bluuebunny/crossref_metadata_2025 huggingface dataset:
The human pathogen
Pseudomonas aeruginosa
utilizes conserved virulence pathways to infect the social amoeba
Dictyostelium discoideum
Authors: Pukatzki Stefan , Kessin Richard H. , Mekalanos John J.
Published: July 2002

Genetically accessible host models are useful for studying microbial pathogenesis because they offer the means to identify novel strategies that pathogens use to evade immune mechanisms, cause cellular injury, and induce disease. We have developed conditions under which the human pathogen
Pseudomonas aeruginosa
infects
Dictyostelium discoideum
, a genetically tractable eukaryotic organism. When
D. discoideum
is plated on nutrient agar plates with different
P. aeruginosa
strains, the bacteria form lawns on these plates with amoebae embedded in them. Virulent
P. aeruginosa
strains kill these amoebae and leave an intact bacterial lawn. A number of
P. aeruginosa
mutants have been identified that are avirulent in this assay. Amoebae feed on these bacteria and form plaques in their bacterial lawns. One avirulent mutant strain carries an insertional mutation in the
lasR
gene. LasR is a transcription factor that controls a number of virulence genes in a density-dependent fashion. Another class of avirulent
P. aeruginosa
mutants is defective in type III secretion. One mutant lacks the PscJ protein, a structural component of the secretion apparatus, suggesting that cytotoxins are injected into the
D. discoideum
cell. One of these cytotoxins is ExoU, and
exoU
mutants are avirulent toward
D. discoideum
. Complementation of the
lasR
and
exoU
mutations restores virulence. Therefore,
P. aeruginosa
uses conserved virulence pathways to kill
D. discoideum
.
Fear recognition in the voice is modulated by unconsciously recognized facial expressions but not by unconsciously recognized affective pictures
Authors: de Gelder Beatrice , Pourtois Gilles , Weiskrantz Lawrence
Published: July 2002

Multisensory integration is a powerful mechanism for increasing adaptive responses, as illustrated by binding of fear expressed in a face with fear present in a voice. To understand the role of awareness in intersensory integration of affective information we studied multisensory integration under conditions of conscious and nonconscious processing of the visual component of an audiovisual stimulus pair. Auditory-event-related potentials were measured in two patients (GY and DB) who were unable to perceive visual stimuli consciously because of striate cortex damage. To explore the role of conscious vision of audiovisual pairing, we also compared audiovisual integration in either naturalistic pairings (a facial expression paired with an emotional voice) or semantic pairings (an emotional picture paired with the same voice). We studied the hypothesis that semantic pairings, unlike naturalistic pairings, might require mediation by intact visual cortex and possibly by feedback to primary cortex from higher cognitive processes. Our results indicate that presenting incongruent visual affective information together with the voice translates as an amplitude decrease of auditory-event-related potentials. This effect obtains for both naturalistic and semantic pairings in the intact field, but is restricted to the naturalistic pairings in the blind field.
Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms
Authors: Espey Michael Graham , Xavier Sandhya , Thomas Douglas D. , Miranda Katrina M. , Wink David A.
Published: July 2002

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 μM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC
50
≈ 20 μM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC
50
> 250 μM). The more physiologically relevant conditions of either peroxynitrite infusion (1 μM/min) or
de novo
formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO
2
) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO
2
can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of NO
2
may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.
A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit
Authors: Belova Larissa , Tenson Tanel , Xiong Liqun , McNicholas Paul M. , Mankin Alexander S.
Published: July 2002

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants ofHalobacterium halobiumcontained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.
Protein deamidation
Authors: Robinson Noah E.
Published: July 2002

A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically relevant phenomenon in a remarkably large percentage of proteins.
Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization
Authors: Yang F.-C. , Atkinson S. J. , Gu Y. , Borneo J. B. , Roberts A. W. , Zheng Y. , Pennington J. , Williams D. A.
Published: July 2002

