bluuebunny/crossref_metadata_2025 huggingface dataset:
Abrogation of disease development in plants expressing animal antiapoptotic genes
Authors: Dickman M. B. , Park Y. K. , Oltersdorf T. , Li W. , Clemente T. , French R.
Published: July 2002

An emerging topic in plant biology is whether plants display
analogous elements of mammalian programmed cell death during
development and defense against pathogen attack. In many
plant–pathogen interactions, plant cell death occurs in both
susceptible and resistant host responses. For example, specific
recognition responses in plants trigger formation of the hypersensitive
response and activation of host defense mechanisms, resulting in
restriction of pathogen growth and disease development. Several studies
indicate that cell death during hypersensitive response involves
activation of a plant-encoded pathway for cell death. Many susceptible
interactions also result in host cell death, although it is not clear
how or if the host participates in this response. We have generated
transgenic tobacco plants to express animal genes that negatively
regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl,
nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable
resistance to several necrotrophic fungal pathogens, suggesting that
disease development required host–cell death pathways. In addition,
the transgenic tobacco plants displayed resistance to a necrogenic
virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function
mutation did not protect against pathogen challenge. We also show that
discrete DNA fragmentation (laddering) occurred in susceptible tobacco
during fungal infection, but does not occur in transgenic-resistant
plants. Our data indicate that in compatible plant–pathogen
interactions apoptosis-like programmed cell death occurs.
Further, these animal antiapoptotic genes function in plants
and should be useful to delineate resistance pathways. These genes also
have the potential to generate effective disease resistance in
economically important crops.
Transmissibility of systemic amyloidosis by a prion-like mechanism
Authors: Lundmark Katarzyna , Westermark Gunilla T. , Nyström Sofia , Murphy Charles L. , Solomon Alan , Westermark Per
Published: July 2002

The generation of amyloid fibrils from an amyloidogenic polypeptide occurs by a nucleation-dependent process initiated
in vitro
by seeding the protein solution with preformed fibrils. This phenomenon is evidenced
in vivo
by the fact that amyloid protein A (AA) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an i.v. injection of protein extracted from AA amyloid-laden mouse tissue. Heretofore, the chemical nature of this “amyloid enhancing factor” (AEF) has not been definitively identified. Here we report that the active principle of AEF extracted from the spleen of mice with silver nitrate-induced AA amyloidosis was identified unequivocally as the AA fibril itself. Further, we demonstrated that this material was extremely potent, being active in doses <1 ng, and that it retained its biologic activity over a considerable length of time. Notably, the AEF was also effective when administered orally. Our studies have provided evidence that AA and perhaps other forms of amyloidosis are transmissible diseases, akin to the prion-associated disorders.
A missense mutation of the Na
+
channel α
II
subunit gene
Na
v
1.2
in a patient with febrile and afebrile seizures causes channel dysfunction
Authors: Sugawara Takashi , Tsurubuchi Yuji , Agarwala Kishan Lal , Ito Masatoshi , Fukuma Goryu , Mazaki-Miyazaki Emi , Nagafuji Hiroshi , Noda Masaharu , Imoto Keiji , Wada Kazumaru , Mitsudome Akihisa , Kaneko Sunao , Montal Mauricio , Nagata Keiichi , Hirose Shinichi , Yamakawa Kazuhiro
Published: July 2002

