bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.
Authors: Coury J E , McFail-Isom L , Williams L D , Bottomley L A
Published: July 2002

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.
NF-kappa B homodimer binding within the HIV-1 initiator region and interactions with TFII-I.
Authors: Montano M A , Kripke K , Norina C D , Achacoso P , Herzenberg L A , Roy A L , Nolan G P
Published: July 2002

We show that the binding of Rel p50 and p52 homodimers at sites within the transcriptional initiation region of HIV-1 provides for their ability to interact with other proteins that bind the initiator. The binding of one such protein, the initiator protein TFII-I, to the initiation region of HIV-1 is augmented in the presence of Rel p50 and Rel p52 homodimers. Consistent with this, in vitro Rel homodimers potentiate HIV-1 transcription in a manner dependent upon TFII-I. The findings suggest that Rel dimers may regulate HIV-1 transcription in two ways. First, through binding at the kappa B enhancer sites at (-104 to -80), NF-kappa B p50:p65 participates in classical transcriptional activation. Second, Rel dimers such as p50 or p52 might bind at initiator sequences to regulate the de novo binding of components of certain preinitiation complexes. These findings, and the existence of Rel binding sites at the initiators of other genes, suggest roles for Rel proteins in early events determining transcriptional control.
Lipophosphoglycan fromLeishmaniasuppresses agonist-induced interleukin 1β gene expression in human monocytes via a unique promoter sequence
Authors: Hatzigeorgiou Dimitrios E. , Geng Jiayuan , Zhu Baixin , Zhang Yihong , Liu Ke , Rom William N. , Fenton Matthew J. , Turco Salvatore J. , Ho John L.
Published: July 2002

Leishmaniaare parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1β (IL-1β) and tumor necrosis factor α are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid ofLeishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1β steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1β mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor α andStaphylococcusinduction of IL-1β gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1β mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the −310 to −57 nucleotide region of the IL-1β promoter was found to mediate LPG’s inhibitory activity. The requirement for the −310 to −57 promoter gene sequence for LPG’s effect is demonstrated by the abrogation of LPG’s inhibitory activity by truncation or deletion of the −310 to −57 promoter gene sequence. Furthermore, the minimal IL-1β promoter (positions −310 to +15) mediated LPG’s inhibitory activity with dose and kinetic profiles that were similar to LPG’s suppression of steady-state IL-1β mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a “gene silencer,” a function, to our knowledge, not previously described. LPG’s inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.
Cloning and characterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone
Authors: Lin Tien-Min , Chang Chawnshang
Published: July 2002

By using mRNA polymerase chain reaction differential display technique (DDPCR), we have identified one early responsive cDNA fragment, TDD5, from a 5α-reductase-deficient T cell hybridoma. The DDPCR profiles of TDD5 suggest that its expression can be repressed by testosterone (T) within 2 hr. More importantly, both DDPCR and Northern blot analysis further demonstrated that the expression of TDD5 was differentially repressed by T and dihydrotestosterone (DHT) at the mRNA level. To our knowledge, this is the first androgen target gene to show a preference in response to T over DHT in cell culture. TDD5 is expressed in several tissues with particular abundance in kidney. Full-length TDD5 cDNA (2,916 bp) encodes a protein with a calculated molecular weight of 42,000. Finally, our animal studies further confirm that TDD5 mRNA levels can be repressed to the basal level 8 hr after DHT administration. The isolation and characterization of the early-responsive androgen target gene TDD5 and the fact that TDD5 mRNA level can be differentially regulated by T and DHT may provide a useful tool to study the molecular mechanism of androgen preference on target gene regulation.
Identification of chemical synapses in the pharynx of
Caenorhabditis elegans
Authors: Li Heyi , Avery Leon , Denk Winfried , Hess George P.
Published: July 2002

