bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic conversion of drug-resistant bacteria to drug sensitivity
Authors: Guerrier-Takada Cecilia , Salavati Reza , Altman Sidney
Published: July 2002

Plasmids that contain synthetic genes coding for small oligoribonucleotides called external guide sequences (EGSs) have been introduced into strains of
Escherichia coli
harboring antibiotic resistance genes. The EGSs direct RNase P to cleave the mRNAs transcribed from these genes thereby converting the phenotype of drug-resistant cells to drug sensitivity. Increasing the EGS-to-target mRNA ratio by changing gene copy number or the number of EGSs complementary to different target sites enhances the efficiency of the conversion process. We demonstrate a general method for the efficient phenotypic conversion of drug-resistant bacterial cultures.
Bordetella bronchiseptica
dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho
Authors: Horiguchi Yasuhiko , Inoue Norimitsu , Masuda Minako , Kashimoto Takashige , Katahira Jun , Sugimoto Nakaba , Matsuda Morihiro
Published: July 2002

Bordetella
dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5′-[γ-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.
Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of
Saccharomyces cerevisiae
splicing factor Prp43
Authors: Gee Sherry , Krauss Sharon Wald , Miller Estelle , Aoyagi Kazuko , Arenas Jaime , Conboy John G.
Published: July 2002

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are ≈65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in
S. cerevisiae
bearing a temperature-sensitive mutation in
prp43
was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes
prp16
or
prp22
. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.
Plasmodium falciparum
antigenic diversity: Evidence of clonal population structure
Authors: Rich Stephen M. , Hudson Richard R. , Ayala Francisco J.
Published: July 2002

Plasmodium falciparum
, the agent of malignant malaria, is one of mankind’s most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite’s rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of
P. falciparum
from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (
i
) the incidence of recombination events does not increase with nucleotide distance; (
ii
) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (
iii
) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (
i
) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (
ii
) the population structure of
P. falciparum
is clonal—a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.
Regulation of ribonuclease III processing by double-helical sequence antideterminants
Authors: Zhang Kejing , Nicholson Allen W.
Published: July 2002

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay.
Escherichia coli
ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson–Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these “disfavored” sequences in a model substrate strongly inhibited cleavage
in vitro
by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNA
Sec
, which acts as an antideterminant of EF-Tu binding to tRNA
Sec
. The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions.
Differential pre- and postsynaptic modulation of chemical transmission in the squid giant synapse by tyrosine phosphorylation
Authors: Llinás Rodolfo , Moreno Herman , Sugimori Mutsuyuki , Mohammadi Moosa , Schlessinger Joseph
Published: July 2002

To assess the role of tyrosine phosphorylation/dephosphorylation balance in synaptic transmission, a set of studies was implemented at the squid giant synapse. Presynaptic induction of tyrosine phosphorylation, following administration of the tyrosine phosphatase inhibitor pervanadate, produced a sizable increase in presynaptic calcium current and a concomitant and paradoxical decrement of the postsynaptic potential amplitude. Presynaptic microinjection of an active protein tyrosine kinase dramatically increased calcium currents and incremented postsynaptic potential amplitude. By contrast, the same procedure at the postsynaptic terminal reduced the size of the postsynaptic potential. This differential effect may be prodromic to long-term plasticity, as postsynaptic sensitivity is momentarily deemphasized, whereas presynaptic second messenger cascades triggered by increased calcium currents are accentuated.
Detection of heterozygous truncating mutations in the
BRCA1
and
APC
genes by using a rapid screening assay in yeast
Authors: Ishioka Chikashi , Suzuki Takao , FitzGerald Michael , Krainer Michael , Shimodaira Hideki , Shimada Akira , Nomizu Tadashi , Isselbacher Kurt J. , Haber Daniel , Kanamaru Ryunosuke
Published: July 2002