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical
subset of febrile seizures (FS), is characterized by frequent episodes
beyond 6 years of age (FS+) and various types of subsequent epilepsy.
Mutations in β1 and α
I
-subunit genes of voltage-gated
Na
+
channels have been associated with GEFS+1 and 2,
respectively. Here, we report a mutation resulting in an amino acid
exchange (R187W) in the gene encoding the α-subunit of neuronal
voltage-gated Na
+
channel type II
(
Na
v
1.2
) in a patient with FS
associated with afebrile seizures. The mutation R187W occurring on
Arg
187
, a highly conserved residue among
voltage-gated Na
+
channels, was not found in 224 alleles of
unaffected individuals. Whole-cell patch clamp recordings on human
embryonic kidney (HEK) cells expressing a rat wild-type
(rNa
v
1.2) and the corresponding mutant channels showed that
the mutant channel inactivated more slowly than wild-type whereas the
Na
+
channel conductance was not affected. Prolonged
residence in the open state of the R187W mutant channel may augment
Na
+
influx and thereby underlie the neuronal
hyperexcitability that induces seizure activity. Even though a small
pedigree could not show clear cosegregation with the disease phenotype,
these findings strongly suggest the involvement of
Na
v
1.2
in a human disease and
propose the R187W mutation as the genetic defect responsible for
febrile seizures associated with afebrile seizures.
A
M
r
34,000 proinflammatory outer membrane protein (
oipA
) of
Helicobacter pylori
Authors: Yamaoka Yoshio , Kwon Dong H. , Graham David Y.
Published: July 2002

The complete genome sequence revealed a family of 32 outer membrane proteins (OMPs) in
Helicobacter pylori.
We examined the effect of four OMPs (HP0638, HP0796, HP1501, and
babA2
) on the production of the proinflammatory cytokine, IL-8. Mutants of the four OMPs, as well as
cagE
and
galE
from
H. pylori
from the U.S. and Japan, were constructed by inserting a chloramphenicol-resistant cassette into the gene. Twenty-two pairs of parental and mutant
H. pylori
strains, as well as 160 clinical isolates (80 from Japanese and 80 from U.S.), were cocultured with gastric cancer cell lines. IL-8 production in the supernatant and adhesion was assayed by ELISA. HP0796, HP1501,
babA2
, and
galE
gene knockouts had no significant effect on IL-8 production. Knockout of the HP0638 gene in 81% of
cag
-positive strains reduced IL-8 production approximately 50%. The three
cag
-positive strains in which IL-8 levels were unchanged by HP0638 knockout had five or seven CT dinucleotide repeats in the 5′ region, resulting in a frame shift and truncation. Strains with naturally inactive HP0638 gene were all from the U.S.; Japanese strains were always “on” and thus, on average, may be more virulent. Although
cag
-negative isolates produced a limited IL-8 response,
cag
-negative strains that contained a functional HP0638 gene produced more than 3-fold greater IL-8 than
cag
-negative nonfunctional HP0638 strains. We hypothesize that functional HP0638 gene may be an important virulence factor in relation to the risk of clinically significant outcomes of
H. pylori
infection. We denote HP0638 gene as outer inflammatory protein (
oipA
).
Role of a pineal cAMP-operated arylalkylamine
N-
acetyltransferase/14-3-3-binding switch in melatonin synthesis
Authors: Ganguly Surajit , Gastel Jonathan A. , Weller Joan L. , Schwartz Christian , Jaffe Howard , Namboodiri M. A. A. , Coon Steven L. , Hickman Alison B. , Rollag Mark , Obsil Tomas , Beauverger Philippe , Ferry Gilles , Boutin Jean A. , Klein David C.
Published: July 2002

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine
N
-acetyltransferase (AANAT; serotonin
N
-acetyltransferase, EC
2.3.1.87
). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the
K
m
for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.
A
Drosophila
PIAS homologue negatively regulates
stat92E
Authors: Betz Aurel , Lampen Nina , Martinek Sebastian , Young Michael W. , Darnell James E.
Published: July 2002

Transcriptional activation by, and therefore the physiologic
impact of, activated tyrosine-phosphorylated STATs
(
s
ignal
t
ransducers and
a
ctivators of
t
ranscription) may be
negatively regulated by proteins termed PIAS (
p
rotein
i
nhibitors of
a
ctivated
s
tats), as shown by previous experiments with mammalian
cells in culture. Here, by using the genetic modifications in
Drosophila
, we demonstrate the
in vivo
functional interaction of the
Drosophila
homologues
stat92E
and a
Drosophila
PIAS gene
(
dpias
). To this end we use a LOF allele and
conditionally overexpressed
dpias
in JAK-STAT pathway
mutant backgrounds. We conclude that the correct
dpias
/
stat92E
ratio is crucial for
blood cell and eye development.
Human lung cancer and
p53
: The interplay between mutagenesis and selection
Authors: Rodin Sergei N. , Rodin Andrew S.
Published: July 2002