The rhythmic contraction of the
Caenorhabditis elegans
pharynx is unique in that the network of 12 neurons, including two M3 neurons, that regulate the contraction is known. The neurotransmitters secreted by these cells, and the target cells responding to these chemical signals, are not known. Here, we describe an approach to obtain this missing information and use the M3 cells as an example. Electrical recordings (electropharyngeograms) were used in conjunction with temporally and spatially defined application of neurotransmitters via photolysis of inactive, photolabile precursors. To illustrate the technique we used pharyngeal preparations in which the two M3 neurons are intact and preparations in which they were removed by laser irradiation. Removal of M3 neurons results in the loss of the small negative peaks in the electropharyngeograms and an increase in time during which the pharynx remains contracted. We demonstrate that the application of glutamate by photolysis of caged glutamate to a pharynx from which the two M3 neurons were removed produces effects similar to those observed before removal of the M3 neurons. In control experiments, photolytic release from photolabile precursors of carbamoylcholine, a stable and well characterized analog of acetylcholine, or of γ-aminobutyric acid, from photolabile precursors did not have this effect. The response depended on the amount of glutamate released. By reducing the size of the photolytic beam, glutamate was released at several different locations of the pharynx. Two areas of the pharynx mainly respond to the application of glutamate; one corresponds to the pm4 muscle cells in the metacorpus, and the other to the junction between muscle cells pm5 in the isthmus and pm6 in the terminal bulb.
Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and
Saccharomyces cerevisiae
Authors: Römisch Karin , Ali Bassam R. S.
Published: July 2002

Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles. In a cell-free system, this process is ATP- and cytosol-dependent. While yeast cytosol promotes the export of glycopeptides from mammalian ER
in vitro
, glycopeptide release cannot be detected in the presence of mammalian cytosol. We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes. Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes. The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.
Tumor induction by a transformation-defective polyoma virus mutant blocked in signaling through Shc
Authors: Bronson Roderick , Dawe Clyde , Carroll John , Benjamin Thomas
Published: July 2002

Transformation of cells in culture by polyoma virus requires integration of signals downstream of middle T–Shc and middle T-phosphatidylinositol 3-kinase interactions, but the same is not true for induction of tumors in the mouse. Thus, a middle T mutant defective in transformation and blocked in binding Shc is able to induce a broad spectrum of tumors after inoculation into newborn mice. The “tumor profile” induced by the mutant shows enhancement of tumors at some sites and reductions at others but otherwise resembles that induced by the wild-type virus. A nontransforming double-mutant blocked in binding phosphatidylinositol 3-kinase as well as Shc is severely affected but still induces some tumors. These results show that pathways that must cooperate to induce full transformation of cells
in vitro
can act independently and are to a large extent redundant in tumor induction.
Characterization of the transposition pattern of the
Ac
element in
Arabidopsis thaliana
using endonuclease I-
Sce
I
Authors: Machida Chiyoko , Onouchi Hitoshi , Koizumi Jun , Hamada Susumu , Semiarti Endang , Torikai Satomi , Machida Yasunori
Published: July 2002

We have investigated physical distances and directions of transposition of the maize transposable element
Ac
in
Arabidopsis thaliana
. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of
Ac
with a site for cleavage by endonuclease I-
Sce
I (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic
Arabidopsis
plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the
Arabidopsis
that carried the gene for
Ac
transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-
Sce
I, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the
Ac
–
Ds
system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of
Ac
in
Arabidopsis
. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.
Genetic interactions with
Rap1
and
Ras1
reveal a second function for the Fat facets deubiquitinating enzyme in
Drosophila
eye development
Authors: Li Qinghong , Hariharan Iswar K. , Chen Fangli , Huang Yongzhao , Fischer Janice A.
Published: July 2002

The
Drosophila
fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the
Drosophila Rap1
and
Ras1
genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.
Folding thermodynamics of a model three-helix-bundle protein
Authors: Zhou Yaoqi , Karplus Martin
Published: July 2002