The detection of inactivating mutations in tumor suppressor genes is critical to their characterization, as well as to the development of diagnostic testing. Most approaches for mutational screening of germ-line specimens are complicated by the fact that mutations are heterozygous and that missense mutations are difficult to interpret in the absence of information about protein function. We describe a novel method using
Saccharomyces cerevisiae
for detecting protein-truncating mutations in any gene of interest. The PCR-amplified coding sequence is inserted by homologous recombination into a yeast URA3 fusion protein, and transformants are assayed for growth in the absence of uracil. The high efficiency of homologous recombination in yeast ensures that both alleles are represented among transformants and achieves separation of alleles, which facilitates subsequent nucleotide sequencing of the mutated transcript. The specificity of translational initiation of the
URA3
gene leads to minimal enzymatic activity in transformants harboring an inserted stop codon, and hence to reliable distinction between specimens with wild-type alleles and those with a heterozygous truncating mutation. This yeast-based stop codon assay accurately detects heterozygous truncating mutations in the
BRCA1
gene in patients with early onset of breast cancer and in the
APC
gene in patients with familial adenomatous polyposis. This approach offers a rapid and reliable method for genetic diagnosis in individuals at high risk for germ-line mutations in cancer susceptibility genes.
Targeting transforming growth factor α expression to discrete loci of the neuroendocrine brain induces female sexual precocity
Authors: Rage Florence , Hill Diane F. , Sena-Esteves Miguel , Breakefield Xandra O. , Coffey Robert J. , Costa Maria E. , McCann Samuel M. , Ojeda Sergio R.
Published: July 2002

Precocious puberty of cerebral origin is a poorly understood disorder of human sexual development, brought about by the premature activation of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation. An increased production of transforming growth factor α (TGFα) in the hypothalamus has been implicated in the mechanism underlying both normal and precocious puberty. We have now used two gene delivery systems to target TGFα overexpression near LHRH neurons in immature female rats. Fibroblasts infected with a retroviral construct in which expression of the human TGFα gene is constitutively driven by the phosphoglycerate kinase promoter, or transfected with a plasmid in which TGFα expression is controlled by an inducible metallothionein promoter, were transplanted into several regions of the hypothalamus. When the cells were in contact with LHRH nerve terminals or in the vicinity of LHRH perikarya, sexual maturation was accelerated. These results suggest that precocious puberty of cerebral origin may result from a focal disorder of TGFα production within the confines of the LHRH neuron microenvironment.
Mass spectrometry of ribosomes and ribosomal subunits
Authors: Benjamin Dennis R. , Robinson Carol V. , Hendrick Joseph P. , Hartl F. Ulrich , Dobson Christopher M.
Published: July 2002

Nanoflow electrospray ionization has been used to introduce intact
Escherichia coli
ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein–protein and protein–RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.
Transdominant genetic analysis of a growth control pathway
Authors: Caponigro Giordano , Abedi Majid R. , Hurlburt Anthony P. , Maxfield Andrew , Judd Weston , Kamb Alexander
Published: July 2002

Genetic selections that use proteinaceous transdominant inhibitors encoded by DNA libraries to cause mutant phenocopies may facilitate genetic analysis in traditionally nongenetic organisms. We performed a selection for random short peptides and larger protein fragments (collectively termed “perturbagens”) that inhibit the yeast pheromone response pathway. Peptide and protein fragment perturbagens that permit cell division in the presence of pheromone were recovered. Two perturbagens were derived from proteins required for pheromone response, and an additional two were derived from proteins that may negatively influence the pheromone response pathway. Furthermore, three known components of the pathway were identified as probable perturbagen targets based on physical interaction assays. Thus, by selection for transdominant inhibitors of pheromone response, multiple pathway components were identified either directly as gene fragments or indirectly as the likely targets of specific perturbagens. These results, combined with the results of previous work [Holzmayer, T. A., Pestov, D. G. & Roninson, I. B. (1992)
Nucl. Acids. Res.
20, 711–717; Whiteway, M., Dignard, D. & Thomas, D. Y. (1992)
Proc. Natl. Acad. Sci. USA
89, 9410–9414; and Gudkov, A. V., Kazarov, A. R., Thimmapaya, R., Axenovich, S. A., Mazo, I. A. & Roninson, I. B. (1994)
Proc. Natl. Acad. Sci. USA
91, 3744–3748], suggest that transdominant genetic analysis of the type described here will be broadly applicable.
3′-Azido-3′-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes
Authors: Ren Jingshan , Esnouf Robert M. , Hopkins Andrew L. , Jones E. Yvonne , Kirby Ian , Keeling James , Ross Carl K. , Larder Brendan A. , Stuart David I. , Stammers David K.
Published: July 2002

