bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin
Authors: Bednar Jan , Horowitz Rachel A. , Grigoryev Sergei A. , Carruthers Lenny M. , Hansen Jeffrey C. , Koster Abraham J. , Woodcock Christopher L.
Published: July 2002

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.
An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control
Authors: Gualberto Antonio , Aldape Kenneth , Kozakiewicz Krystyna , Tlsty Thea D.
Published: July 2002

Although it is well-established that
p53
functions as a tumor suppressor gene, certain mutations exhibit gain-of-function activities that increase oncogenic transformation. We have found a common class of p53 missense mutation that exhibits a dominant, gain-of-function activity that generates genomic instability. Fibroblasts from Li–Fraumeni syndrome heterozygotes with such mutations generate polyploid cells when exposed to spindle depolymerizing agents. Expression of such mutant alleles in normal fibroblasts yields the same phenotype. This class of dominant, gain-of-function p53 mutation (p53
RSC
, relaxed spindle checkpoint allele) does not require the transcriptional activation function of p53 for this behavior. Thus p53 mutations can contribute to progression of a cancer cell not only by absence of p53 tumor suppressor activity but also by the presence of an activity that promotes genetic instability.
Structure and function of a membrane-bound murine MHC class I molecule
Authors: Celia Hervé , Wilson-Kubalek Elizabeth , Milligan Ronald A. , Teyton Luc
Published: July 2002

MHC molecules are expressed at the surface of nucleated cells to present peptides to T cells. Structural information on MHC molecules has been gathered by x-ray crystallography techniques by using soluble proteins. Although relationships between MHC molecules and cell membranes have not been studied in detail, they are of critical importance for T cell recognition. Using a chemically modified lipid, we have been able to capture and orient histidine-tagged MHC molecules on lipid membranes. Surface plasmon resonance experiments show that the protein binds to the nickel lipid in a specific manner and in an oriented fashion, which allows T cell receptor binding. Similar lipid surfaces have been used to grow two-dimensional crystals and to determine the structure of a membrane-anchored murine H-2K
b
MHC class I molecule. The docking of the crystallographic structure into the three-dimensional reconstructed structure derived from the two-dimensional crystals allows us to determine that the histidine tag is near the membrane surface and that the MHC molecule is in an upright position, exposing the peptide/α1-α2 domains toward the T cell.
Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression
Authors: Brewer Joseph W. , Hendershot Linda M. , Sherr Charles J. , Diehl J. Alan
Published: July 2002

Alterations in normal protein biogenesis and the resulting accumulation of improperly folded proteins in the endoplasmic reticulum (ER) trigger a stress response that up-regulates the expression of ER chaperones, while coordinately repressing overall protein synthesis and causing cell-cycle arrest. Activation of this unfolded protein response (UPR) in mouse NIH 3T3 fibroblasts with the glycosylation inhibitor tunicamycin led to a decline in cyclin D- and E-dependent kinase activities and to G
1
phase arrest. Cyclin D1 protein synthesis was rapidly inhibited by tunicamycin treatment. However, the drug did not significantly affect the mitogen-dependent activities of the extracellular signal-activated protein kinases ERK1 and ERK2 or the level of cyclin D1 mRNA until much later in the response. Therefore, the UPR triggers a signaling pathway that blocks cyclin D1 translation despite continuous mitogenic stimulation. Enforced overexpression of cyclin D1 in tunicamycin-treated cells maintained cyclin D- and E-dependent kinase activities and kept cells in cycle in the face of a fully activated UPR. Translational regulation of cyclin D1 in response to ER stress is a mechanism for checkpoint control that prevents cell-cycle progression until homeostasis is restored.
Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity
Authors: Luo Zhao-Qing , Farrand Stephen K.
Published: July 2002

TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer. The protein requires as coinducer an acyl-homoserine lactone signal molecule called AAI (
Agrobacterium
a
uto
i
nducer) that is produced by the bacteria themselves. TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription. To assess this, we converted TraR into a repressor by placing a copy of the
tra
box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the −10 region of a promoter driving expression of
lacZ
. Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of
Vibrio fischeri
. C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions. The C-terminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator. Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAI-dependent manner. We conclude that TraR recognizes the
tra
box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding.
Evidence for a conserved system for iron metabolism in the mitochondria of
Saccharomyces cerevisiae
Authors: Schilke Brenda , Voisine Cindy , Beinert Helmut , Craig Elizabeth
Published: July 2002

nifU
of nitrogen-fixing bacteria is involved in the synthesis of the Fe–S cluster of nitrogenase. In a synthetic lethal screen with the mitochondrial heat shock protein (HSP)70,
SSQ1
, we identified a gene of
Saccharomyces cerevisiae
,
NFU1
, which encodes a protein with sequence identity to the C-terminal domain of NifU. Two other yeast genes were found to encode proteins related to the N-terminal domain of bacterial NifU. They have been designated
ISU1
and
ISU2
. Isu1, Isu2, and Nfu1 are located in the mitochondrial matrix.
ISU
genes of yeast carry out an essential function, because a
Δisu1Δisu2
strain is inviable. Growth of
Δnfu1Δ isu1
cells is significantly compromised, allowing assessment of the physiological roles of Nfu and Isu proteins. Mitochondria from
Δnfu1Δisu1
cells have decreased activity of several respiratory enzymes that contain Fe–S clusters. As a result,
Δnfu1Δisu1
cells grow poorly on carbon sources requiring respiration.
Δnfu1Δisu1
cells also accumulate abnormally high levels of iron in their mitochondria, similar to
Δssq1
cells, indicating a role for these proteins in iron metabolism. We suggest that
NFU1
and
ISU1
gene products play a role in iron homeostasis, perhaps in assembly, insertion, and/or repair of mitochondrial Fe–S clusters. The conservation of these protein domains in many organisms suggests that this role has been conserved throughout evolution.
Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor
Authors: Andrade-Gordon Patricia , Maryanoff Bruce E. , Derian Claudia K. , Zhang Han-Cheng , Addo Michael F. , Darrow Andrew L. , Eckardt Annette J. , Hoekstra William J. , McComsey David F. , Oksenberg Donna , Reynolds Elwood E. , Santulli Rosemary J. , Scarborough Robert M. , Smith Charles E. , White Kimberly B.
Published: July 2002

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeitnotin human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses inbothcell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.
Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells
Authors: Teichmann Martin , Wang Zhengxin , Martinez Ernest , Tjernberg Agneta , Zhang Di , Vollmer Frank , Chait Brian T. , Roeder Robert G.
Published: July 2002

The TATA-binding protein (TBP)-related factor TRF1, has been
described in
Drosophila
and a related protein, TRF2, has
been found in a variety of higher eukaryotes. We report that human
(h)TRF2 is encoded by two mRNAs with common protein coding but distinct
5′ nontranslated regions. One mRNA is expressed ubiquitously
(hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted
expression pattern and is extremely abundant in testis. In addition, we
show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but
not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2.
Neither recombinant human (rh)TRF2 nor the native flag⋅hTRF2-TFIIA
complex is able to replace TBP or TFIID in basal or activated
transcription from various RNA polymerase II promoters. Instead,
rhTRF2, but not the flag⋅hTRF2–TFIIA complex, moderately inhibits
basal or activated transcription in the presence of rhTBP or
flag⋅TFIID. This effect is either completely (TBP-mediated
transcription) or partially (TFIID-mediated transcription) counteracted
by addition of free TFIIA. Neither rhTRF2 nor flag⋅hTRF2–TFIIA
has any effect on the repression of TFIID-mediated transcription by
negative cofactor-2 (NC2) and neither substitutes for TBP in RNA
polymerase III-mediated transcription.
Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase
Authors: Gladyshev Vadim N. , Stadtman Thressa C. , Hatfield Dolph L. , Jeang Kuan-Teh
Published: July 2002