The calculated folding thermodynamics of a simple off-lattice three-helix-bundle protein model under equilibrium conditions shows the experimentally observed protein transitions: a collapse transition, a disordered-to-ordered globule transition, a globule to native-state transition, and the transition from the active native state to a frozen inactive state. The cooperativity and physical origin of the various transitions are explored with a single “optimization” parameter and characterized with the Lindemann criterion for liquid versus solid-state dynamics. Below the folding temperature, the model has a simple free energy surface with a single basin near the native state; the surface is similar to that calculated from a simulation of the same three-helix-bundle protein with an all-atom representation [Boczko, E. M. & Brooks III, C. L. (1995) Science 269, 393–396].
Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia,
Oreochromis mossambicus
Authors: Shepherd Brian S. , Sakamoto Tatsuya , Nishioka Richard S. , Richman Nurney H. , Mori Ikue , Madsen Steffen S. , Chen Thomas T. , Hirano Tetsuya , Bern Howard A. , Grau E. Gordon
Published: July 2002

It is increasingly clear that growth hormone (GH) has growth-promoting effects in fishes, which are mediated in part by the insulin-like growth factor (IGF)-I. Growth-promoting actions of prolactin (PRL) have been reported in higher vertebrates, but are less well established in teleosts. We examined the effects of injecting homologous GH or the two homologous tilapia PRLs (tPRL
177
and tPRL
188
) on the
in vitro
incorporation of [
35
S]sulfate (extracellular matrix synthesis) and [
3
H]thymidine (DNA synthesis) by ceratobranchial cartilage explants and on IGF-I mRNA levels in tilapia liver. Tilapia GH (tGH) and tPRL
177
stimulated sulfate uptake at the highest doses examined. Thymidine incorporation was stimulated by tPRL
177
. tPRL
188
was without these effects. Consistent with its somatotropic actions, tGH elevated IGF-I mRNA levels in the liver. tPRL
177
also elevated liver IGF-I levels. Consistent with the previously described osmoregulatory actions of GH and PRL in teleosts, we observed that tGH elevated and tPRL
177
and tPRL
188
lowered levels of gill Na
+
,K
+
-ATPase activity. High-affinity, low-capacity binding sites for tGH in the tilapia liver were identified. tPRL
177
binds with lower affinity than tGH to these sites but can displace
125
I-labeled tGH from its receptor. The ability of tPRL
177
to displace tGH was similar to that of ovine GH. tPRL
188
did not displace
125
I-labeled tGH binding. Collectively, this work suggests that tPRL
177
may possess somatotropic actions similar to tGH, but only in freshwater tilapia where tPRL
177
levels are sufficiently high for it to act as a competitive ligand for GH receptors.
Molecular characterization of
TUB, TULP1
, and
TULP
2, members of the novel tubby gene family and their possible relation to ocular diseases
Authors: North Michael A. , Naggert Juergen K. , Yan Yingzhuo , Noben-Trauth Konrad , Nishina Patsy M.
Published: July 2002

Tubby, an autosomal recessive mutation, mapping to mouse chromosome 7, was recently found to be the result of a splicing defect in a novel gene with unknown function. Database searches revealed that sequences corresponding to the C terminus of the
tub
protein were highly conserved across a number of species including humans, mice,
Caenorhabditis elegans, Arabidopsis,
rice, and maize, and that
tub
was a member of a gene family. We describe here,
TUB
, the human homolog of mouse
tub
, and two newly characterized family members,
TULP1
for tubby like protein 1 and
TULP2
. These three family members, which differ in the N-terminal half of the protein, share 60–90% amino acid identity across their conserved C-terminal region and have distinct tissue expression patterns. Alternatively spliced transcripts with 5′ variable sequences, three of which have been identified for the tubby gene, may mediate tissue specific expression. We also report that
TUB, TULP1
, and
TULP2
map to human chromosomes 11p15.4, 6p21.3, and 19q13.1, respectively.
TULP1
and
TULP2
map within the minimal intervals identified for retinitis pigmentosa 14 on chromosome 6p21.3 and cone-rod dystrophy on chromosome 19q13.1.
TULP1
and
TULP2
, which are expressed in the retina, make excellent candidates for these ocular diseases as a mutation within the
tub
gene is known to lead to early progressive retinal degeneration.
Tn
552
transposase catalyzes concerted strand transfer
in vitro
Authors: Leschziner Andres E. , Griffin Thomas J. , Grindley Nigel D. F.
Published: July 2002

