bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of potent and selective human cathepsin K inhibitors that span the active site
Authors: Thompson Scott K. , Halbert Stacie M. , Bossard Mary J. , Tomaszek Thaddeus A. , Levy Mark A. , Zhao Baoguang , Smith Ward W. , Abdel-Meguid Sherin S. , Janson Cheryl A. , D’Alessio Karla J. , McQueney Michael S. , Amegadzie Bernard Y. , Hanning Charles R. , DesJarlais Renee L. , Briand Jacques , Sarkar Susanta K. , Huddleston Michael J. , Ijames Carl F. , Carr Steven A. , Garnes Keith T. , Shu Art , Heys J. Richard , Bradbeer Jeremy , Zembryki Denise , Lee-Rykaczewski Liz , James Ian E. , Lark Michael W. , Drake Fred H. , Gowen Maxine , Gleason John G. , Veber Daniel F.
Published: July 2002

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both
in vitro
and
in vivo
and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.
Identification of the B cell superantigen-binding site of HIV-1 gp120
Authors: Karray Saoussen , Zouali Moncef
Published: July 2002

Previous studies showed that the gp120 envelope protein of HIV-1 is able to crosslink membrane IgM on normal human B cells and to induce their activation in a VH3 immunoglobulin gene-family-specific manner. Because this VHgene family is the largest in the human repertoire, this superantigen (SAg) property is thought to have deleterious consequences for the host, including a progressive decline of B cells with progression of the HIV-1-induced disease. Here, we have identified the sequence motifs on gp120 involved in SAg binding to normal Igs. We show that this SAg-binding activity is present in gp120s from highly divergent isolates of HIV-1 belonging to clades derived from various geographical origins, and that carbohydrate residues are not essential for its expression. The SAg-binding site is formed by protein sequences from two regions of the gp120 molecule. The core motif is a discontinuous epitope spanning the V4 variable domain and the amino-terminal region flanking the C4 constant domain. The most critical residues appear to be Leu395–Asp397and Ile425–Gln427. Residues from the C2 constant domain (positions 252–272) also seem to play an accessory role in SAg binding of gp120 to normal human Igs. These findings are important in the design of a successful gp120-based vaccine against HIV-1.
Multiple promoter elements differentially regulate the expression of the mouse tenascin gene
Authors: Copertino Donald W. , Edelman Gerald M. , Jones Frederick S.
Published: July 2002

Tenascin (TN) is an extracellular matrix glycoprotein that is expressed in a characteristic spatiotemporal pattern during development and is up-regulated in the adult during tumorigenesis, wound healing, and nerve regeneration. In previous studies, we identified a promoter within the proximal 250 bp upstream of the mouse TN gene that contains several putative regulatory elements that are conserved among vertebrate TN genes. We have identified four different DNA elements within this promoter and show that they contribute in different ways to TN gene expression in NIH 3T3 fibroblasts, C6 glioma cells, and N2A neuroblastoma cells. These elements comprise a binding site for Krox proteins, one for nuclear factor 1, an octamer motif that binds POU-homeodomain proteins, and a novel TN control element. The nuclear factor 1 and TN control element had positive effects on TN promoter activity and formed similar DNA–protein complexes with nuclear extracts from all three cell lines. The Krox element had a negative effect on TN promoter activity in N2A cells, a positive effect in C6 cells, and no effect in NIH 3T3 cells. Two DNA binding complexes, one correlated with the negative and the other with the positive activities of the Krox element, were found to contain the protein Krox24. In cotransfection experiments, the octamer motif was required for induction of TN promoter activity by the POU-homeodomain protein Brn2 in N2A cells but was inactive in C6 cells. Consistent with these findings, N2A cells transfected with Brn2 formed octamer-binding complexes containing N-Oct3, the transcriptionally active form of Brn2, whereas complexes formed in C6 cells contained only N-Oct5A and N-Oct5B. Our results provide a striking example of the diversity of regulatory mechanisms that can be called forth by combining different promoter motifs with transcriptional activators or repressors.
Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol
Authors: Rapak Andrzej , Falnes Pål Ø. , Olsnes Sjur
Published: July 2002