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT- and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.
Endogenous E2F-1 promotes timely G
0
exit of resting mouse embryo fibroblasts
Authors: Wang Zhiyan M. , Yang Hong , Livingston David M.
Published: July 2002

Much evidence strongly suggests a positive role for one or more E2F species in the control of exit from G
0
/G
1
. Results described here provide direct evidence that endogenous E2F-1, as predicted, contributes to progression from G
0
to S. By contrast, cycling cells lacking an intact E2F-1 gene demonstrated normal cell cycle distribution. Therefore, E2F-1 exerts a unique function leading to timely G
0
exit of resting cultured primary cells, while at the same time being unnecessary for normal G
1
to S phase progression of cycling cells.
Autoreactivity of T cells from nonobese diabetic mice: An I-Ag7-dependent reaction
Authors: Kanagawa Osami , Martin Steven M. , Vaupel Barbara A. , Carrasco-Marin Eugenio , Unanue Emil R.
Published: July 2002

Mice bearing the I-Ag7class II major histocompatibility complex molecules contain a high number of spontaneous autoreactive T cells, as estimated by limiting-dilution assays. We found this autoreactivity in various strains that bear the I-Ag7molecule, such as the nonobese diabetic (NOD) mouse strain, which spontaneously develops autoimmune diabetes. However, NOD mice strains that do not express the I-Ag7molecule, but instead express I-Ab, do not have a high incidence of autoreactive T cells. About 15% of the autoreactive T cells also recognize the I-Ag7molecule expressed in the T2 line, which is defective in the processing of protein antigens. We interpret this to mean that some of the T cells may interact with class II molecules that are either devoid of peptides or contain a limited peptide content. We also find a high component of autoreactivity among antigen-specific T cell clones. These T cell clones proliferate specifically to protein antigens but also have a high level of reactivity to antigen-presenting cells not pulsed with antigen. Thus, the library of T cell receptors in NOD mice is skewed to autoreactivity, which we speculate is based on the weak peptide-binding properties of I-Ag7molecules.
Biotechnology: Enhancing human nutrition in developing and developed worlds
Authors: Kishore Ganesh M. , Shewmaker Christine
Published: July 2002

While the last 50 years of agriculture have focused on meeting the food, feed, and fiber needs of humans, the challenges for the next 50 years go far beyond simply addressing the needs of an ever-growing global population. In addition to producing more food, agriculture will have to deal with declining resources like water and arable land, need to enhance nutrient density of crops, and achieve these and other goals in a way that does not degrade the environment. Biotechnology and other emerging life sciences technologies offer valuable tools to help meet these multidimensional challenges. This paper explores the possibilities afforded through biotechnology in providing improved agronomic “input” traits, differentiated crops that impart more desirable “output” traits, and using plants as green factories to fortify foods with valuable nutrients naturally rather than externally during food processing. The concept of leveraging agriculture as green factories is expected to have tremendous positive implications for harnessing solar energy to meet fiber and fuel needs as well. Widespread adaptation of biotech-derived products of agriculture should lay the foundation for transformation of our society from a production-driven system to a quality and utility-enhanced system.
Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation
Authors: Tien Ming , Berlett Barbara S. , Levine Rodney L. , Chock P. Boon , Stadtman Earl R.
Published: July 2002

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO
2
.
At physiological concentrations of CO
2
(1.3 mM), the generation of carbonyl groups (0.2–0.4 equivalents/subunit) is little affected by pH over the range of 7.2–9.0, but, in the absence of CO
2
, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO
2
.
However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases ≈65% with increasing pH over the range of 7.2–8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4–8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO
2
at both high and low pH values. These results emphasize the importance of controlling the pH and CO
2
concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO
2
concentrations.
Relative rates of insertion and deletion mutations in a microsatellite sequence in cultured cells
Authors: Twerdi Christine D. , Boyer Jayne C. , Farber Rosann A.
Published: July 2002