It is an almost consensus opinion that the major carcinogenic risk
of tobacco smoke is in its direct mutagenic action on DNA of
cancer-related genes. The key data supposedly linking smoke-induced
mutations to lung cancer were obtained from the adduct spectrum of the
p53
tumor suppressor gene. Results of our analysis of
p53 mutations compiled from the International Agency for Research on
Cancer
p53
database (April 1999 update) and from the
literature point to a different causative link. Our new analytical
tests focused on complementary base substitutions and showed that it is
strand-specific repair of primary lesions and site-specific selection
of the resultant mutations that determine the lung cancer-specific hot
spots of G:C to T:A transversions along the
p53
gene and
also their increased abundance in lung tissues as compared with
smoke-inaccessible tissues. However, on each of the two strands of p53
DNA, our tests revealed no significant difference between smokers and
nonsmokers, either in the frequency of different types of mutations or
in the frequency of their occurrence along the
p53
gene.
Moreover, in both smokers and nonsmokers, there was the same frequency
of lung tumors with silent p53 mutations. Accordingly, we offer here a
selection-based explanation of why lung cancers with nonsilent p53
mutations are more common in smokers than in nonsmokers. We conclude
that physiological stresses (not necessarily genotoxic) aggravated by
smoking are the leading risk factor in the p53-associated etiology of
lung cancer.
Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34
+
cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27
Kip
, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity
Authors: Jiang Yuehua , Zhao Robert C. H. , Verfaillie Catherine M.
Published: July 2002

β
1
-integrin engagement on normal (NL) CD34
+
cells increases levels of the cyclin-dependent kinase inhibitor (cdki), p27
Kip
, decreases cdk2 activity, and inhibits G
1
/S-phase progression. In contrast, β
1
-integrin engagement on chronic myelogenous leukemia (CML) CD34
+
cells does not inhibit G
1
/S progression. We now show that, in CML, baseline p27
Kip
levels are significantly higher than in NL CD34
+
cells, but adhesion to fibronectin (FN) does not increase p27
Kip
levels. p27
Kip
mRNA levels are similar in CML and NL CD34
+
cells and remain unchanged after adhesion, suggesting posttranscriptional regulation. Despite the elevated p27
Kip
levels, cdk2 kinase activity is similar in CML and NL CD34
+
cells. In NL CD34
+
cells, >90% of p27
Kip
is located in the nucleus, where it binds to cdk2 after integrin engagement. In CML CD34
+
cells, however, >80% of p27
Kip
is located in the cytoplasm even in FN-adherent cells, and significantly less p27
Kip
is bound to cdk2. Thus, presence of BCR/ABL induces elevated levels of p27
Kip
and relocation of p27
Kip
to the cytoplasm, which contributes to the loss of integrin-mediated proliferation inhibition, characteristic of CML.
Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry
Authors: Amexis Georgios , Oeth Paul , Abel Kenneth , Ivshina Anna , Pelloquin Francois , Cantor Charles R. , Braun Andreas , Chumakov Konstantin
Published: July 2002

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest.
Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2
Authors: Huang H.-C. , Nguyen Truyen , Pickett Cecil B.
Published: July 2002