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na
2
35
SO
4
, the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.
Human tau becomes phosphorylated and forms filamentous deposits when overexpressed in lamprey central neurons
in situ
Authors: Hall Garth F. , Yao Jun , Lee Gloria
Published: July 2002

Microinjection of plasmids encoding human tau (htau) protein into identified lamprey reticulospinal neurons (anterior bulbar cells, or ABCs)
in situ
induces chronic htau expression. htau protein is transported to both the axon and dendrites of expressing ABCs by mechanisms that require the C-terminal domain of htau protein but do not require directed htau mRNA transport. htau becomes phosphorylated at the PHF-1 (Ser-396/404) and TAU-1/AT8 (Ser-199/202) epitopes throughout ABCs with heavy htau accumulations; many such cells also exhibit degenerative changes, which include the development of extracellular htau deposits. Finally, expression of htau protein fused to green fluorescent protein induced the somatodendritic accumulation of filaments containing htau when examined by immunoelectron microscopy. These results suggest that chronic expression of htau in lamprey ABCs may be useful for studying cellular mechanisms underlying tau hyperphosphorylation and filament formation in vertebrate central neurons
in situ
.
Mutations in the
MRE11
,
RAD50
,
XRS2
, and
MRE2
genes alter chromatin configuration at meiotic DNA double-stranded break sites in premeiotic and meiotic cells
Authors: Ohta Kunihiro , Nicolas Alain , Furuse Munenori , Nabetani Akira , Ogawa Hideyuki , Shibata Takehiko
Published: July 2002

In the yeast
Saccharomyces cerevisiae
, meiotic recombination is initiated by DNA double-stranded breaks (DSBs) occurring in micrococcal nuclease (MNase)-hypersensitive regions of the chromatin. MNase-sensitive sites also undergo meiosis-specific alterations in chromatin structure prior to the appearance of DSBs. DSB formation requires the products of numerous genes. Herein we have examined the effects of mutations in four such genes,
MRE11
,
RAD50
,
XRS2
, and
MRE2
, on MNase sensitivity at DSB sites in premeiotic and meiotic cells. Disruption mutations in each of four genes confer greater than wild-type levels of MNase sensitivity in premeiotic cells. In meiotic prophase, all of these mutations affect MNase sensitivity at DSB sites and fall into two distinct phenotypic classes. The type 1 mutations (
mre2
and
mre11
) confer a reduction in MNase sensitivity relative to the wild-type level. The type 2 mutations (
rad50
and
xrs2
) permit a meiotic increase in the MNase sensitivity to reach a final level higher than that observed in wild-type cells. An
mre11
disruption mutation (type 1) is epistatic to a
rad50
null mutation (type 2) with respect to its meiotic effects on MNase sensitivity, suggesting that the events observed in the type 2 mutants during meiosis are dependent upon type 1 functions. One interpretation of these results is that
Mre11
,
Rad50
,
Xrs2
, and possibly
Mer2
(whose splicing is
Mre2
-dependent) form a complex at recombination hot spots and establish a chromatin/DNA configuration favorable for the induction of DSBs.
Filament structure as an essential factor for regulation of
Dictyostelium
myosin by regulatory light chain phosphorylation
Authors: Liu Xiong , Ito Kohji , Morimoto Sayuri , Hikkoshi-Iwane Atsuko , Yanagida Toshio , Uyeda Taro Q. P.
Published: July 2002

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of
Dictyostelium
myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of
Dictyostelium
myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of
Dictyostelium
heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.
HIV type-1 infection of the cotton rat (Sigmodon fulviventerandS. hispidus)
Authors: Langley Raymond J. , Prince Gregory A. , Ginsberg Harold S.
Published: July 2002