It has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using
75
Se-labeled human Jurkat T cells, we show that the levels of four
75
Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells. These major selenoproteins migrated as 57-, 26-, 21-, and 15-kDa species on SDS/PAGE gels. In our earlier studies, the 57-kDa protein was purified from T cells and identified as a subunit of thioredoxin reductase. The 26- and 21-kDa proteins were identified in immunoblot assays as the glutathione peroxidase (cGPX or GPX1) subunit and phospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4), respectively. We recently purified the 15-kDa protein and characterized it as a selenoprotein of unknown function. In contrast to selenoproteins, low molecular mass [
75
Se]compounds accumulated during HIV infection and migrated as a diffuse band near the front of SDS/PAGE gels.
sqv
mutants of
Caenorhabditis elegans
are defective in vulval epithelial invagination
Authors: Herman Tory , Hartwieg Erika , Horvitz H. Robert
Published: July 2002

By screening for mutations that perturb the invagination of the vulva of the
Caenorhabditis elegans
hermaphrodite, we have isolated 25 mutations that define eight genes. We have named these genes
sqv-1
to
sqv-8
(squashed vulva). All 25 mutations cause the same vulval defect, an apparent partial collapse of the vulval invagination and an elongation of the central vulval cells. Most
sqv
mutations also cause an oocyte or somatic gonad defect that results in hermaphrodite sterility, and some
sqv
mutations cause maternal-effect lethality. We propose that the
sqv
genes affect a pathway common to vulval invagination, oocyte development, and embryogenesis.
Identification of
SSF1, CNS1,
and
HCH1
as multicopy suppressors of a
Saccharomyces cerevisiae
Hsp90 loss-of-function mutation
Authors: Nathan Debra F. , Vos Melissa Harju , Lindquist Susan
Published: July 2002

Hsp90 functions in a multicomponent chaperone system to promote the
maturation and maintenance of a diverse, but specific, set of target
proteins that play key roles in the regulation of cell growth and
development. To identify additional components of the Hsp90 chaperone
system and its targets, we searched for multicopy suppressors of
various temperature-sensitive mutations in the yeast Hsp90 gene,
HSP82
. Three suppressors were isolated for one Hsp90 mutant
(glutamate → lysine at amino acid 381). Each exhibited a unique,
allele-specific pattern of suppression with other Hsp90 mutants and had
unique structural and biological properties.
SSF1
is a
member of an essential gene family and functions in the response to
mating pheromones.
CNS1
is an essential gene that encodes a
component of the Hsp90 chaperone machinery. The role of
HCH1
is unknown; its sequence has no strong homology to any
protein of known function.
SSF1
and
CNS1
were
weak suppressors, whereas
HCH1
restored wild-type growth
rates at all temperatures tested to cells expressing the E381K mutant.
Overexpression of
CNS1
or
HCH1
, but not
SSF1
, enhanced the maturation of a heterologous Hsp90
target protein, p60
v-src
. These results suggest that like
Cns1p, Hch1p is a general modulator of Hsp90 chaperone functions,
whereas Ssf1p likely is either an Hsp90 target protein or functions in
the same pathway as an Hsp90 target protein.
Vibrio cholerae
O1 El Tor: Identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation
Authors: Yildiz Fitnat H. , Schoolnik Gary K.
Published: July 2002

The rugose colony variant of
Vibrio cholerae
O1, biotype El Tor, is shown to produce an exopolysaccharide, EPS
ETr
, that confers chlorine resistance and biofilm-forming capacity. EPS
ETr
production requires a chromosomal locus,
vps
, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPS
ETr
may enable the survival of
V. cholerae
O1 within environmental aquatic habitats between outbreaks of human disease.
BglG, the transcriptional antiterminator of the
bgl
system, interacts with the β′ subunit of the
Escherichia coli
RNA polymerase
Authors: Nussbaum-Shochat Anat , Amster-Choder Orna
Published: July 2002