A coordinated cellular response to oxidative stress occurs in part
through transcriptional regulation via a cis-acting sequence known as
the antioxidant response element (ARE). NF-E2-related factor 2 (Nrf2),
a member of the Cap'n'Collar family of basic region-leucine zipper
(bZIP) transcription factors, has been implicated as an essential
component of an ARE-binding transcriptional complex, but the signaling
pathway leading to its activation has remained unclear. Using a
reporter gene assay, we found that ARE-directed transcription was
activated by phorbol 12-myristate 13-acetate (PMA), but completely
suppressed by staurosporine and Ro-32–0432, selective inhibitors of
protein kinase C (PKC). Immunocytochemistry and subcellular
fractionation revealed that PMA, like
tert
-butylhydroquinone (tBHQ), promoted the nuclear
localization of Nrf2, a process that was blocked by staurosporine or
Ro-32–0432. We showed that Nrf2, a previously unidentified kinase
target, was phosphorylated in HepG2 cells. PMA transiently activated
Nrf2 phosphorylation, whereas the addition of tBHQ or
β-naphthoflavone (βNF) led to a persistent stimulation, which was
abolished by staurosporine, but not by U0126 and SB203580, respective
inhibitors of MEK and p38 kinases. Purified Nrf2 was phosphorylated
in vitro
by the catalytic subunit of PKC, or by PKC
immunoprecipitated from cell lysates. Significantly, PKC precipitated
from tBHQ- or βNF-treated cells showed enhanced activity against
Nrf2. These findings indicate an important role of the PKC pathway in
the ARE-mediated gene expression, and suggest that PKC-directed
phosphorylation of Nrf2 may be a critical event for the nuclear
translocation of this transcription factor in response to oxidative
stress.
Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix–loop–helix factor PIF3 in a reaction requiring the PAS domain of PIF3
Authors: Zhu Yuxian , Tepperman James M. , Fairchild Craig D. , Quail Peter H.
Published: July 2002

The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix–loop–helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previousin vivodata from PIF3-deficientArabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.
COMPASS: A complex of proteins associated with a trithorax-related SET domain protein
Authors: Miller Trissa , Krogan Nevan J. , Dover Jim , Erdjument-Bromage H. , Tempst Paul , Johnston Mark , Greenblatt Jack F. , Shilatifard Ali
Published: July 2002

The
trithorax
genes encode an evolutionarily conserved family of proteins that function to maintain specific patterns of gene expression throughout cellular development. Members of this protein family contain a highly conserved 130- to 140-amino acid motif termed the SET domain. We report the purification and molecular identification of the subunits of a protein complex in the yeast
Saccharomyces cerevisiae
that includes the trithorax-related protein Set1. This protein complex, which we have named COMPASS (Complex Proteins Associated with Set1), consists of seven polypeptides ranging from 130 to 25 kDa. The same seven proteins were identified in COMPASS purified either by conventional biochemical chromatography or tandem-affinity tagging of the individual subunits of the complex. Null mutants missing any one of the six nonessential subunits of COMPASS grow more slowly than wild-type cells under normal conditions and demonstrate growth sensitivity to hydroxyurea. Furthermore, gene expression profiles of strains missing either of two nonessential subunits of COMPASS are altered in similar ways, suggesting these proteins have similar roles in gene expression
in vivo
. Molecular characterization of trithorax complexes will facilitate defining the role of this class of proteins in the regulation of gene expression and how their misregulation results in the development of human cancer.
Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity
Authors: Persson Egon , Kjalke Marianne , Olsen Ole H.
Published: July 2002

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC
3.4.21.21
), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TF-induced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.
In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.
Authors: Ren M , Zeng J , De Lemos-Chiarandini C , Rosenfeld M , Adesnik M , Sabatini D D
Published: July 2002

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.
Antisense ribosomes: rRNA as a vehicle for antisense RNAs.
Authors: Sweeney R , Fan Q , Yao M C
Published: July 2002

Although rRNA has a conserved core structure, its size varies by more than 2000 bases between eubacteria and vertebrates, mostly due to the size variation of discrete variable regions. Previous studies have shown that insertion of foreign sequences into some of these variable regions has little effect on rRNA function. These properties make rRNA a potentially very advantageous vehicle to carry other RNA moieties with biological activity, such as "antisense RNAs." We have explored this possibility by inserting antisense RNAs targeted against one essential and two nonessential genes into a site within a variable region in the Tetrahymena thermophila large subunit rRNA gene. Expression of each of the three genes tested can be drastically reduced or eliminated in transformed T. thermophila lines containing these altered rRNAs. In addition, we found that only antisense rRNAs containing RNA sequences complementary to the 5' untranslated region of the targeted mRNA were effective. Lines containing antisense rRNAs targeted against either of the nonessential genes grow well, indicating that the altered rRNAs fulfill their functions within the ribosome. Since functional rRNA is extremely abundant and stable and comes into direct contact with translated mRNAs, it may prove to be an unparalleled vehicle for enhancing the activity of functional RNAs that act on mRNAs.
RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.
Authors: Ye F , Signer E R
Published: July 2002