A cell culture system has been used to determine the relative rates of insertions and deletions of integral numbers of dinucleotide repeats in a microsatellite sequence. A plasmid was constructed that contained 17 repeats of poly(dC-dA)⋅poly(dG-dT) near the 5′ end of a bacterial neomycin-resistance (
neo)
gene, such that the
neo
gene was translated in the (+1) reading frame. The plasmid was introduced into mismatch-repair-proficient and mismatch-repair-deficient mammalian cell lines. Rates of mutation to resistance to the neomycin analogue G418 were measured, and the nature of the mutations was determined. The mutations were all gains or losses of integral numbers of repeats, and mutations involving a single repeat greatly predominated over those involving multiple repeats. The data obtained from these studies were compared with results previously obtained with cells transfected with a similar plasmid in which the sequence of the oligonucleotide insert placed the
neo
gene in the (−1) reading frame. This experimental design made it possible to make direct comparisons between insertions and deletions of a single repeat unit. A significant excess of insertions over deletions was found in both repair-proficient and repair-deficient cell lines, although the few mutations involving more than two repeats were deletions.
Signal transfer from rhodopsin to the G-protein: Evidence for a two-site sequential fit mechanism
Authors: Kisselev Oleg G. , Meyer Christoph K. , Heck Martin , Ernst Oliver P. , Hofmann Klaus Peter
Published: July 2002

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein–protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor’s signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt γ-subunit, Gtγ(50–71)farnesyl, can replace the holoprotein in stabilizing rhodopsin’s active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtβγ complex in direct activation assays. Competition by Gtγ(50–71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtβγ in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt α-subunit, Gtα(340–350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R* by Gtα(340–350) and Gtγ(50–71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.
The cell surface-associated intercellular C-signal induces behavioral changes in individual
Myxococcus xanthus
cells during fruiting body morphogenesis
Authors: Jelsbak Lars , Søgaard-Andersen Lotte
Published: July 2002

Fruiting body formation in
Myxococcus xanthus
depends on ordered changes in cell movements from swarming to aggregation in response to starvation. We show that appropriately starved individual cells change behavior during fruiting body formation. Specifically, from the time of initiation of aggregation, individual wild-type cells began to move with increased gliding speeds, the duration of the mean gliding interval increased, and the stop frequency decreased whereas the duration of the mean stop interval and the reversal frequency remained unchanged. Mutants lacking the cell surface-associated, intercellular C-signal (
csgA
mutants) failed to aggregate. Likewise, appropriately starved individual
csgA
cells did not change their behavior during development. In the absence of other cell–cell interactions, the motility defect of individual
csgA
cells was corrected in a time- and concentration-dependent manner after C-signaling was reestablished by exogenous MalE-CsgA protein. The C-signal-induced stimulation of motility depended on the cytoplasmic Frz signal transduction system. We propose that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development.
Ultraviolet pigments in birds evolved from violet pigments by a single amino acid change
Authors: Yokoyama Shozo , Radlwimmer F. Bernhard , Blow Nathan S.
Published: July 2002

UV vision has profound effects on the evolution of organisms by affecting such behaviors as mating preference and foraging strategies. Despite its importance, the molecular basis of UV vision is not known. Here, we have transformed the zebra finch UV pigment into a violet pigment by incorporating one amino acid change, C84S. By incorporating the reverse mutations, we have also constructed UV pigments from the orthologous violet pigments of the pigeon and chicken. These results and comparative amino acid sequence analyses of the pigments in vertebrates demonstrate that many avian species have achieved their UV vision by S84C.
Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F
0
F
1
-ATPase
Authors: Salomon Arthur R. , Voehringer David W. , Herzenberg Leonard A. , Khosla Chaitan
Published: July 2002