The
Escherichia coli
BglG protein antiterminates transcription at two terminator sites within the
bgl
operon in response to the presence of β-glucosides in the growth medium. BglG was previously shown to be an RNA-binding protein that recognizes a specific sequence located just upstream of each of the terminators and partially overlapping with them. We show here that BglG also binds to the
E. coli
RNA polymerase, both
in vivo
and
in vitro
. By using several techniques, we identified the β′ subunit of RNA polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the N-terminal region of β′. The β′ subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase β′ subunit are discussed.
TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family
Authors: Rabenstein Mark D. , Zhou Sharleen , Lis John T. , Tjian Robert
Published: July 2002

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the “basal” transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in
Drosophila
. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans,
Drosophila
,
Caenorhabditis
elegans
, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2’s primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on
Drosophila
chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.
Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into β-sheet A
Authors: Stratikos Efstratios , Gettins Peter G. W.
Published: July 2002

To determine the location of the proteinase in the covalent serpin-proteinase complex we prepared seven single-cysteine-containing variants of the Pittsburgh variant of the serpin α
1
-proteinase inhibitor, and we labeled each cysteine with the dansyl fluorophore. The dansyl probes were used to determine proximity of the proteinase trypsin in covalent and noncovalent complexes with the serpin, both by direct perturbation and by fluorescence energy transfer from tryptophans in trypsin to dansyl. Large direct effects on dansyl fluorophores were seen for only two positions in covalent complex and one position in noncovalent complex. Distances ranging from <14 Å to 64 Å were used to severely constrain possible structures for the complex. The structure consistent with both distance constraints and direct perturbations of the dansyl fluorophores placed the proteinase at the distal end of the serpin from the initial docking site. This position for the proteinase requires complete translocation of the proteinase from one end of the serpin to the other and full insertion of the reactive center loop into β-sheet A to form the kinetically trapped complex. The consequent tight juxtapositioning of serpin and proteinase could explain how distortion of the proteinase active site can occur and hence how many combinations of serpin and proteinase can be inhibited by a common conformational change mechanism.
The γ-aminobutyric acid type A receptor (GABA
A
R)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse
Authors: Kneussel Matthias , Haverkamp Silke , Fuhrmann Jens C. , Wang Hongbing , Wässle Heinz , Olsen Richard W. , Betz Heinrich
Published: July 2002

γ-Aminobutyric acid type A receptors (GABA
A
Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA
A
R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA
A
R subunits with gephyrin have not been reported. Recently, the GABA
A
R-associated protein GABARAP was found to bind to the γ2 subunit of GABA
A
Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP–gephyrin interactions are not important for postsynaptic GABA
A
R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.
Intratumoral injection of adenoviral vectors encoding tumor-targeted immunoconjugates for cancer immunotherapy
Authors: Hu Zhiwei , Garen Alan
Published: July 2002

The efficacy and safety of a new immunotherapy protocol for cancer were tested in a severe combined immunodeficient mouse model of human skin and metastatic lung melanoma. The protocol involves intratumoral injections of replication-incompetent adenoviral vectors encoding immunoconjugates composed of the Fc region of an IgG1 immunoglobulin conjugated to a tumor-targeting domain. One targeting domain is factor VII that binds to tissue factor expressed on endothelial cells lining the tumor neovasculature and on tumor cells; the active site of factor VII was mutated to inhibit the initiation of blood coagulation. Another targeting domain is a single-chain Fv antibody that binds to a cognate antigen expressed on human melanoma cells. The adenoviral vectors infect mainly the cells of the injected tumor, which synthesize and secrete the immunoconjugates. The bloodborne immunoconjugates induce a cytolytic immune response against the targeted neovasculature endothelial cells and tumor cells. The mouse model experiments showed that intratumoral delivery of the factor VII immunoconjugate, either alone or together with the single-chain Fv immunoconjugate, resulted in growth inhibition and regression of the injected tumor, and also of distant metastatic tumors, without evidence of damage to normal organs. There was extensive destruction of the tumor neovasculature, presumably mediated by the factor VII immunoconjugate bound to tissue factor on neovasculature endothelial cells. Because tissue factor is generally expressed on neovascular endothelial cells and tumor cells, a factor VII immunoconjugate could be used for immunotherapy against a broad range of human solid tumors.
Antagonistic actions of analogs related to growth hormone-releasing hormone (GHRH) on receptors for GHRH and vasoactive intestinal peptide on rat pituitary and pineal cells
in vitro
Authors: Rekasi Zoltan , Varga Jozsef L. , Schally Andrew V. , Halmos Gabor , Groot Kate , Czompoly Tamas
Published: July 2002