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.
Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.
Authors: Mazzaccaro R J , Gedde M , Jensen E R , van Santen H M , Ploegh H L , Rock K L , Bloom B R
Published: July 2002

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.
The
Schizosaccharomyces pombe
spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex
Authors: He Xiangwei , Patterson Thomas E. , Sazer Shelley
Published: July 2002

The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated
mad2
, a spindle checkpoint component in fission yeast, and shown that
mad2
overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that
mad2
-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis.
Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α
Authors: Su Fei , Schneider Robert J.
Published: July 2002

Persistent infection with hepatitis B virus (HBV) is a leading cause of human liver disease and is strongly associated with hepatocellular carcinoma, one of the most prevalent forms of human cancer. Apoptosis (programmed cell death) is an important mediator of chronic liver disease caused by HBV infection. It is demonstrated that the HBV HBx protein acutely sensitizes cells to apoptotic killing when expressed during viral replication in cultured cells and in transfected cells independently of other HBV genes. Cells that were resistant to apoptotic killing by high doses of tumor necrosis factor α (TNFα), a cytokine associated with liver damage during HBV infection, were made sensitive to very low doses of TNFα by HBx. HBx induced apoptosis by prolonged stimulation of N-Myc and the stress-mediated mitogen-activated-protein kinase kinase 1 (MEKK1) pathway but not by up-regulating TNF receptors. Cell killing was blocked by inhibiting HBx stimulation of N-Myc or mitogen-activated-protein kinase kinase 1 using dominant-interfering forms or by retargeting HBx from the cytoplasm to the nucleus, which prevents HBx activation of cytoplasmic signal transduction cascades. Treatment of cells with a mitogenic growth factor produced by many virus-induced tumors impaired induction of apoptosis by HBx and TNFα. These results indicate that HBx might be involved in HBV pathogenesis (liver disease) during virus infection and that enhanced apoptotic killing by HBx and TNFα might select for neoplastic hepatocytes that survive by synthesizing mitogenic growth factors.
Electric field-induced reorganization of two-component supported bilayer membranes
Authors: Groves Jay T. , Boxer Steven G. , McConnell Harden M.
Published: July 2002

Application of electric fields tangent to the plane of a confined
patch of fluid bilayer membrane can create lateral concentration
gradients of the lipids. A thermodynamic model of this steady-state
behavior is developed for binary systems and tested with experiments in
supported lipid bilayers. The model uses Flory’s approximation for the
entropy of mixing and allows for effects arising when the components
have different molecular areas. In the special case of equal area
molecules the concentration gradient reduces to a Fermi–Dirac
distribution. The theory is extended to include effects from charged
molecules in the membrane. Calculations show that surface charge on the
supporting substrate substantially screens electrostatic interactions
within the membrane. It also is shown that concentration profiles can
be affected by other intermolecular interactions such as clustering.
Qualitative agreement with this prediction is provided by comparing
phosphatidylserine- and cardiolipin-containing membranes.
Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry
Authors: Brügger B. , Erben G. , Sandhoff R. , Wieland F. T. , Lehmann W. D.
Published: July 2002