Critical to homeostasis of blood cell production by hematopoietic
stem/progenitor (HSC/P) cells is the regulation of HSC/P
retention within the bone marrow microenvironment and migration between
the bone marrow and the blood. Key extracellular regulatory elements
for this process have been defined (cell–cell adhesion, growth
factors, chemokines), but the mechanism by which HSC/P cells
reconcile multiple external signals has not been elucidated. Rac and
related small GTPases are candidates for this role and were studied in
HSC/P deficient in Rac2, a hematopoietic cell-specific family member.
Rac2 appears to be critical for HSC/P adhesion both
in
vitro
and
in vivo
, whereas a compensatory
increase in Cdc42 activation regulates HSC/P migration. This genetic
analysis provides physiological evidence of cross-talk between GTPase
proteins and suggests that a balance of these two GTPases controls
HSC/P adhesion and mobilization
in vivo
.
Changes in the prevalence of chronic disability in the United States black and nonblack population above age 65 from 1982 to 1999
Authors: Manton Kenneth G. , Gu XiLiang
Published: July 2002

Survey evidence through the early 1990s generally suggests a reduction in disability in the elderly population of the United States. Because the evidence is not fully consistent, several authors have speculated about whether disability declines will continue. This paper reports results from the 1999 National Long-Term Care Survey on disability trends from 1982 through 1999. It is found that disability continued to decline in the 1994 to 1999 period, and that the decline was greater in the 1990s than in the 1980s. The disability decline from 1982 to 1989 was 0.26% per year, from 1989 to 1994 it was 0.38% per year, and from 1994 to 1999 it was 0.56% per year. In addition, disability declined by a greater percentage for blacks than for nonblacks over the 1989 to 1999 period.
Computational subunits of visual cortical neurons revealed by artificial neural networks
Authors: Lau Brian , Stanley Garrett B. , Dan Yang
Published: July 2002

A crucial step toward understanding visual processing is to obtain a comprehensive description of the relationship between visual stimuli and neuronal responses. Many neurons in the visual cortex exhibit nonlinear responses, making it difficult to characterize their stimulus–response relationships. Here, we recorded the responses of primary visual cortical neurons of the cat to spatiotemporal random-bar stimuli and trained artificial neural networks to predict the response of each neuron. The random initial connections in the networks consistently converged to regular patterns. Analyses of these connection patterns showed that the response of each complex cell to the random-bar stimuli could be well approximated by the sum of a small number of subunits resembling simple cells. The direction selectivity of each complex cell measured with drifting gratings was also well predicted by the combination of these subunits, indicating the generality of the model. These results are consistent with a simple functional model for complex cells and demonstrate the usefulness of the neural network method for revealing the stimulus–response transformations of nonlinear neurons.
Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: A soluble, secreted CD30 homologue
Authors: Panus Joanne Fanelli , Smith Craig A. , Ray Caroline A. , Smith Terri Davis , Patel Dhavalkumar D. , Pickup David J.
Published: July 2002

Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus. Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene,vCD30,encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus. The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51–59% identity with highly conserved extracellular segments of both mouse and human CD30. A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions. Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses. Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases.
The relaxation dynamics of the excited electronic states of retinal in bacteriorhodopsin by two-pump-probe femtosecond studies
Authors: Logunov S. L. , Volkov V. V. , Braun M. , El-Sayed M. A.
Published: July 2002

We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S
1
) populated by different modes. In the first mode, a direct (S
0
→ S
1
) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S
1
state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S
0
→ S
1
transition whereas the second pulse excites the S
1
→ S

n

transition. The nonradiative relaxation from the S
n
state populates the lowest excited state. Our results suggest that the S
1
state relaxes faster when populated nonradiatively from the S

n

state than when pumped directly by the S
0
→ S
1
excitation. Additionally, the S
n
→ S
1
nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the S
n
state populates a different surface (called S′
1
) than the one being directly excited (S
1
). The possible involvement of the A
g
type states, the J intermediate, and the conical intersection leading to the S
0
or to the isomerization product (K intermediate) are discussed in the framework of the proposed model.
Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses
Authors: Stevenson Philip G. , Efstathiou Stacey , Doherty Peter C. , Lehner Paul J.
Published: July 2002

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8
+
cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4
+
T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.
Effects of DNA sequence and structure on binding of RecA to single-stranded DNA
Authors: Bar-Ziv R. , Libchaber A.
Published: July 2002