Cotton rats (Sigmodon hispidusandS. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat andS. fulviventeris more susceptible thanS. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorlyin vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 × 105cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. NeonateS. fulviventerwere not more susceptible to infection than adults.In vitroculture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase–PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improvedin vivo.
Studying the recruitment of Sp1 to the β-globin promoter with an
in vivo
method: Protein position identification with nuclease tail (PIN*POINT)
Authors: Lee Jong-Soo , Lee Chang-Hun , Chung Jay H.
Published: July 2002

Transcription is thought to be regulated by recruitment of transcription factors, adaptors, and certain enzymes to cis-acting elements through protein–DNA interactions and protein–protein interactions. To better understand transcription, a method with the capability to detect
in vivo
recruitment of these individual proteins will be essential. Toward this end, we use a previously undescribed
in vivo
method that we term protein position identification with nuclease tail (PIN*POINT). In this method, a fusion protein composed of a chosen protein linked to a nonsequence-specific nuclease is expressed
in vivo
, and the binding of the protein to DNA is made detectable by the nuclease-induced cleavage near the binding site. In this article, we used the technique protein position identification with nuclease tail to study the effect of the β-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Sp1 to the β-globin promoter. We present evidence that the hypersensitive sites of the LCR synergistically enhance the recruitment of a multimeric Sp1 complex to the β-globin promoter and that this may be accomplished by protein–protein interactions with proteins bound to the LCR, the upstream activator region, and, possibly, general transcription factors bound near the “TATA” box.
Mitochondrial Lon of
Saccharomyces cerevisiae
is a ring-shaped protease with seven flexible subunits
Authors: Stahlberg Henning , Kutejová Eva , Suda Kitaru , Wolpensinger Bettina , Lustig Ariel , Schatz Gottfried , Engel Andreas , Suzuki Carolyn K.
Published: July 2002

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from
Saccharomyces cerevisiae
identify Lon as a flexible ring-shaped heptamer. In the presence of ATP or 5′-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease.
Allele-specific activators and inhibitors for kinesin
Authors: Kapoor Tarun M. , Mitchison Timothy J.
Published: July 2002

Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in
in vitro
assays should clarify the role of individual motor proteins in complex cellular processes.
Heme oxygenase induction mediates the photoimmunoprotective activity of UVA radiation in the mouse
Authors: Reeve Vivienne E. , Tyrrell Rex M.
Published: July 2002

In contrast to the immunosuppressive potential of UVB (280–320 nm) radiation in experimental animals and humans, UVA (320–400 nm) radiation at environmentally relevant doses appears to be immunologically inert. However, such exposure to UVA radiation has been observed unexpectedly to induce resistance to UVB-induced immunosuppression in mice, by a mechanism resulting in the inactivation ofcis-urocanic acid (UCA), an epidermal immunosuppressive UV photoproduct. In this study in mice, we show that the immunoprotective activity of UVA radiation, against the effects of both UVB radiation andcis-UCA, can be attributed to the induction of cutaneous heme oxygenase (HO; EC1.14.99.3). Cell-mediated immune function was assessedin vivoby the contact hypersensitivity response induced to oxazolone at an unirradiated skin site, and HO enzyme activity was measured in cutaneous microsomal preparations from treated mice. There was a progressive increase in HO enzyme activity for at least 3 days after UVA irradiation. However HO activity, both constitutive and UVA radiation-induced, was sensitive to the effects of injecting mice with the specific HO inhibitor, tin protoporphyrin (Sn [IV] protoporphyrin IX; SnPP). We observed, in addition, that in SnPP-injected mice, the immunoprotective effect of UVA radiation against either UVB radiation orcis-UCA was abrogated. Because SnPP injection did not affect normal contact hypersensitivity responsiveness but did inhibit the constitutive HO enzyme activity, it appeared that only the inducible HO was active in modulating immune function. This finding indicates that UVA-induced HO activity is a major player in the skin defenses against UVB immunosuppression.
The molecular clock of HIV-1 unveiled through analysis of a known transmission history
Authors: Leitner Thomas , Albert Jan
Published: July 2002