Recently, a family of polyketide inhibitors of
F
0
F
1
-ATPase, including apoptolidin, ossamycin,
and oligomycin, were shown to be among the top 0.1% most cell line
selective cytotoxic agents of 37,000 molecules tested against the 60
human cancer cell lines of the National Cancer Institute. Many cancer
cells maintain a high level of anaerobic carbon metabolism even in the
presence of oxygen, a phenomenon that is historically known as the
Warburg effect. A mechanism-based strategy to sensitize such cells to
this class of potent small molecule cytotoxic agents is presented.
These natural products inhibit oxidative phosphorylation by targeting
the mitochondrial F
0
F
1
ATP synthase. Evaluation
of gene expression profiles in a panel of leukemias revealed a strong
correlation between the expression level of the gene encoding subunit 6
of the mitochondrial F
0
F
1
ATP synthase (known
to be the binding site of members of this class of macrolides) and
their sensitivity to these natural products. Within the same set of
leukemia cell lines, comparably strong drug–gene correlations were
also observed for the genes encoding two key enzymes involved in
central carbon metabolism, pyruvate kinase, and aspartate
aminotransferase. We propose a simple model in which the mitochondrial
apoptotic pathway is activated in response to a shift in
balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of
both lactate formation and carbon flux through the Embden–Meyerhof
pathway significantly sensitized apoptolidin-resistant tumors to this
drug. Nine different cell lines derived from human leukemias and
melanomas, and colon, renal, central nervous system, and ovarian tumors
are also sensitized to killing by apoptolidin.
Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)
Authors: Behrens Axel , Sabapathy Kanaga , Graef Isabella , Cleary Mike , Crabtree Gerald R. , Wagner Erwin F.
Published: July 2002

The Jun N-terminal kinases (JNKs) recently have been shown to be
required for thymocyte apoptosis and T cell differentiation
and/or proliferation. To investigate the molecular targets of JNK
signaling in lymphoid cells, we used mice in which the serines
phosphorylated by JNK in c-Jun were replaced by homologous
recombination with alanines (
junAA
mice). Lymphocytes
from these mice showed no phosphorylation of c-Jun in response to
activation stimuli, whereas c-Jun was rapidly phosphorylated in
wild-type cells. Despite the fact that
c-jun
is
essential for early development,
junAA
mice develop
normally; however, c-Jun N-terminal phosphorylation was required for
efficient T cell receptor-induced and tumor necrosis factor-α-induced
thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK
is not required for T cell proliferation or differentiation. Because
jnk2
−/− T cells display a proliferation defect, we
concluded that JNK2 must have other substrates required for lymphocyte
function. Surprisingly,
jnk2
−/− T cells showed
reduced NF-AT DNA-binding activity after activation. Furthermore,
overexpression of JNK2 in Jurkat T cells strongly enhanced
NF-AT-dependent transcription. These results demonstrate that JNK
signaling differentially uses c-Jun and NF-AT as molecular effectors
during thymocyte apoptosis and T cell proliferation.
Tyrosine Phosphorylation of Eps15 Is Required for Ligand-Regulated, but Not Constitutive, Endocytosis
Authors: Confalonieri Stefano , Salcini Anna Elisabetta , Puri Claudia , Tacchetti Carlo , Di Fiore Pier Paolo
Published: July 2002

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.
Aggresomes Resemble Sites Specialized for Virus Assembly
Authors: Heath Colin M. , Windsor Miriam , Wileman Thomas
Published: July 2002

The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; García-Mata, R., Z. Bebök, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239–1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.
Evasion of Cytotoxic T Lymphocyte (CTL) Responses by Nef-dependent Induction of Fas Ligand (CD95L) Expression on Simian Immunodeficiency Virus–infected Cells
Authors: Xu Xiao-Ning , Screaton Gavin R. , Gotch Frances M. , Dong Tao , Tan Rusung , Almond Neil , Walker Barry , Stebbings Richard , Kent Karen , Nagata Shigekazu , Stott Jim E. , McMichael Andrew J.
Published: July 2002