Fluorescence anisotropy is used to follow the binding of RecA to short single-stranded DNA (ssDNA) sequences (39 bases) at low DNA and RecA concentration where the initial phase of polymerization occurs. We observe that RecA condensation is extremely sensitive to minute changes in DNA sequences. RecA binds strongly to sequences that are rich in pyrimidines and that lack significant secondary structure and base stacking. We find a correlation between the DNA folding free energy and the onset concentration for RecA binding. These results suggest that the folding of ssDNA and base stacking represent a barrier for RecA binding. The link between secondary structure and binding affinity is further analyzed with two examples: discrimination between two naturally occurring polymorphisms differing by one base and RecA binding on a molecular beacon. A self-assembly model is introduced to explain these observations. We propose that RecA may be used to sense ssDNA sequence and structure.
A genomic island in
Pseudomonas aeruginosa
carries the determinants of flagellin glycosylation
Authors: Arora Shiwani K. , Bangera Mahalaxmi , Lory Stephen , Ramphal Reuben
Published: July 2002

Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in
Pseudomonas aeruginosa
PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30
P. aeruginosa
isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.
DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in
Arabidopsis
Authors: Härtel Heiko , Dörmann Peter , Benning Christoph
Published: July 2002

The galactolipids, mono- and digalactosyldiacylglycerol (DGDG), are the most common nonphosphorous lipids in the biosphere and account for 80% of the membrane lipids found in green plant tissues. These lipids are major constituents of photosynthetic membranes (thylakoids), and a large body of evidence suggests that galactolipids are associated primarily with plastid membranes in seed plants. A null-mutant of
Arabidopsis
(
dgd1
), which lacks the DGDG synthase (DGD1) resulting in a 90% reduction in the amount of DGDG under normal growth conditions, accumulated DGDG after phosphate deprivation up to 60% of the amount present in the wild type. This observation suggests the existence of a DGD1-independent pathway of galactolipid biosynthesis. The fatty acid composition of the newly formed DGDG was distinct, showing an enrichment of 16-carbon fatty acids in the C-1 position of the glycerol backbone of DGDG. Roots with their rudimentary plastids accumulated large amounts of DGDG after phosphate deprivation, suggesting that this galactolipid may be located in extraplastidic membranes. Corroborating evidence for this hypothesis was obtained directly by fractionation of subcellular membranes from leaf tissue and indirectly by lipid analysis of the phosphate-deprived
fad3
mutant primarily deficient in extraplastidic fatty acid desaturation. The discovery of extraplastidic DGDG biosynthesis induced by phosphate deprivation has revealed a biochemical mechanism for plants to conserve phosphate. Apparently, plants replace phospholipids with nonphosphorous galactolipids if environmental conditions such as phosphate deprivation require this for survival.
Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law
Authors: Movileanu Liviu , Bayley Hagan
Published: July 2002

The dependence of the rate on polymer mass was examined for the reaction of four sulfhydryl-directed poly(ethylene glycol) reagents with cysteine residues located in the lumen of the staphylococcal α-hemolysin pore. The logarithms of the apparent rate constants for a particular site in the lumen were proportional to
N
, the number of repeat units in a polymer chain. The proportionality constant was −(
a
/
D
)
5/3
, where
a
is the persistence length of the polymer (≈3.5Å) and
D
is the diameter of the pore. Despite some incongruencies with the assumptions of the derivation, the result suggests that the polymers partition into the lumen of the pore according to the simple scaling law of Daoud and de Gennes,
c
pore
/
c
solution
= exp(−
N
(
a
/
D
)
5/3
). Therefore, the measured reaction rates yield an estimate of the diameter of the pore and might be applied to determine the approximate dimensions of cavities within other similar proteins.
An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease
Authors: Mochizuki Hideki , Hayakawa Hideki , Migita Makoto , Shibata Mamoru , Tanaka Ryota , Suzuki Asuka , Shimo-Nakanishi Yumi , Urabe Takao , Yamada Masanori , Tamayose Kenji , Shimada Takashi , Miura Masayuki , Mizuno Yoshikuni
Published: July 2002