Peptide analogs of growth hormone-releasing hormone (GHRH) can potentially interact with vasoactive intestinal peptide (VIP) receptors (VPAC
1
-R and VPAC
2
-R) because of the structural similarities of these two hormones and their receptors. We synthesized four new analogs related to GHRH (JV-1–50, JV-1–51, JV-1–52, and JV-1–53) with decreased GHRH antagonistic activity and increased VIP antagonistic potency. To characterize various peptide analogs for their antagonistic activity on receptors for GHRH and VIP, we developed assay systems based on superfusion of rat pituitary and pineal cells. Receptor-binding affinities of peptides to the membranes of these cells were also evaluated by radioligand competition assays. Previously reported GHRH antagonists JV-1–36, JV-1–38, and JV-1–42 proved to be selective for GHRH receptors, because they did not influence VIP-stimulated VPAC
2
receptor-dependent prolactin release from pituitary cells or VPAC
1
receptor-dependent cAMP efflux from pinealocytes but strongly inhibited GHRH-stimulated growth hormone (GH) release. Analogs JV-1–50, JV-1–51, and JV-1–52 showed various degrees of VPAC
1
-R and VPAC
2
-R antagonistic potency, although also preserving a substantial GHRH antagonistic effect. Analog JV-1–53 proved to be a highly potent VPAC
1
and VPAC
2
receptor antagonist, devoid of inhibitory effects on GHRH-evoked GH release. The antagonistic activity of these peptide analogs on processes mediated by receptors for GHRH and VIP was consistent with the binding affinity. The analogs with antagonistic effects on different types of receptors expressed on tumor cells could be utilized for the development of new approaches to treatment of various human cancers.
Adaptive responses and apoptosis in endothelial cells exposed to carbon monoxide
Authors: Thom Stephen R. , Fisher Donald , Xu Y. Anne , Notarfrancesco Kathy , Ischiropoulos Harry
Published: July 2002

Prior studies have shown that exposure to carbon monoxide (CO) will elevate the steady-state concentration of nitric oxide (⋅NO) in several cell types and body organs and that some toxic effects of CO are directed toward endothelial cells. Studies reported in this paper were conducted with bovine pulmonary artery endothelial cells exposed to 10 to 100 ppm CO to achieve concentrations between 11 and 110 nM in air-saturated buffer. Exposure to 11 nM CO increased synthesis of manganous superoxide dismutase and conferred resistance against the lethal effects of 110 nM CO. At concentrations of 88 nM CO or more, exposures for 1 h or longer caused cell death that became apparent 18 h after the exposure ceased. Caspase-1 was activated in response to CO, and cell death was inhibited by a caspase-1 inhibitor. Alteration of proteolytic pathways by CO was indicated by the presence of ubiquitin-containing intracellular inclusion bodies. Morphological changes and caspase activation indicated that cell death was an apoptotic process. Cells exposed to 110 nM CO had higher concentrations of manganous superoxide dismutase and heme oxygenase-1 but no changes in glutathione peroxidase, glucose-6-phosphate dehydrogenase, thiols, or catalase. Elevated levels of antioxidant enzymes and apoptosis were inhibited by the nitric oxide synthase inhibitor, S-isopropylisothiourea, and the peroxynitrite scavenger, selenomethionine. These results show that biochemical effects of CO occur at environmentally relevant concentrations, that apoptotic cell death follows exposure to relatively high concentrations of CO, and that these actions of CO are mediated by nitric oxide.
In situ
laser-Raman imagery of Precambrian microscopic fossils
Authors: Kudryavtsev Anatoliy B. , Schopf J. William , Agresti David G. , Wdowiak Thomas J.
Published: July 2002