Detailed knowledge about the rate and mode of the genetic variation
is vital for understanding how HIV-1 induces disease and develops
resistance as well as for studies on the molecular epidemiology and
origin of the virus. To unveil the molecular clock of HIV-1 we analyzed
a unique set of viruses from a known transmission history with
separation times between samples of up to 25 years. The
env
V3 and p17
gag
regions of the genome were sequenced, and
genetic distances were estimated by using the true tree and a
nucleotide substitution model based on a general reversible Markov
process with a gamma distribution to account for differences in
substitution rates among sites. Linear regression analysis showed that
separation times were significantly correlated with synonymous as well
as nonsynonymous nucleotide distances in both V3 and p17, giving strong
support for the existence of a molecular clock. The estimated rate of
nucleotide substitution was 6.7 ± 2.1 × 10
−3
substitutions/site per year in V3 and 2.7 ± 0.5 ×
10
−3
in p17. Importantly, the regression analyses showed
that there was a significant genetic distance at zero divergence times.
This pretransmission interval exists because the ramifications in the
phylogenetic trees do not correspond to time of transmission, but
rather to the coalescence time of the most recent common ancestor of
the viruses carried by the transmitter and the recipient. Simulation
experiments showed that neither the V3 nor the p17 clocks were
overdispersed, which indicates that the introduction of nucleotide
substitutions can be described adequately by a simple stochastic
Poisson process.
Reptilian chemistry: Characterization of dianeackerone, a secretory product from a crocodile
Authors: Whyte Authrine , Yang Zhi-Cai , Tiyanont Kittichoat , Weldon Paul J. , Eisner Thomas , Meinwald Jerrold
Published: July 2002

The major volatile component in the paracloacal glandular secretion of the adult African dwarf crocodile (
Osteolaemus tetraspis
) was isolated and characterized as a 19-carbon aromatic ketone, dianeackerone (3,7-diethyl-9-phenyl-2-nonanone). This ketone is absent from the secretion of immatures. Careful examination of dianeackerone samples isolated from individual adults revealed that this ketone occurs as both the (3
S
, 7
S
) and (3
S
, 7
R
) stereoisomers, with different individuals presenting strikingly different ratios of the isomeric forms. Our initial suspicion that the stereoisomeric dianeackerones might be indicators of gender proved untenable, leaving the role of these glandular constituents a challenge for future study.
Reptilian chemistry: Characterization of a family of dianeackerone-related steroidal esters from a crocodile secretion
Authors: Yang Zhicai , Whyte Authrine , Attygalle Athula B. , Weldon Paul J. , Eisner Thomas , Meinwald Jerrold
Published: July 2002

The African dwarf crocodile,
Osteolaemus tetraspis
(Crocodilidae, Reptilia), possesses a pair of skin glands, the paracloacal glands, the secretion of which is thought to be used to mark nest sites or attract mates. Ten aromatic steroidal esters were isolated from this secretion and characterized on the basis of NMR spectroscopic investigations, electrospray ionization-MS analyses, and chemical degradation. These esters, which account for more than 90% of the paracloacal glandular secretion, are derived from either cholesterol or cholestanol, esterified with a C-20 or C-22 acid closely related to dianeackerone, the only significant volatile compound found in this secretion.
Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes
Authors: McDowell T. L. , Gibbons R. J. , Sutherland H. , O’Rourke D. M. , Bickmore W. A. , Pombo A. , Turley H. , Gatter K. , Picketts D. J. , Buckle V. J. , Chapman L. , Rhodes D. , Higgs D. R.
Published: July 2002

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the
hATRX
gene cause severe syndromal mental retardation associated with α-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the
Drosophila
heterochromatic protein HP1
in vivo
, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the
ATRX
gene.
Intra-ring variability of Cr, As, Cd, and Pb in red oak revealed by secondary ion mass spectrometry: Implications for environmental biomonitoring
Authors: Brabander D. J. , Keon N. , Stanley R. H. R. , Hemond H. F.
Published: July 2002