Adeno-associated virus (AAV) vector delivery of an Apaf-1-dominant negative inhibitor was tested for its antiapoptotic effect on degenerating nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. The wild-type caspase recruitment domain of Apaf-1 was used as a dominant negative inhibitor of Apaf-1 (rAAV-Apaf-1-DN-EGFP). An AAV virus vector was used to deliver it into the striatum of C57 black mice, and the animals were treated with MPTP. The number of tyrosine hydroxylase-positive neurons in the substantia nigra was not changed on the rAAV-Apaf-1-DN-EGFP injected side compared with the noninjected side. We also examined the effect of a caspase 1 C285G mutant as a dominant negative inhibitor of caspase 1 (rAAV-caspase-1-DN-EGFP) in the same model. However, there was no difference in the number of tyrosine hydroxylase-positive neurons between the rAAV-caspase-1-DN-EGFP injected side and the noninjected side. These results indicate that delivery of Apaf-1-DN by using an AAV vector system can prevent nigrostriatal degeneration in MPTP mice, suggesting that it could be a promising therapeutic strategy for patients with Parkinson's disease. The major mechanism of dopaminergic neuronal death triggered by MPTP seems to be the mitochondrial apoptotic pathway.
Predicting the clinical status of human breast cancer by using gene expression profiles
Authors: West Mike , Blanchette Carrie , Dressman Holly , Huang Erich , Ishida Seiichi , Spang Rainer , Zuzan Harry , Olson John A. , Marks Jeffrey R. , Nevins Joseph R.
Published: July 2002

Prognostic and predictive factors are indispensable tools in
the treatment of patients with neoplastic disease. For the most part,
such factors rely on a few specific cell surface, histological, or
gross pathologic features. Gene expression assays have the potential to
supplement what were previously a few distinct features with many
thousands of features. We have developed Bayesian regression models
that provide predictive capability based on gene expression data
derived from DNA microarray analysis of a series of primary breast
cancer samples. These patterns have the capacity to discriminate breast
tumors on the basis of estrogen receptor status and also on the
categorized lymph node status. Importantly, we assess the utility and
validity of such models in predicting the status of tumors in
crossvalidation determinations. The practical value of such approaches
relies on the ability not only to assess relative probabilities of
clinical outcomes for future samples but also to provide an honest
assessment of the uncertainties associated with such predictive
classifications on the basis of the selection of gene subsets for each
validation analysis. This latter point is of critical importance in the
ability to apply these methodologies to clinical assessment of tumor
phenotype.
Solid-state NMR studies of the secondary structure of a mutant prion protein fragment of 55 residues that induces neurodegeneration
Authors: Laws David D. , Bitter Hans-Marcus L. , Liu Kai , Ball Haydn L. , Kaneko Kiyatoshi , Wille Holger , Cohen Fred E. , Prusiner Stanley B. , Pines Alexander , Wemmer David E.
Published: July 2002

The secondary structure of a 55-residue fragment of the mouse prion
protein, MoPrP(89–143), was studied in randomly aggregated (dried from
water) and fibrillar (precipitated from water/acetonitrile) forms by
13
C solid-state NMR. Recent studies have shown that the
fibrillar form of the P101L mutant of MoPrP(89–143) is capable of
inducing prion disease in transgenic mice, whereas unaggregated or
randomly aggregated samples do not provoke disease. Through analysis of
13
C chemical shifts, we have determined that both wild-type
and mutant sequence MoPrP(89–143) form a mixture of β-sheet and
α-helical conformations in the randomly aggregated state although the
β-sheet content in MoPrP(89–143, P101L) is significantly higher
than in the wild-type peptide. In a fibrillar state, MoPrP(89–143,
P101L) is completely converted into β-sheet, suggesting that the
formation of a specific β-sheet structure may be required for the
peptide to induce disease. Studies of an analogous peptide from Syrian
hamster PrP verify that sequence alterations in residues 101–117
affect the conformation of aggregated forms of the peptides.
Estrogen receptors and proliferation markers in primary and recurrent breast cancer
Authors: Jensen Elwood V. , Cheng Guojun , Palmieri Carlo , Saji Shigehira , Mäkelä Sari , Van Noorden Susan , Wahlström Torsten , Warner Margaret , Coombes R. Charles , Gustafsson Jan-Åke
Published: July 2002