The Tn
552
transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer
in vitro
, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn
552
termini. As is the case with transposition
in vivo
, the insertions recovered from the reaction
in vitro
are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg
2+
. Although it stimulates strand transfer overall, Mn
2+
promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn
552
transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.
Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers
Authors: Lee JinKeun , Lentz Barry R.
Published: July 2002

Activation energies for the individual steps of secretory and viral fusion are reported to be large [Oberhauser, A. F., Monck, J. R. & Fernandez, J. M. (1992)
Biophys. J.
61, 800–809; Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990)
Biochemistr
y 29, 1303–1308]. Understanding the cause for these large activation energies is crucial to defining the mechanisms of these two types of biological membrane fusion. We showed recently that the fusion of protein-free model lipid bilayers mimics the sequence of steps observed during secretory and viral fusion, suggesting that these processes may involve common lipid, rather than protein, rearrangements. To test for this possibility, we determined the activation energies for the three steps that we were able to distinguish as contributing to the fusion of protein-free model lipid bilayers. Activation energies for lipid rearrangements associated with formation of the reversible first intermediate, with conversion of this to a semi-stable second intermediate, and with irreversible fusion pore formation were 37 kcal/mol, 27 kcal/mol, and 22 kcal/mol, respectively. The first and last of these were comparable to the activation energies observed for membrane lipid exchange (42 kcal/mol) during viral fusion and for the rate of fusion pore opening during secretory granule release (23 kcal/mol). This striking similarity suggests strongly that the basic molecular processes involved in secretory and viral fusion involve a set of lipid molecule rearrangements that also are involved in model membrane fusion.
The role of SOS and flap processing in microsatellite instability in
Escherichia coli
Authors: Morel Patrice , Reverdy Céline , Michel Bénédicte , Ehrlich S. Dusko , Cassuto Era
Published: July 2002

Mutations affecting mismatch repair result in elevated frequencies of microsatellite length alteration in prokaryotes and eukaryotes. However, the finding that microsatellite instability is found often in cells with a functional mismatch repair system prompted a search for other factors of tract alteration. In the present report, we show that, in
Escherichia coli
, poly(AC/TG) tracts are destabilized by mutations that induce SOS. These observations may have implications for eukaryotic cells because recent results suggest the existence of a mammalian SOS response analogous to that in prokaryotes. In addition, a defect in the 5′–3′ exonuclease domain of DNA polymerase I, homologous to the mammalian FEN1 and the yeast RAD27 nucleases, leads to a marked increase in repeat expansions characteristic of several genetic disorders. Finally, we found that the combination of a proofreading defect with mismatch repair deficiency results in extreme microsatellite instability.
Ethylene-mediated phenotypic plasticity in root nodule development on
Sesbania rostrata
Authors: Fernández-López Manuel , Goormachtig Sofie , Gao Mengsheng , D’Haeze Wim , Van Montagu Marc , Holsters Marcelle
Published: July 2002

Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region.
Sesbania rostrata
was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type.
In situ
hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial
nod
and
nif
gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene.
Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation
Authors: Bogue Molly A. , Jhappan Chamelli , Roth David B.
Published: July 2002

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.
Genome projects and gene pools: New germplasm for plant breeding?
Authors: Lee Michael
Published: July 2002

