pnina , Bockova Jana , Altmann Daniel M. , Cohen Irun R.
Published: July 2002

Allograft rejection is a process of immune reactivity triggered by foreign transplantation antigens. We now demonstrate that the 60-kDa heat shock protein (hsp60), a molecule that is identical in the donor and the recipient, can regulate allograft immunity. In wild-type mice, hsp60 expression was greatly enhanced in allografts being rejected. By using MHC class II (Eα) promoter hsp60 transgenic mice either as donors of skin with enhanced expression of hsp60, or as allograft recipients with decreased hsp60 autoimmunity, we found that augmented expression of mouse hsp60 in the allograft accelerated its rejection, whereas reduced autoimmunity to mouse hsp60 in graft recipients delayed the process. Moreover, in nontransgenic mice, therapeutic administration of hsp60 or hsp60 peptides, known to modulate naturally occurring hsp60 autoimmunity, led to delayed allograft rejection. Thus, we demonstrate that hsp60 expression and hsp60 autoimmunity can influence and modify the immune response to foreign antigens. Hence, autoimmunity to self-hsp60 epitopes is not necessarily an aberration, but may serve physiologically and therapeutically to modulate foreign immunity.