Reconstructing the history of ambient levels of metals by using tree-ring chemistry is controversial. This controversy can be resolved in part through the use of selective microanalysis of individual wood cells. Using a combination of instrumental neutron activation analysis and secondary ion mass spectrometry, we have observed systematic inhomogeneity in the abundance of toxic metals (Cr, As, Cd, and Pb) within annual growth rings of
Quercus rubra
(red oak) and have characterized individual xylem members responsible for introducing micrometer-scale gradients in toxic metal abundances. These gradients are useful for placing constraints on both the magnitude and the mechanism of heavy metal translocation within growing wood. It should now be possible to test, on a metal-by-metal basis, the suitability of using tree-ring chemistries for deciphering long-term records of many environmental metals.
The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-κB pathways
Authors: Costello Patrick S. , Walters Alice E. , Mee P. Joseph , Turner Martin , Reynolds Lucinda F. , Prisco Antonella , Sarner Nitza , Zamoyska Rose , Tybulewicz Victor L. J.
Published: July 2002

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav−/−) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiateIL-2gene transcription. Furthermore, we analyze TCR-proximal signaling pathways inVav−/−T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-κB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.
Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells
Authors: Chung Chang Y. , Lee Susan , Briscoe Celia , Ellsworth Charlene , Firtel Richard A.
Published: July 2002

We have used the chemotactic ability of
Dictyostelium
cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active
Dictyostelium
Rac1B
61L
or disruption of DdRacGAP1, which encodes a
Dictyostelium
Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B
61L
and
Ddracgap1
null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B
17N
do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.
Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase
Authors: Tachedjian Gilda , Aronson Hans-Erik G. , Goff Stephen P.
Published: July 2002

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization.
In vitro
binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.
Solution to Darwin's dilemma: Discovery of the missing Precambrian record of life
Authors: Schopf J. William
Published: July 2002

In 1859, in
On the Origin of Species
, Darwin
broached what he regarded to be the most vexing problem facing his
theory of evolution—the lack of a rich fossil record predating the
rise of shelly invertebrates that marks the beginning of the Cambrian
Period of geologic time (≈550 million years ago), an
“inexplicable” absence that could be “truly urged as a valid
argument” against his all embracing synthesis. For more than 100
years, the “missing Precambrian history of life” stood out as one
of the greatest unsolved mysteries in natural science. But in recent
decades, understanding of life's history has changed markedly as the
documented fossil record has been extended seven-fold to some 3,500
million years ago, an age more than three-quarters that of the planet
itself. This long-sought solution to Darwin's dilemma was set in
motion by a small vanguard of workers who blazed the trail in the 1950s
and 1960s, just as their course was charted by a few pioneering
pathfinders of the previous century, a history of bold pronouncements,
dashed dreams, search, and final discovery.
Precambrian animal diversity: Putative phosphatized embryos from the Doushantuo Formation of China
Authors: Chen Jun-Yuan , Oliveri Paola , Li Chia-Wei , Zhou Gui-Qing , Gao Feng , Hagadorn James W. , Peterson Kevin J. , Davidson Eric H.
Published: July 2002

Putative fossil embryos and larvae from the Precambrian phosphorite rocks of the Doushantuo Formation in Southwest China have been examined in thin section by bright field and polarized light microscopy. Although we cannot completely exclude a nonbiological or nonmetazoan origin, we identified what appear to be modern cnidarian developmental stages, including both anthozoan planula larvae and hydrozoan embryos. Most importantly, the sections contain a variety of small (≤200 μm) structures that greatly resemble gastrula stage embryos of modern bilaterian forms.
De novo
design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions
Authors: Ramagopal Udupi A. , Ramakumar Suryanarayanarao , Sahal Dinkar , Chauhan Virander S.
Published: July 2002