Crop gene pools have adapted to and sustained the demands of agricultural systems for thousands of years. Yet, very little is known about their content, distribution, architecture, or circuitry. The presumably shallow elite gene pools often continue to yield genetic gains while the exotic pools remain mostly untapped, uncharacterized, and underutilized. The concept and content of a crop’s gene pools are being changed by advancements in plant science and technology. In the first generation of plant genomics, DNA markers have refined some perceptions of genetic variation by providing a glimpse of a primary source, DNA polymorphism. The markers have provided new and more powerful ways of assessing genetic relationships, diversity, and merit by infusing genetic information for the first time in many scenarios or in a more comprehensive manner for others. As a result, crop gene pools may be supplemented through more rapid and directed methods from a greater variety of sources. Previously limited by the barriers of sexual reproduction, the native gene pools will soon be complemented by another gene pool (transgenes) and perhaps by other native exotic gene pools through comparative analyses of plants’ biological repertoire. Plant genomics will be an important force of change for crop improvement. The plant science community and crop gene pools may be united and enriched as never before. Also, the genomes and gene pools, the products of evolution and crop domestication, will be reduced and subjected to the vagaries and potential divisiveness of intellectual property considerations. Let the gains begin.
The cleaved peptide of the thrombin receptor is a strong platelet agonist
Authors: Furman Mark I. , Liu Longbin , Benoit Stephen E. , Becker Richard C. , Barnard Marc R. , Michelson Alan D.
Published: July 2002

Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41-aa peptide and generating a new amino terminus that acts as a tethered ligand for the receptor. Peptides corresponding to the new amino terminal end of the proteolyzed seven-transmembrane domain thrombin receptor [TR
42–55,
SFLLRNPNDKYEPF, also known as TRAP (thrombin receptor-activating peptide)], previously have been demonstrated to activate the receptor. In this study, we demonstrate that the 41-aa cleaved peptide, TR
1–41
(MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR) is a strong platelet agonist. TR
1–41
induces platelet aggregation. In whole-blood flow cytometric studies, TR
1–41
was shown to be more potent than TR
42–55
and almost as potent as thrombin, as determined by the degree of increase in: (
i
) platelet surface expression of P-selectin (reflecting α granule secretion); (
ii
) exposure of the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex; and (
iii
) fibrinogen binding to the activated GPIIb-IIIa complex. As determined by experiments with inhibitors [prostaglandin I
2
, staurosporine, wortmannin, the endothelium-derived relaxing factor congener
S
-nitroso-
N
-acetylcysteine (SNAC), EDTA, EGTA, and genestein], and with Bernard-Soulier or Glanzmann’s platelets, we demonstrated that TR
1–41
-induced platelet activation is: (
i
) inhibited by cyclic AMP; (
ii
) mediated by protein kinase C, phosphatidyl inositol-3-kinase, myosin light chain kinase, and intracellular protein tyrosine kinases; (
iii
) dependent on extracellular calcium; and (
iv
) independent of the GPIb-IX and GPIIb-IIIa complexes. TR
1–41
-induced platelet activation was synergistic with TR
42–55
. In summary, the cleaved peptide of the seven-transmembrane domain TR (TR
1–41
) is a strong platelet agonist.
Synthetic activation of caspases: Artificial death switches
Authors: MacCorkle Rebecca A. , Freeman Kevin W. , Spencer David M.
Published: July 2002

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal “suicide switch” would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1β converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases.
Specificity in cholesterol regulation of gene expression by coevolution of sterol regulatory DNA element and its binding protein
Authors: Athanikar Jyoti N. , Osborne Timothy F.
Published: July 2002