Laser-Raman imagery is a sensitive, noninvasive, and nondestructive
technique that can be used to correlate directly chemical composition
with optically discernable morphology in ancient carbonaceous fossils.
By affording means to investigate the molecular makeup of specimens
ranging from megascopic to microscopic, it holds promise for providing
insight into aspects of organic metamorphism and biochemical evolution,
and for clarifying the nature of ancient minute fossil-like objects of
putative but uncertain biogenicity.
Neurovascular damage in experimental allergic encephalomyelitis: a target for pharmacological control
Authors: Bolton C.
Published: July 2002

The blood‐brain barrier (BBB) is composed of a continuous endothelial layer with pericytes and astrocytes in close proximity to offer homeostatic control to the neurovasculature. The human demyelinating disease multiple sclerosis and the animal counterpart experimental allergic encephalomyelitis (EAE) are characterized by enhanced permeability of the BBB facilitating oedema formation and recruitment of systemically derived inflammatory‐type cells into target tissues to mediate eventual myelin loss and neuronal dysfunction. EAE is considered a useful model for examining the pathology which culminates in loss of BBB integrity and the disease is now proving valuable in assessing compounds for efficacy in limiting damage at neurovascular sites. The precise mechanisms culminating in EAE‐induced BBB breakdown are unclear although several potentially disruptive mediators have been implicated and have been previously identified as potent effectors of cerebrovascular damage in non‐disease related conditions of the central nervous system. The review considers evidence that common mechanisms may mediate cerebrovascular permeability changes irrespective of the initial insult and discusses therapeutic approaches for the control of BBB leakage in the demyelinating diseases.
Pulmonary hypertension in infants with congenital heart defects: are leukotrienes involved?
Authors: Serraf A. , Gascard J-P. , Bruniaux J. , Labat C. , Planche C. , Brink C.
Published: July 2002

The circulating levels of leukotriene E4 in infants with congenital heart defects, increased pulmonary blood flow and pulmonary arterial hypertension, were determined and compared with infants with decreased pulmonary blood flow (Tetralogy of Fallot). There was no correlation (r=0.38) between the pulmonary arterial pressure
(56 ± 4 mmHg) and the leukotriene E4 levels (1.37 ± 0.67 ng/ml blood) measured in peripheral blood samples from the hypertensive group prior to surgery. There was considerable variation in the detectable leukotriene E4 levels in blood samples from different patients. The levels detected in the blood samples between the two groups of patients was similar. These data suggest that neither the surgical repair during cardiopulmonary bypass nor the pulmonary hypertension appeared to modify the leukotriene E4 blood levels in the small number of patients studied.
Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies
Authors: Mouithys-Mickalad A. , Deby-Dupont G. , Hoebeke M. , Mathy-Hartert M. , Lamy M. , Deby C.
Published: July 2002

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12‐myristate 13‐acetate (PMA, 5 × 10-7M) stimulated PMN (6 × 106 cells). By ESR with spin trapping (100 mM DMPO: 5,5‐dimethyl‐1‐pyrroline‐Noxide), we showed that SPN (5 to 8 × 10-6M), C2‐ceramide (N‐acetyl SPN) and C6‐ceramide (N‐hexanoyl SPN) at the final concentration of 2 × 10-5 and 2 × 10-4M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO‐superoxide anion spin adduct. Inhibition by 5 × 10-6M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 × 10-6M) has no effect
on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N‐acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 × 10-6 to 10-5M), but N‐hexanoyl SPN was less active (from 2 × 10-5 to 2 × 10-4M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH‐oxidase, the key enzyme responsible for production of oxygen‐derived free radicals.
The effect of ozone exposure on the release of eicosanoids in guinea‐pig BAL fluid in relation to cellular damage and inflammation
Authors: Van Hoof H. J. M. , Zijlstra F. J. , Voss H-P. , Garrelds I. M. , Dormans J. A. M. A. , Van Bree L. , Bast A.
Published: July 2002

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2α and 6‐ketoPGF1α whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone‐induced lung function changes.
Dexamethasone reduces cell surface levels of CD11b on human eosinophils
Authors: Das A. M. , Lim L. H. K. , Flower R. J. , Perretti M.
Published: July 2002