Design of helical super secondary structural motifs is expected to
provide important scaffolds to incorporate functional sites, thus
allowing the engineering of novel miniproteins with function. An
α,β-dehydrophenylalanine containing 21-residue apolar peptide was
designed to mimic the helical hairpin motif by using a simple
geometrical design strategy. The synthetic peptide folds into the
desired structure as assessed crystallographically at 1.0-Å
resolution. The two helices of the helical-hairpin motif, connected by
a flexible (Gly)
4
linker, are docked to each other by the
concerted influence of weak interactions. The folding of the peptide
without binary patterning of amino acids, disulfide bonds, or metal
ions is a remarkable observation. The results demonstrate that
preferred interactions among the hydrophobic residues selectively
discriminate their putative partners in space, leading to the unique
folding of the peptide, also a hallmark of the unique folding of
hydrophobic core in globular proteins. We demonstrate here the
engineering of molecules by using weak interactions pointing to their
possible further exploitation in the
de novo
design of
protein super secondary structural elements.
Insoluble glycogen, a metabolizable internal adsorbent, decreases the lethality of endotoxin shock in rats
Authors: Sipka S. , Bot G. , Gergely P. , Bertók L. , Csongor J. , Sápy P. , Szappanos M. , Nemes J. , Duda E. , Szegedi G.
Published: July 2002

Insoluble glycogen is an enzymatically modified form of naturally occurring soluble glycogen with a great adsorbing capacity. It can be metabolized by phagocytes to glucose. In this study we used insoluble glycogen intravenously in the experimental endotoxin shock of rats. Wistar male rats were sensitized to endotoxin by Pb acetate. The survival of rats were compared in groups of animals endotoxin shock treated and non‐treated with insoluble glycogen. Furthermore, we have determined in vitro the binding capacity of insoluble glycogen for endotoxin, tumour necrosis factor alpha, interleukin‐1 and secretable phospholipase A2. Use of 10 mg/kg dose of insoluble glycogen could completely prevent the lethality of shock induced by LD50 quantity of endotoxin in rats. All animals treated survived. Insoluble glycogen is a form of ‘metabolizable internal adsorbents’. It can potentially be
used for treatment of septic shock.
Economic policy and development in Austrian Lombardy, 1815–1859
Authors: Pichler Rupert
Published: July 2002

The question of economic integration is not new in Europe. Historically, the birth and construction of nation-states was important in stimulating interest in the systematic relationships between political and economic integration. In the case of the multinational structure of the Habsburg monarchy in the nineteenth century, the result was an economic policy that, for political reasons, aimed to unite the material interests of a state that was completely heterogeneous in other respects. Lombardy was a case in point. Traditionally the region had been in the economic vanguard in central Europe. When it again became part of Austria in 1815 it also became subject to the imperial policy of political integration. As a result its economic priorities were partially reformulated. On the one hand, Austria had a protectionist system aimed at autarky which made incentives to industrial production a priority. Lombardy's purely mercantilist outlook, on the other hand, was based around the production of a few highly specialized goods, most notably silk, for export. Conflict between economic interests in Lombardy was the inevitable result. Nevertheless, the imperial government had to take account of the fact that it was impossible to restrict Lombardy's international trade relations exclusively to the Austrian market. And the problems that beset any effort to tie the Lombard economy into a denser network of relationships with the Austrian market were not due to the political formation of the Italian nation because Northern Italy, and Lombardy in particular, continued to occupy an anomalous position within the context of the Italian economy.
Serum prolactin and response to treatment among cocaine‐dependent individuals
Authors: Patkar Ashwin A. , Hill Kevin P. , Sterling Robert C. , Gottheil Edward , Berrettini Wade H. , Weinstein Stephen P.
Published: July 2002