When demand for cholesterol rises in mammalian cells, the sterol regulatory element (SRE) binding proteins (SREBPs) are released from their membrane anchor through proteolysis. Then, the N-terminal region enters the nucleus and activates genes of cholesterol uptake and biosynthesis. Basic helix–loop–helix (bHLH) proteins such as SREBPs bind to a palindromic DNA sequence called the E-box (5′-CANNTG-3′). However, SREBPs are special because they also bind direct repeat elements called SREs. Importantly, sterol regulation of all promoters studied thus far is mediated by SREBP binding only to SREs. To study the reason for this we converted the direct repeat SRE from the sterol-regulated low-density lipoprotein receptor promoter into an E-box. In this report we show that SREBPs are still able to bind and activate this promoter however, sterol regulation is lost. The results are consistent with the mutant promoter being a target for promiscuous activation by constitutively expressed E-box binding bHLH proteins that are not regulated by cholesterol. Kim and coworkers [Kim, J. B., Spotts, G. D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H. C. & Spiegelman, B. M. (1995)
Mol. Cell. Biol.
15, 2582–2588] demonstrated that the dual DNA binding specificity of SREBPs is caused by a specific tyrosine in the conserved basic region of the DNA binding domain that corresponds to an arginine in all other bHLH proteins that recognize only E-boxes. Taken together the data suggest an evolutionary mechanism where a DNA binding protein along with its recognition site have coevolved to ensure maximal specificity and sensitivity in a crucial nutritional regulatory response.
Catalysis of peptide dissociation from class II MHC-peptide complexes
Authors: Schmitt Lutz , Kratz Johannes R. , Davis Mark M. , McConnell Harden M.
Published: July 2002

Certain peptides such as dynorphin A [dynA-(1–13)] enhance the release of antigenic peptides bound to class II MHC molecules at neutral pH. This enhanced release has been termedpush off.Previous work has shown that the antigenic pigeon cytochromecpeptide PCC-(89–104) has at leasttwoconformational isomers when bound to the class II MHC protein I-Ek. We have accordingly studied the push off of PCC-(89–104) from the complex PCC-(89–104)/I-Ekto see whether these isomeric conformations are distinguished by the push-off effect. A comparison of the association and dissociation kinetics of PCC-(89–104)/I-Ekin the presence of dynA-(1–13) shows that dynA-(1–13) does not simply replace PCC-(89–104) but rather acts catalytically. The major product is peptide-free I-Ek, which is receptive to further peptide binding. Evidence is presented that a two peptide—one MHC complex is formed in solution. This ternary complex represents the first step of the mechanism of push off.19F NMR data are presented that indicate that dynA-(1–13) interacts specifically with only one of the two isomeric complexes of PCC-(89–104) and I-Ek. A push-off mechanism is proposed in which dynA-(1–13) binds outside the peptide binding groove. In a second step, the dissociation of one of the two isomers is specifically enhanced. Thus the push-off effect may be useful for identifying conformational isomers and for separating them.
Canine sexual dimorphism in Egyptian Eocene anthropoid primates:
Catopithecus
and
Proteopithecus
Authors: Simons Elwyn L. , Plavcan J. Michael , Fleagle John G.
Published: July 2002

Two very small late Eocene anthropoid primates,
Catopithecus browni
and
Proteopithecus sylviae,
from Fayum, Egypt show evidence of substantial sexual dimorphism in canine teeth. The degree of dimorphism suggests that these early anthropoids lived in social groups with a polygynous mating system and intense male–male competition.
Catopithecus
and
Proteopithecus
are smaller in estimated body size than any living primates showing canine dimorphism. The origin of canine dimorphism and polygyny in anthropoids was not associated with the evolution of large body size.
Identification of profilin and src homology 3 domains as binding partners for
Drosophila
Enabled
Authors: Ahern-Djamali Shawn M. , Bachmann Christiane , Hua Ping , Reddy Srinevas K. , Kastenmeier Andrew S. , Walter Ulrich , Hoffmann F. M.
Published: July 2002