Overnight incubation of human eosinophils (Eøs) with the glucocorticoid hormone dexamethasone (DEX; 0.1 μM) resulted in lower expression of the CD11b, but not CD49d, antigen on their plasma membrane, as assessed by flow cytometry. DEX produced a consistent inhibitory effect (ranging from 16% to 20%) when tested at a concentration of 0.1 μM. Eø stimulation with 100 ng/ml eotaxin produced an increase in CD11b (+26%), but not CD11c, levels and concomitantly a reduction (–25%) on CD62L expression. The inhibition exerted by DEX upon CD11b levels was also evident following eotaxin upregulation, with a degree of inhibition similar to that seen on basal levels. These data highlight a novel mechanism of action by which glucocorticoid hormones may be effective in reducing Eø accumulation during allergic inflammation in man.
Antisocial Alcoholic Patients Show as Much Improvement at 14‐Month Follow‐up as Non‐Antisocial Alcoholic Patients
Authors: Verheul Roel , Brink Wim van den , Koeter Maarten W.J. , Hartgers Christina
Published: July 2002

The authors investigated the impact of DSM‐III‐R adult criteria for antisocial personality disorder (and co‐occurrence of childhood conduct or mood disorder) on one‐year changes of multi‐domain problem severity in 309 alcoholic patients. Adult antisocial traits were associated with more drug, legal, and psychiatric problems at baseline and with more drug problems at follow‐up. However, patients with antisocial traits showed at least as much improvement from baseline through follow‐up as their non‐antisocial counterparts. Furthermore, the co‐occurrence of childhood conduct disorder or mood disorder among the antisocial alcoholics did not define prognostically relevant subgroups. These findings suggest that antisocial alcoholics benefit from treatment at least as much as non‐antisocial alcoholics.
Phonological systems of a set of Putonghua‐speaking twins
Authors: Hua Zhu , Dodd Barbara
Published: July 2002

Impaired phonology is reported to be the most salient characteristic of the communication profiles of twin children. However, little is known about the phonological development of twins speaking languages other than English. This case study described the phonological systems of a set of Putonghua‐speaking twins, using quantitative and qualitative measures. The analyses indicated that the phonological systems of the co‐twins were not identical, though both evidenced characteristics of delayed or disordered phonological development and shared some error patterns. They comprehended both adult and their sibling's phonological forms, suggesting a dual phonological representation in their mental lexicon for some words. These results confirmed Dodd and McEvoy's (1994) research findings for English‐speaking twins.
Ribonuclease from Cobra Snake Venom: Purification by Affinity Chromatography and Further Characterization
Authors: Mahalakshmi Y. V. , Jagannadham M. V. , Pandit M. W.
Published: July 2002

A ribonuclease from cobra snake venom was isolated and purified to homogeneity using antibody‐affinity chromatography, increasing the yield fourfold. The purified enzyme showed cytidylic acid specificity, as reported earlier. Further, the effects of temperature, pH, metal ions, inhibitors, and urea on the enzyme activity were studied. Snake venom RNase exhibited salt‐dependent reversible association?dissociation behaviour. Immunological studies indicate that this enzyme shares one of the antigenic sites of RNase A. The partial N‐terminal sequence of the enzyme showed considerable homology with phospholipases from snake venom; however, the enzyme itself did not show any phospholipase activity.
Retinoic Acid Receptor α Function in Vertebrate Limb Skeletogenesis: a Modulator of Chondrogenesis
Authors: Cash David E. , Bock Cheryl B. , Schughart Klaus , Linney Elwood , Underhill T. Michael
Published: July 2002