To elucidate the clinical importance of estrogen receptor (ER) β
in breast cancer, 29 archival primary breast cancer specimens, six
locally recurrent cancers, and five benign mammary tumors were examined
histochemically for ERα, ERβ and the proliferation markers Ki67 and
cyclin A. In benign tumors, most epithelial cells contained
ERβ, but ERα was rare. In primary cancers, both ERα and ERβ
occurred in epithelial cells, the presence of ERβ being associated
with elevated expression of Ki67 and cyclin A, and ERα with decreased
levels. Thus, the highest content of proliferation markers was seen in
primary cancers that were ERα
−
ERβ
+
. Most
Ki67-containing cells coexpressed ERβ, but few showed ERα. In
locally recurring cancers, ERα, ERβ, and Ki67 were more highly
expressed than in the corresponding primary tumors, and many cells
containing ERβ, but few with ERα, expressed Ki67. Surprisingly,
ERβ, but not ERα, was seen in the stromal cells of both primary and
recurrent cancers. Because the response of breast cancers to tamoxifen
therapy is correlated with the presence of ERα, cancer cells that
lack ERα but contain ERβ and proliferation markers represent a
novel population of apparently proliferating cells that probably are
not targeted by the current antiestrogens. Thus, appropriate
ERβ-specific ligands, perhaps in combination with tamoxifen, may be
useful in improving the treatment of breast cancers.
Detection of glutamic acid decarboxylase-activated T cells with I-Ag7tetramers
Authors: Liu Chih-Pin , Jiang Kai , Wu Chun-Hua , Lee Wen-Hui , Lin Wei-Jen
Published: July 2002

CD4+T cells selected by the type 1 diabetes associated class II MHC I-Ag7molecules play a critical role in the disease process. Multivalent MHC/peptide tetramers have been used to directly detect antigen-specific T cells. Detection of autoantigen-activated CD4+T cells with tetramers should be very helpful in the study of the roles of these cells in diabetes. We report here the generation of tetramers of I-Ag7covalently linked to two glutamic acid decarboxylase (GAD) peptides and the detection of GAD peptide-activated T cells from nonobese diabetic (NOD) mice. The I-Ag7heterodimers can form stable complexes with a covalently bound GAD peptide and can stimulate antigen specific T cells. Furthermore, I-Ag7/GAD peptide tetramer can detect most if not all of the antigen-specific CD4+T cells from immunized NOD mice. Antigen-specific T cells detected by the tetramers can up-regulate their CD4 expression on the cell surface after being restimulated with the GAD peptidesin vitro. In contrast, the tetramers can detect a percentage of T cells in lymph nodes and spleens and T cells infiltrating islets from nonimmunized mice that is not significantly above the background. Therefore, T cells specific for the GAD peptides are present in NOD mice at a frequency too low to be detected, but immunization of NOD mice can facilitate their detection by tetramers.
Bell's theorem and the problem of decidability between the views of Einstein and Bohr
Authors: Hess Karl , Philipp Walter
Published: July 2002

Einstein, Podolsky, and Rosen (EPR) have designed a gedanken
experiment that suggested a theory that was more complete than quantum
mechanics. The EPR design was later realized in various forms, with
experimental results close to the quantum mechanical prediction. The
experimental results by themselves have no bearing on the EPR claim
that quantum mechanics must be incomplete nor on the existence of
hidden parameters. However, the well known inequalities of Bell are
based on the assumption that local hidden parameters exist and, when
combined with conflicting experimental results, do appear to prove that
local hidden parameters cannot exist. This fact leaves only
instantaneous actions at a distance (called “spooky” by
Einstein) to explain the experiments. The Bell inequalities are
based on a mathematical model of the EPR experiments. They have no
experimental confirmation, because they contradict the results of all
EPR experiments. In addition to the assumption that hidden parameters
exist, Bell tacitly makes a variety of other assumptions; for instance,
he assumes that the hidden parameters are governed by a single
probability measure independent of the analyzer settings. We argue that
the mathematical model of Bell excludes a large set of local hidden
variables and a large variety of probability densities. Our set of
local hidden variables includes time-like correlated parameters and a
generalized probability density. We prove that our extended space of
local hidden variables does permit derivation of the quantum result and
is consistent with all known experiments.
Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy
Authors: Gomes Marcelo D. , Lecker Stewart H. , Jagoe R. Thomas , Navon Ami , Goldberg Alfred L.
Published: July 2002