Abstract Considerable evidence indicates that dopaminergic mechanisms may modulate the central effects of cocaine. We investigated whether basal serum prolactin, a measure of central dopamine activity, differed between cocaine‐dependent subjects and controls, and whether prolactin levels among cocaine patients were related to their response to treatment. Eighty‐six African‐American cocaine‐dependent outpatients and 35 African‐American controls were studied. Prolactin concentrations in fasting blood samples were assayed by radioimmunoassay. The outcome measures were: number of negative urine drug screens, retention in treatment, counselor ratings of improvement and discharge status. Chi‐square tests and independent t ‐tests were used for data analyses. The basal prolactin(ng/ml) among cocaine patients (9.12 ± 4.12) was significantly higher compared to controls (7.14 ± 3.36) (t = 2.52, p < 0.02). Furthermore, the higher prolactin subjects (median prolactin = 7.71) had significantly fewer negative urine screens (p < 0.05) and received less favorable ratings of improvement by counselors (p < 0.01) compared to the lower prolactin group. However, the two groups did not differ significantly in treatment retention (p = 0.13) or discharge status (p = 0.08). The higher basal prolactin among cocaine patients relative to controls may reflect changes in DA activity among cocaine patients. Moreover, higher prolactin seems to be related negatively with certain measures of response to treatment among cocaine patients.
Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly
Authors: Becker K. David , Gottshall Kim R. , Hickey Reed , Perriard Jean-Claude , Chien Kenneth R.
Published: July 2002

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.
KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion
Authors: Beh Christopher T. , Brizzio Valeria , Rose Mark D.
Published: July 2002

KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.
A Structural Model for Phosphorylation Control of Dictyostelium Myosin II Thick Filament Assembly
Authors: Liang Wenchuan , Warrick Hans M. , Spudich James A.
Published: July 2002

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3×Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3×Asp myosin II fails to rescue myosin II–null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II–null cells containing 3×Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled–coil structure.
The Tetraspanin Cd9 Associates with Transmembrane TGF-α and Regulates TGF-α–Induced Egf Receptor Activation and Cell Proliferation
Authors: Shi Wen , Fan Huizhou , Shum Lillian , Derynck Rik
Published: July 2002

Transforming growth factor-α (TGF-α) is a member of the EGF growth factor family. Both transmembrane TGF-α and the proteolytically released soluble TGF-α can bind to the EGF/TGF-α tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-α physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-α in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-α. CD9 expression strongly decreased the growth factor– and PMA- induced proteolytic conversions of transmembrane to soluble TGF-α and strongly enhanced the TGF- α–induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-α. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-α resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-α decreased the autocrine growth stimulatory effect of TGF-α in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21CIP1. These data reveal that the association of CD9 with transmembrane TGF-α regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.
Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport
Authors: Waterman-Storer Clare , Duey Devin Y. , Weber Kari L. , Keech John , Cheney Richard E. , Salmon E.D. , Bement William M.
Published: July 2002

Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.
The ER Repeat Protein Yt521-B Localizes to a Novel Subnuclear Compartment
Authors: Nayler Oliver , Hartmann Annette M. , Stamm Stefan
Published: July 2002

The characterization of distinct subnuclear domains suggests a dynamic nuclear framework supporting gene expression and DNA replication. Here, we show that the glutamic acid/arginine-rich domain protein YT521-B localizes to a novel subnuclear structure, the YT bodies. YT bodies are dynamic compartments, which first appear at the beginning of S-phase in the cell cycle and disperse during mitosis. Furthermore, in untreated cells of the human cell line MCF7 they were undetectable and appeared only after drug- induced differentiation. YT bodies contain transcriptionally active sites and are in close contact to other subnuclear structures such as speckles and coiled bodies. YT bodies disperse upon actinomycin D treatment, whereas other transcriptional inhibitors such as α-amanitin or DRB have little effect. On the basis of our experiments, we propose that YT521-B may participate in the assembly of genes into transcription centers, thereby allowing efficient regulation of gene expression.
Autocrine Stimulation of Human Pancreatic Duct–Like Development by Soluble Isoforms of Epimorphin in Vitro
Authors: Lehnert Lasse , Lerch Markus M. , Hirai Yohei , Kruse Marie-Luise , Schmiegel Wolff , Kalthoff Holger
Published: July 2002

Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5–6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.