Drosophila
Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(
l
-proline) residues with similarity to both profilin and src homology 3 binding sites. Here, we show that Ena can bind directly to the
Drosophila
homolog of profilin, chickadee. Furthermore, Ena and profilin were colocalized in spreading cultured cells. We report that the proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing
Drosophila
embryo.
A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy
Authors: Bönnemann Carsten G. , Cox Gerald F. , Shapiro Frederic , Wu Jiann-Jiu , Feener Chris A. , Thompson Teryl G. , Anthony Douglas C. , Eyre David R. , Darras Basil T. , Kunkel Louis M.
Published: July 2002

Multiple epiphyseal dysplasia (MED) is a degenerative cartilage condition shown in some cases to be caused by mutations in genes encoding cartilage oligomeric matrix protein or type IX collagen. We studied a family with autosomal dominant MED affecting predominantly the knee joints and a mild proximal myopathy. Genetic linkage to the
COL9A3
locus on chromosome 20q13.3 was established with a peak log
10
odds ratio for linkage score of 3.87 for markers D20S93 and D20S164. Reverse transcription–PCR performed on the muscle biopsy revealed aberrant mRNA lacking exon 3, which predicted a protein lacking 12 amino acids from the COL3 domain of α3(IX) collagen. Direct sequencing of genomic DNA confirmed the presence of a splice acceptor mutation in intron 2 of the
COL9A3
gene (intervening sequence 2, G-A, -1) only in affected family members. By electron microscopy, chondrocytes from epiphyseal cartilage exhibited dilated rough endoplasmic reticulum containing linear lamellae of alternating electron-dense and electron-lucent material, reflecting abnormal processing of mutant protein. Type IX collagen chains appeared normal in size and quantity but showed defective cross-linking by Western blotting. The novel phenotype of MED and mild myopathy is likely caused by a dominant-negative effect of the exon 3-skipping mutation in the
COL9A3
gene. Patients with MED and a waddling gait but minimal radiographic hip involvement should be evaluated for a primary myopathy and a mutation in type IX collagen.
Unusual resistance of ALR/Lt mouse β cells to autoimmune destruction: Role for β cell-expressed resistance determinants
Authors: Mathews Clayton E. , Graser Robert T. , Savinov Alexei , Serreze David V. , Leiter Edward H.
Published: July 2002

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target β cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the β-cell level. ALR islets were found to be remarkably resistant to two different combinations of β-cytotoxic cytokines (IL-1β, tumor necrosis factor α, and IFN-γ) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kdand H2-Ag7identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determinedin vivoby means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR × NOD)F1recipients with NOD bone marrow. In allin vivosystems, ALR and F1female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the β cell level.
Armed Defiance and Biblical Appropriation: Assimilation and the Transformation of Mordvin Resistance, 1740–1810*
Authors: Werth Paul W.
Published: July 2002

If until recently Western investigations of “the nationalities question” in Russia and the Soviet Union focused almost exclusively on the larger and more visible “nations” that enjoyed union-republic status in the Soviet period, scholars have now begun to devote more sustained collective attention to the history of smaller ethnic groups that received only “autonomous” units within the Russian republic itself. For many of these peoples, subjected to Russian imperial rule and cultural domination for the entirety of their modern history and endowed with fewer of the opportunities for national development available to titular nationalities in the union republics, the problem of maintaining their particularity and of articulating a vision of collective cohesion has been especially acute both historically and in more recent times. Yet the fact that some of these groups are now threatened with eventual disappearance as distinct linguistic and cultural communities should not blind us to the complex, contingent, and inherently messy nature of their assimilation. Indeed, close scrutiny reveals that the very processes of assimilation contain within themselves possibilities for the emergence of hybrid cultural configurations and the appropriation of dominant conceptions for the transformation of indigenous culture along new trajectories.
Osmotic effects of ethanol on lymphocytes
Authors: Greig J. E. , Keast D. , Palmer T. N.
Published: July 2002