Muscle wasting is a debilitating consequence of fasting,
inactivity, cancer, and other systemic diseases that results primarily
from accelerated protein degradation by the ubiquitin-proteasome
pathway. To identify key factors in this process, we have used cDNA
microarrays to compare normal and atrophying muscles and found a
unique gene fragment that is induced more than ninefold in
muscles of fasted mice. We cloned this gene, which is expressed
specifically in striated muscles. Because this mRNA also markedly
increases in muscles atrophying because of diabetes, cancer, and renal
failure, we named it atrogin-1. It contains a functional F-box domain
that binds to Skp1 and thereby to Roc1 and Cul1, the other components
of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization
sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels
increase specifically in skeletal muscle and before atrophy occurs.
Atrogin-1 is one of the few examples of an F-box protein or Ub-protein
ligase (E3) expressed in a tissue-specific manner and appears to be a
critical component in the enhanced proteolysis leading to muscle
atrophy in diverse diseases.
Building highly sensitive dye assemblies for biosensing from molecular building blocks
Authors: Jones Robert M. , Lu Liangde , Helgeson Roger , Bergstedt Troy S. , McBranch Duncan W. , Whitten David G.
Published: July 2002

Fluorescence superquenching is investigated for polyelectrolytes
consisting of cyanine dye pendant polylysines ranging in number of
polymer repeat units (N
PRU
) from 1 to 900, both in solution
and after adsorption onto silica nanoparticles. As
N
PRU
increases, the absorption and fluorescence evolve from
monomer spectra to red-shifted features indicative of molecular J
aggregates. In solution, the superquenching sensitivity toward an
anionic electron acceptor increases by more than a millionfold over the
N
PRU
range from 1 to 900. The dramatic increase is
attributed to enhanced equilibrium constants for binding the quenchers,
and the amplified quenching of a delocalized exciton of ≈100 polymer
repeat units. The self-assembly of monomer onto silica and clay
nanoparticles leads to formation of J aggregates, and surface-activated
superquenching enhanced 10,000× over the monomer in solution,
indicating the formation of “self-assembled polymers” on the
nanoparticle surface. Utilization of these self-assembled polymers as
high-sensitivity biosensors is demonstrated.
A receptor tyrosine kinase from choanoflagellates: Molecular insights into early animal evolution
Authors: King Nicole , Carroll Sean B.
Published: July 2002

The evolution of the Metazoa from protozoans is one of the
major milestones in life's history. The genetic and developmental
events involved in this evolutionary transition are unknown but may
have involved the evolution of genes required for signaling and gene
regulation in metazoans. The genome of animal ancestors may be
reconstructed by identification of animal genes that are shared with
related eukaryotes, particularly those that share a more recent
ancestry and cell biology with animals. The choanoflagellates have long
been suspected to be closer relatives of animals than are fungi, the
closest outgroup of animals for which comparative genomic information
is available. Phylogenetic analyses of choanoflagellate and animal
relationships based on small subunit rDNA sequence, however,
have yielded ambiguous and conflicting results. We find that analyses
of four conserved proteins from a unicellular choanoflagellate,
Monosiga brevicollis
, provide robust support for a close
relationship between choanoflagellates and Metazoa, suggesting that
comparison of the complement of expressed genes from choanoflagellates
and animals may be informative concerning the early evolution of
metazoan genomes. We have discovered in
M. brevicollis
the first receptor tyrosine kinase (RTK), to our knowledge,
identified outside of the Metazoa, MBRTK1. The architecture of MBRTK1,
which includes multiple extracellular ligand-binding domains, resembles
that of RTKs in sponges and humans and suggests the ability to receive
and transduce signals. Thus, choanoflagellates express genes involved
in animal development that are not found in other eukaryotes and that
may be linked to the origin of the Metazoa.
Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells
Authors: Christensen Julie L. , Weissman Irving L.
Published: July 2002