Nano-electrospray tandem mass spectrometry allows qualitative and quantitative analysis of complex membrane lipid mixtures at the subpicomole level. We have exploited this technique to selectively detect individual classes of phospholipids from unprocessed total cellular lipid extracts by either precursor ion or neutral loss scanning. This way phosphatidylcholine, sphingomyelin, phosphatidylinositol and -phosphates, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and their plasmalogen analogues can be detected. The optimized ionization and fragmentation conditions described together with the principle of internal standardization by nonnatural analogues allow the rapid and quantitative determination of membrane lipid compositions down to sample amounts of 1000 cells.
Obesity increases sensitivity to endotoxin liver injury: Implications for the pathogenesis of steatohepatitis
Authors: Yang Shi Qi , Lin Hui Zhi , Lane M. Daniel , Clemens Mark , Diehl Anna Mae
Published: July 2002

Genetically obese fatty/fatty rats and obese/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS). Among obese animals, females are more sensitive to endotoxin liver injury than males. LPS induction of tumor necrosis factor α (TNFα), the proven affecter of endotoxin liver injury, is no greater in the livers, white adipose tissues, or sera of obese animals than in those of lean controls. Indeed, the lowest serum concentrations of TNF occur in female obese rodents, which exhibit the most endotoxin-induced liver injury. Several cytokines that modulate the biological activity of TNF are regulated abnormally in the livers of obese animals. After exposure to LPS, mRNA of interferon γ, which sensitizes hepatocytes to TNF toxicity, is overexpressed, and mRNA levels of interleukin 10, a TNF inhibitor, are decreased. The phagocytic activity of liver macrophages and the hepatic expression of a gene encoding a macrophage-specific receptor are also decreased in obesity. This new animal model of obesity-associated liver disease demonstrates that hepatic macrophage dysfunction occurs in obesity and suggests that this might promote steatohepatitis by sensitizing hepatocytes to endotoxin.
The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production
Authors: Dong Yanjie , Zeng Carl Q.-Y. , Ball Judith M. , Estes Mary K. , Morris Andrew P.
Published: July 2002

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl−secretion in young mice. The cellular basis of this phenomenon was investigated in anin vitrocell line model for the human intestine. Intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 μM) induced both Ca2+release from intracellular stores and plasmalemma Ca2+influx. During NSP4-induced [Ca2+]imobilization, [Na+]ihomeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+entry into these cells. The ED50of the NSP4 effect on peak [Ca2+]imobilization was 4.6 ± 0.8 nM. Pretreatment of cells with either 2.3 × 10−3units/ml trypsin or 4.4 × 10−2units/ml chymotrypsin for 1–10 min abolished the NSP4-induced [Ca2+]imobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+]iin human intestinal cells through receptor-mediated phospholipase C activation and IP3production.
Effects of folding on metalloprotein active sites
Authors: Winkler Jay R. , Wittung-Stafshede Pernilla , Leckner Johan , Malmström Bo G. , Gray Harry B.
Published: July 2002

Experimental data for the unfolding of cytochrome
c
and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome
c
, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome
c
are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome
c
folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of Cu
I
in the unfolded protein is linear or trigonal, whereas Cu
II
prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline)
2
2+/+
reference system.
The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion
Authors: Yuan Chao-Xing , Ito Mitsuhiro , Fondell Joseph D. , Fu Zheng-Yuan , Roeder Robert G.
Published: July 2002

Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor α (TRα) from ligand-treated HeLa (α-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRα, and these interactions are mediated through the C terminus of TRα and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor α, retinoid X receptor α, peroxisome proliferation-activated receptor (PPAR) α, PPARγ and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor α, PPARα, and PPARγ. Consistent with these results, TRAP220 moderately stimulates human TRα-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRα) and in cell free transcription systems (TRα and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRα during the function of the TRα–TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.
Altered peptide ligands induce quantitatively but not qualitatively different intracellular signals in primary thymocytes
Authors: Smyth Lesley A. , Williams Owen , Huby Russell D. J. , Norton Trisha , Acuto Oreste , Ley Steven C. , Kioussis Dimitris
Published: July 2002