Retinoic acid is a signaling molecule involved in the regulation of growth and morphogenesis during development. There are three types of nuclear receptors for all-trans retinoic acid in mammals, RARα, RARβ, and RARγ, which transduce the retinoic acid signal by inducing or repressing the transcription of target genes (Leid, M., P. Kastner, and P. Chambon. 1992. Trends Biochem. Sci. 17:427–433). While RARα, RARβ, and RARγ are expressed in distinct but overlapping patterns in the developing mouse limb, their exact role in limb development remains unclear. To better understand the role of retinoic acid receptors in mammalian limb development, we have ectopically expressed a modified RARα with constitutive activity (Balkan, W., G.K. Klintworth, C.B. Bock, and E. Linney. 1992. Dev. Biol. 151:622–625) in the limbs of transgenic mice. Overexpression of the transgene was associated with marked pre- and postaxial limb defects, particularly in the hind limb, where expression of the transgene was consistently seen across the whole anteroposterior axis. The defects displayed in these mice recapitulate, to a large degree, many of the congenital limb malformations observed in the fetuses of dams administered high doses of retinoic acid (Kochhar, D.M. 1973. Teratology. 7:289–295). Further analysis of these transgenic animals showed that the defect in skeletogenesis resided at the level of chondrogenesis. Comparison of the expression of the transgene relative to that of endogenous RARα revealed that downregulation of RARα is important in allowing the chondrogenic phenotype to be expressed. These results demonstrate a specific function for RARα in limb development and the regulation of chondroblast differentiation.
Multiple Exocytotic Pathways in Pancreatic β Cells
Authors: Takahashi Noriko , Kadowaki Takashi , Yazaki Yoshio , Miyashita Yasushi , Kasai Haruo
Published: July 2002

Ca2+-dependent exocytotic pathways in mouse pancreatic β cells were investigated using both capacitance measurement and amperometric detection of vesicular contents. Serotonin was preloaded into large dense-core vesicles for the amperometry. Exocytosis was induced by rapid elevation of cytosolic Ca2+ concentrations using caged-Ca2+ compounds. Capacitance measurement revealed two major components of exocytosis, and only the slow component was accompanied by amperometric events reflecting quantal serotonin secretion. Moreover, the fast and slow exocytoses induced the two forms of endocytosis that were reported to follow the exocytoses of small-clear and large dense-core vesicles, respectively. Interestingly, we recorded two types of responses of quantal events: in the type-1 response, most quantal events occurred with a delay of 0.2 s and were rapidly exhausted with a time constant of 1.7 s, while, in the type-2 response, quantal events occurred with a delay of 2.5 s and were sustained. This suggests the existence of two pathways or modes of the exocytosis involving large dense-core vesicles. Thus, we have revealed three exocytotic pathways with divergent fusion kinetics in β cells, which provide a new basis for the understanding of the physiology and pathology of β cells.
Nuclear–Cytoplasmic Shuttling of the Focal Contact Protein, Zyxin: A Potential Mechanism for Communication between Sites of Cell Adhesion and the Nucleus
Authors: Nix David A. , Beckerle Mary C.
Published: July 2002

Integrin-dependent cell adhesion to specific extracellular matrix molecules has been demonstrated to trigger dramatic changes in gene expression that can affect cell fate. However, little is understood about the molecular mechanism by which events at sites of cell– substratum adhesion are communicated to the cell interior to regulate the transcriptional apparatus. By analogy to classical mechanisms of cell surface receptor function, it seems likely that some components of the integrin-activated signal transduction machinery will be colocalized with cell adhesion molecules. Zyxin is a low abundance phosphoprotein that accumulates with integrins at sites of cell–substratum attachment. Here we show that zyxin exhibits a functional nuclear export signal that is required to keep zyxin concentrated in the cytoplasm and is sufficient to direct nuclear proteins to the cytosol. Furthermore, we demonstrate that native zyxin shuttles between the nucleus and sites of cell adhesion in fibroblasts and is thus an excellent candidate for relaying information between these two compartments.
Leukemia Inhibitory Factor–dependent Transcriptional Activation in Embryonic Stem Cells
Authors: Boeuf Hélène , Hauss Charlotte , Graeve Fabienne De , Baran Nathalie , Kedinger Claude
Published: July 2002

STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.
Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function
Authors: Porter Joanna C. , Hogg Nancy
Published: July 2002

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg2+, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.