Clonogenic multipotent mouse hematopoietic stem cells (HSCs)
and progenitor cells are contained within the c-kit
+
(K)
lineage
−/lo
(L) Sca-1
+
(S) population of
hematopoietic cells; long-term (LT) and short-term (ST) HSCs are
Thy-1.1
lo
. c-kit is a member of the receptor tyrosine
kinase family, a class of receptors that are important in the
proliferation and differentiation of hematopoietic cells. To establish
whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the
most primitive LT-HSCs, we sorted highly purified multipotent stem and
progenitor cells on the basis of Flk-2 surface expression and used them
in competitive reconstitution assays. Low numbers of
Flk-2
−
HSCs gave rise to long-term multilineage
reconstitution in the majority of recipients, whereas the transfer of
Flk-2
+
multipotent cells resulted in mostly short-term
multilineage reconstitution. The KLS subset of adult mouse bone marrow
was analyzed for Flk-2 and Thy-1.1 expression. Three
phenotypically and functionally distinct populations were isolated:
Thy
lo
Flk-2
−
(LT-HSCs), Thy
lo
Flk-2
+
(ST-HSCs), and Thy
−
Flk-2
+
multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2
expression marks the loss of self-renewal in HSC maturation. The
addition of Flk-2 antibody to the lineage mix allows direct isolation
of LT-HSC from adult bone marrow as c-kit
+
lin
−
Sca-1
+
Flk-2
−
from many
strains of mice. Fetal liver HSCs are contained within
Flk-2
−
and Flk-2
+
KTLS cells.
Onconeural antigens and the paraneoplastic neurologic disorders: at the intersection of cancer, immunity, and the brain.
Authors: Darnell R B
Published: July 2002

Paraneoplastic neurologic disorders (PNDs) are believed to be autoimmune neuronal degenerations that develop in some patients with systemic cancer. A series of genes encoding previously undiscovered neuronal proteins have been cloned using antiserum from PND patients. Identification of these onconeural antigens suggests a reclassification of the disorders into four groups: those in which neuromuscular junction proteins, nerve terminal/vesicle-associated proteins, neuronal RNA binding proteins, or neuronal signal-transduction proteins serve as target antigens. This review considers insights into basic neurobiology, tumor immunology, and autoimmune neuronal degeneration offered by the characterization of the onconeural antigens.
The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.
Authors: Dreyling M H , Martinez-Climent J A , Zheng M , Mao J , Rowley J D , Bohlander S K
Published: July 2002

The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line.
Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.
Authors: Roth M , Eickelberg O , Kohler E , Erne P , Block L H
Published: July 2002

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.
Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.
Authors: Grano M , Galimi F , Zambonin G , Colucci S , Cottone E , Zallone A Z , Comoglio P M
Published: July 2002

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.
An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.
Authors: Bacher M , Metz C N , Calandra T , Mayer K , Chesney J , Lohoff M , Gemsa D , Donnelly T , Bucala R
Published: July 2002

The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.
Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block.
Authors: Kupper J , Ascher P , Neyton J
Published: July 2002

Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.
Iron chelates bind nitric oxide and decrease mortality in an experimental model of septic shock.
Authors: Kazmierski W M , Wolberg G , Wilson J G , Smith S R , Williams D S , Thorp H H , Molina L
Published: July 2002

The hydroxamic acid siderophore ferrioxamine B [FeIII(HDFB)+] and the iron complex of diethylenetri-aminepentaacetic acid [FeIII(DTPA)2-] protected mice against death by septic shock induced by Corynebacterium parvum + lipopolysaccharide. Although FeIII(DTPA)2- was somewhat more effective than FeIII(HDFB)+, the iron-free ligand H4DFB+ was significantly more effective than DTPA. The hydroxamic acid chelator has a much higher iron affinity than the amine carboxylate, allowing for more efficient formation of the FeIII(HDFB)+ complex upon administration of the iron-free ligand. Electrochemical studies show that FeIII(DTPA)2- binds NO stoichiometrically upon reduction to iron(II) at biologically relevant potentials to form a stable NO adduct. In contrast, FeIII(HDFB)+ is a stable and efficient electrocatalyst for the reduction of NO to N2O at biologically relevant potentials. These results suggest that the mechanism of protection against death by septic shock involves NO scavenging and that particularly effective drugs that operate a low dosages may be designed based on the principle of redox catalysis. These complexes constitute a new family of drugs that rely on the special ability of transition metals to activate small molecules. In addition, the wealth of information available on siderophore chemistry and biology provides an intellectual platform for further development.
Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.
Authors: Schonberger O , Knox C , Bibi E , Pines O
Published: July 2002

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.