Interaction of the T cell receptor (TCR) with peptide/major histocompatibility complexes (MHC) in the thymus is of critical importance for developing thymocytes. In a previous study, we described an antagonist peptide that inhibited negative selection of transgenic thymocytes induced by an agonist peptide. In this study we show that this antagonist peptide can induce positive selection of CD8+thymocytes more efficiently than the agonist or the weak agonist peptides, whereas the opposite is true for their ability to cause negative selection. The intracellular signals induced in thymocytes by such peptides after TCR ligation was examined in CD4+8+double-positive thymocytes from F5/β2mo/Rag-1otransgenic mice. TCR ligation with either the agonist, weak agonist, or antagonist peptide variants resulted in hyperphosphorylation of CD3ζ, CD3ɛ, ZAP-70, Syk, Vav, SLP-76, and pp36–38. The extent of phosphorylation of these intracellular proteins correlated with the efficiency with which the peptide analogs induced apoptosis of immature thymocytes. Unexpectedly, there was no correlation between the upstream TCR signaling pathways analyzed and the capacity of the different peptides to induce positive selection.
Ribonuclease A variants with potent cytotoxic activity
Authors: Leland Peter A. , Schultz L. Wayne , Kim Byung-Moon , Raines Ronald T.
Published: July 2002

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI–RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.
Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes
Authors: al-Ramadi Basel K. , Zhang Hui , Bothwell Alfred L. M.
Published: July 2002

By using antisense RNA, Lck-deficient transfectants of a T helper 2 (Th2) clone have been derived and shown to have a qualitative defect in the T cell receptor signaling pathway. A striking feature observed only in Lck-deficient T cells was the presence of a constitutively tyrosine-phosphorylated 32-kDa protein. In the present study, we provide evidence that this aberrantly hyperphosphorylated protein is p34cdc2(cdc2) a key regulator of cell-cycle progression. Lck-deficient transfectants expressed high levels of cdc2 protein and its regulatory units, cyclins A and B. The majority of cdc2, however, was tyrosine-phosphorylated and therefore enzymatically inactive. The transfectants were significantly larger than the parental cells and contained 4NDNA. These results establish that a deficiency in Lck leads to a cell-cycle arrest in G2. Moreover, transfected cells were hypersusceptible to apoptosis when activated through the T cell receptor. Importantly, however, this hypersusceptibility was largely reversed in the presence of T cell growth factors. These findings provide evidence that, in mature T lymphocytes, cell-cycle progression through the G2–M check point requires expression of the Src-family protein tyrosine kinase, Lck. This requirement is Lck-specific; it is observed under conditions in which the closely related Fyn kinase is expressed normally, evincing against a redundancy of function between these two kinases.
Myosin II localization during cytokinesis occurs by a mechanism that does not require its motor domain
Authors: Zang Ji-Hong , Spudich James A.
Published: July 2002

Myosin II generates force for the division of eukaryotic cells. The molecular basis of the spatial and temporal localization of myosin II to the cleavage furrow is unknown, although models often imply that interaction between myosin II and actin filaments is essential. We examined the localization of a chimeric protein that consists of the green fluorescent protein fused to the N terminus of truncated myosin II heavy chain in
Dictyostelium
cells. This chimera is missing the myosin II motor domain, and it does not bind actin filaments. Surprisingly, it still localizes to the cleavage furrow region during cytokinesis. These results indicate that myosin II localization during cytokinesis occurs through a mechanism that does not require it to be the force-generating element or to interact with actin filaments directly.
ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles
Authors: Stamnes Mark , Schiavo Giampietro , Stenbeck Gudrun , Söllner Thomas H. , Rothman James E.
Published: July 2002

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise—in fact they decline—during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996)J. Cell Biol.134, 295–306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.
PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein
Authors: Zheng Peizhong , Eastman Jean , Vande Pol Scott , Pimplikar Sanjay W.
Published: July 2002

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a
ba
solateral
s
orting
s
ignal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer’s disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (
p
rotein interacting with
A
PP
t
ail
1
), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.
Adenovirus E4orf6 oncoprotein modulates the function of the p53-related protein, p73
Authors: Higashino Fumihiro , Pipas James M. , Shenk Thomas
Published: July 2002

Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.