The alterations in the immune system caused by ethanol appear to be a complex combination of direct and indirect effects. The role of ethanol as an osmolyte has previously been studied in this laboratory with rat splenocytes. In the present study the osmotic effects of ethanol were investigated in lymphocytes from human normal subjects and alcohol abusers. Mitogen‐stimulated lymphocytes were cultured in vitro with ethanol in hyperosmotic isotonic or iso‐osmotic hypotonic conditions. The former conditions mimic the physiological situation where ethanol increases osmolality in an electrolyte‐balanced environment. Under these conditions, lymphocyte proliferation was unaffected. Ethanol addition in iso‐osmotic hypotonic conditions, where there is electrolyte imbalance, was associated with inhibition of T‐lymphocyte proliferation. Hyperosmotic hypertonic solutions in the absence of ethanol also resulted in inhibition of T‐lymphocyte proliferation. Electron microscopy and measurement of cell viability and metabolic activity (lactate and ATP levels) indicated that the decreased proliferation associated with NaCl‐induced hyperosmotic hypertonic conditions was at least partially attributable to cell death together with, and possibly caused by, detrimental effects on mitochondria. Conversely, decreased T‐lymphocyte proliferation in iso‐osmotic hypotonic high ethanol solutions, appeared not to be due to changes in cell viability, nor alterations to energy metabolism. It is proposed that ion fluxes involved in the maintenance of cell volume, in particular K + movement, may be important in facilitating normal lymphocyte proliferation in the presence of ethanol in pathological conditions associated with electrolyte imbalance.
The effects of age and gender on laryngeal aerodynamics
Authors: Goozée Justine V. , Murdoch Bruce E. , Theodoros Deborah G. , Thompson Elizabeth C.
Published: July 2002

A computerized airflow/air pressure analysis system, the Aerophone II Model 6800 (Kay Elemetrics Corp.), was used to assess the effects of age and gender on laryngeal aerodynamics. A sample of 56 male and 53 female normal speaking subjects was divided into six age groups (20‐30; 31‐40; 41‐50; 51‐60; 61‐70 and 71‐80 years). The laryngeal aerodynamic parameters measured included phonatory (mean) flow rate, estimated subglottal pressure, laryngeal airway resistance, phonatory sound pressure level, phonatory power, and phonatory efficiency. Most comfortable phonation, vocal efficiency, and running speech tasks were used to collect the aerodynamic data. Comfortable pitch and loudness levels were used for each of these tasks. Age and gender effects were found for a number of the phonatory (mean) flow rate and phonatory sound pressure level values. Results failed, however, to indicate age or gender effects for the estimated subglottal pressure, laryngeal airway resistance, phonatory power and phonatory efficiency parameters. High intersubject variability was found for the phonatory flow rate, laryngeal airway resistance, phonatory power and phonatory efficiency values. Estimated subglottal pressure values, however, appeared to vary the least among subjects. The results are discussed with respect to factors that might influence laryngeal aerodynamics, such as underlying laryngeal anatomical and physiological age‐related changes and gender‐related differences. The clinical implications of the findings for the assessment and treatment of individuals with voice disorders using the Aerophone II are also discussed.
The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells
Authors: Malliri Angeliki , Symons Marc , Hennigan Robert F. , Hurlstone Adam F.L. , Lamb Richard F. , Wheeler Tricia , Ozanne Bradford W.
Published: July 2002

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.
Direct Membrane Insertion of Voltage-dependent Anion-selective Channel Protein Catalyzed by Mitochondrial Tom20
Authors: Schleiff Enrico , Silvius John R. , Shore Gordon C.
Published: July 2002

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent β-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP.  Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.
Human Myoblast Fusion Requires Expression of Functional Inward Rectifier Kir2.1 Channels
Authors: Fischer-Lougheed Jacqueline , Liu Jian-Hui , Espinos Estelle , Mordasini David , Bader Charles R. , Belin Dominique , Bernheim Laurent
Published: July 2002

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through α1H T channels.