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four–amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4+ and CD8+ populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.
Interleukin 2–mediated Uncoupling of T Cell Receptor α/β from CD3 Signaling
Authors: Haughn Loralee , Leung Bernadine , Boise Lawrence , Veillette André , Thompson Craig , Julius Michael
Published: July 2002

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56lck is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2–dependent, antigen-specific CD4+ T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3ε-specific monoclonal antibody. Survival of this IL-2–dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR–induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4+ T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70–pp21ζ complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.
Targeted Gene Disruption Reveals an Adhesin Indispensable for Pathogenicity of Blastomyces dermatitidis
Authors: Tristan Brandhorst T. , Wüthrich Marcel , Warner Thomas , Klein Bruce
Published: July 2002

Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood. In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products. In Blastomyces dermatitidis, the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor. DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer. These findings establish the pivotal role of WI-1 in adherence and virulence of B. dermatitidis yeasts. To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms.
Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47
Authors: Bain Gretchen , Quong Melanie W. , Soloff Rachel S. , Hedrick Stephen M. , Murre Cornelis
Published: July 2002

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.
The interpretation of experimental scattering cross sections with pseudopotential calculations for the alkali-mercury interaction
Authors: R Duren
Published: July 2002

For the example of collisions of alkali metals with mercury the potentials for the ground state and the lowest excited states are determined by a comparison of a pseudopotential calculation with experiments. The potentials for Na, K, Rb and Cs are described by one common pair of parameters in the pseudopotential. From this example a new method for the evaluation of scattering data is derived, which should allow a uniform description of the potentials of a class of collision systems.
Total cross sections for electron scattering by He
Authors: F J de Heer  , R H J Jansen
Published: July 2002

A set of total cross sections for scattering of electrons by He has been evaluated over the energy range of zero to 3000 eV by means of the analysis of experiments and theories on total cross sections for elastic scattering, ionisation and excitation, and on differential cross sections for elastic and inelastic scattering. Between 0 and 19.8 eV, where no inelastic processes occur, the total cross sections for scattering are equal to those for elastic scattering. Above 19.8 eV the authors have evaluated total cross sections for scattering of electrons by adding those for ionisation, excitation and elastic scattering. The total cross sections thus obtained are probably accurate to about 5% over a large part of the energy range. They appear to be in good agreement with the experimental results of Blaauw et al. (1977).
A semiclassical study of the body-fixed approximation for rotational excitation in atom-molecule collisions
Authors: A S Dickinson  , D Richards
Published: July 2002

Approximations similar to those made in deriving the coupled-states approximation have been studied using the strong-coupling correspondence principle. It is shown that the validity of this approximation depends upon the properties of certain fundamental trajectory integrals and a detailed study of these integrals has been made. This approximation to the strong-coupling correspondence principle has been used to calculate cross sections for Ne-N2, Ar-N2and Ar-TlF collisions. Comparison with other calculations shows that the approximation is most successful for Ar-N2and least successful for Ar-TlF collisions.
Rotational transitions in collisions between polar molecules: an application of the semiclassical strong-coupling method to HCl-HCl collisions
Authors: S S Bhattacharyya  , S Saha  , A K Barua
Published: July 2002

Probabilities and cross sections for simultaneous rotational transitions in collision between HCl molecules have been calculated for a relative kinetic energy of 0.025 eV. The calculations have been made for two sets of initial states, j10=0, j20=1 and j10=0, j20=0. All energetically accessible rotational channels were included in the semiclassical strong-coupling formalism. The special features of the resonant transition have been shown. The oscillatory behaviour of various transition probabilities with impact parameters have been physically interpreted as the consequence of interference between different intermediate transitions leading to the same final state, as well as between different terms of the coupling potential.
Theoretical study of the elastic and charge exchange processes in H++Cs collision
Authors: V Sidis  , C Kubach
Published: July 2002

A projected valence bond method is used to obtain adiabatic and diabatic states for the study of the H++Cs collisional system. By freezing an adjusted Hartree Fock type Cs+core the electronic problem is reduced to that of treating the outer electron of the (H-Cs)+quasimolecule. The present treatment is designed to correct some serious defects of previous theoretical approaches. The calculated well in the adiabatic potential energy curve correlated with H++Cs satisfactorily accounts for the rainbow structures observed recently by Scheidt and colleagues (1977, 1978) in the measured elastic differential cross sections at low energy.
Electron density measurements using the Stark-broadened line wings of hydrogenic ions in laser-produced plasmas
Authors: C C Smith  , N J Peacock
Published: July 2002

It is shown that the measurement of wing intensities of those Stark-broadened Lyman lines, for which the upper level is thermally populated with respect to the free electrons and fully stripped ions, enables accurate deduction of electron density in a high temperature plasma. The parameters which characterise the plasma microfield distributions are defined in such a way as to allow the use of existing tabulated values of the electric microfield to construct accurate theoretical profiles. The method is illustrated using emission lines from laser-irradiated solid polymer targets.
A quantum theory of ionisation in fast collisions between ions and atomic systems
Authors: Dz Belkic
Published: July 2002

Ionisation of atomic systems by fast ions is considered. A second-order quantum theory is derived which correctly accounts for all distortions of the initial- and final-state wavefunctions arising from Coulomb interactions between three charged particles. The present transition amplitude is the rigorous first-order (or lowest order) approximation to the exact scattering amplitude for ionisation treated by the Faddeev equations in the distorted wave formalism. The problem is briefly studied by the impact parameter method as well, and its equivalence with the full quantal treatment is proved (for projectiles mainly scattered forward) in the limit of very large values of the reduced mass. Ionisation of atomic hydrogen by proton impact is examined in considerable detail at incident energies between 25 and 200 keV. Comprehensive comparisons of available experimental data with singly differential as well as total cross sections for electron ejection are made and the validity of the present method is assessed.
Model-potential methods for the calculation of atom-rare-gas interaction: application to the H-He system
Authors: P Valiron  , R Gayet  , R McCarroll  , F Masnou-Seeuws  , M Philippe
Published: July 2002

The theory of model-potential methods is re-examined with particular reference to the calculation of the interaction of one-valence (or Rydberg) electron atoms with rare gases. It is shown that the use of a purely local potential to represent the rare-gas core may lead to spurious curve crossings in the molecular calculation. Such difficulties can be avoided by the introduction of a non-local term which simulates the Pauli principle, a procedure equivalent to constraining the model-potential wavefunction to be orthogonal to the one-electron orbitals of the inner shells. The theory is applied to obtain a model potential for He, optimised to the best available electron scattering data. This model potential is subsequently used to calculate the long and intermediate range interaction of the x2Sigma ground state of H-He. The potential well depth (0.39 meV) at 7 a0is in excellent agreement with the best experimental and theoretical determinations.
Deflection functions for curve-crossing collisions
Authors: A Barany
Published: July 2002

An exact parametrisation of the two-state close-curve-crossing reduced scattering matrix for the BNO or DT model is used to define semiclassical deflection functions for impact parameters close to and beyond the crossing radius. These deflection functions connect smoothly with the ones defined from the Stueckelberg scattering matrix for smaller impact parameters and are shown to behave classically as the impact parameter increases through the crossing region.
The drift of thermal electrons in dense gases
Authors: V Dallacasa
Published: July 2002

The author considers the solution of the Boltzmann equation for the drift velocity of electrons in gases, at high number densities N and low E/N values. The result is shown to coincide with that obtained by a quantum-mechanical Kubo-like approach presented recently. The correction term, with respect to the conventional form, is worked out in detail by means of phaseshift and dispersion relation analyses and discussed in connection with recent investigations.
Circularly polarised resonance fluorescence of85Rb excited with pi polarised D2line
Authors: M Krainska-Miszczak
Published: July 2002

Circularly polarised components of the resonance fluorescence of85Rb excited with the pi polarised D2line are investigated in a magnetic field in the range 0-170 G. A difference in the intensities of the sigma-and sigma+components is reported. As a result orientation can be obtained in the ground state of alkali atoms.