bluuebunny/crossref_metadata_2025 huggingface dataset:
Heteromeric association creates a P2Y-like adenosine receptor
Authors: Yoshioka Kazuaki , Saitoh Osamu , Nakata Hiroyasu
Published: July 2002

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G
i/o
protein-coupled A
1
adenosine receptor (A
1
R) and G
q
protein-coupled P2Y
1
receptor (P2Y
1
R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A
1
R and P2Y
2
receptor, but not A
1
R and dopamine D
2
receptor, also were found to coimmunoprecipitate in cotransfected cells. A
1
R agonist and antagonist binding to cell membranes were reduced by coexpression of A
1
R and P2Y
1
R, whereas a potent P2Y
1
R agonist adenosine 5′-
O
-(2-thiotriphosphate) (ADPβS) revealed a significant potency to A
1
R binding only in the cotransfected cell membranes. Moreover, the A
1
R/P2Y
1
R coexpressed cells showed an ADPβS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A
1
R antagonist, indicating that ADPβS binds A
1
R and inhibits adenylyl cyclase activity via G
i/o
proteins. Also, a high degree of A
1
R and P2Y
1
R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A
1
R with P2Y
1
R generates A
1
R with P2Y
1
R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.
Bacterial-type DNA Holliday junction resolvases in eukaryotic viruses
Authors: Garcia Alonzo D. , Aravind L. , Koonin Eugene V. , Moss Bernard
Published: July 2002

Homologous DNA recombination promotes genetic diversity and the maintenance of genome integrity, yet no enzymes with specificity for the Holliday junction (HJ)—a key DNA recombination intermediate—have been purified and characterized from metazoa or their viruses. Here we identify critical structural elements of RuvC, a bacterial HJ resolvase, in uncharacterized open reading frames from poxviruses and an iridovirus. The putative vaccinia virus resolvase was expressed as a recombinant protein, affinity purified, and shown to specifically bind and cleave a synthetic HJ to yield nicked duplex molecules. Mutation of either of two conserved acidic amino acids abrogated the catalytic activity of the A22R protein without affecting HJ binding. The presence of bacterial-type enzymes in metazoan viruses raises evolutionary questions.
A maize sesquiterpene cyclase gene induced by insect herbivory and volicitin: Characterization of wild-type and mutant alleles
Authors: Shen Binzhang , Zheng Zhenwei , Dooner Hugo K.
Published: July 2002

Plants can defend themselves from herbivorous insects by emitting
volatile chemical signals that attract natural enemies of the
herbivore. For example, maize seedlings attacked by beet armyworm
larvae (
Spodoptera exigua
) produce a mixture of
terpenoid and indole volatiles that serve to attract parasitic wasps. A
key step in terpenoid biosynthesis is the conversion of acyclic prenyl
diphosphates to terpenoid compounds by specific terpenoid synthases
(cyclases). We have cloned a maize sesquiterpene cyclase gene,
stc1
, by transposon tagging and have identified two
deletion mutations of the gene. The
stc1
gene is located
on chromosome 9S and does not seem to have a closely related ortholog
in the maize genome. It is induced 15- to 30-fold in maize leaves by
beet armyworm larvae feeding or by application of purified volicitin,
the insect-derived elicitor, at a mechanically wounded site.
stc1
induction is systemic, because undamaged leaves of
the same plant show a similar increase in
stc1
transcription. Analysis of volatiles from volicitin-treated seedlings
revealed that a major naphthalene-based sesquiterpene was present in
wild-type seedlings but absent in the
Ac
-insertion and
x-ray-deletion mutants. Therefore, we have identified a maize gene that
responds to caterpillar herbivory by producing a chemical defense
signal that most likely serves to attract natural enemies of the
herbivore.
The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.
Authors: Choi T , Fukasawa K , Zhou R , Tessarollo L , Borror K , Resau J , Vande Woude G F
Published: July 2002

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.
Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.
Authors: Smith T L , Sauer R T
Published: July 2002

In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc. The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.
Coordinated morphogenesis of epithelia during development of the Caenorhabditis elegans uterine-vulval connection.
Authors: Newman A P , Sternberg P W
Published: July 2002

Development of the nematode egg-laying system requires the formation of a connection between the uterine lumen and the developing vulval lumen, thus allowing a passage for eggs and sperm. This relatively simple process serves as a model for certain aspects of organogenesis. Such a connection demands that cells in both tissues become specialized to participate in the connection, and that the specialized cells are brought in register. A single cell, the anchor cell, acts to induce and to organize specialization of the epidermal and uterine epithelia, and registrates these tissues. The inductions act via evolutionarily conserved intercellular signaling pathways. The anchor cell induces the vulva from ventral epithelial cells via the LIN-3 growth factor and LET-23 transmembrane tyrosine kinase. It then induces surrounding uterine intermediate precursors via the receptor LIN-12, a founding member of the Notch family of receptors. Both signaling pathways are used multiple times during development of Caenorhabditis elegans. The outcome of the signaling is context-dependent. Both inductions are reciprocated. After the anchor cell has induced the vulva, it stretches toward the induced vulval cells. After the anchor cell has induced specialized uterine intermediate precursor cells, it fuses with a subset of their progeny.
Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates.
Authors: Du S , Traktman P
Published: July 2002

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of > or = 200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.
Organization of the Hox gene cluster in the grasshopper,
Schistocerca gregaria
Authors: Ferrier David E. K. , Akam Michael
Published: July 2002

The conserved organization of the Hox genes throughout the animal
kingdom has become one of the major paradigms of evolutionary
developmental biology. We have examined the organization of the Hox
genes of the grasshopper,
Schistocerca gregaria
. We find
that the grasshopper Hox cluster is over 700 kb long, and is not split
into equivalents of the Antennapedia complex and the bithorax complex
of
Drosophila melanogaster
.
SgDax
and probably
also
Sgzen
, the grasshopper homologues of
fushi-tarazu
(
ftz
) and
Zerknüllt
(
zen
), respectively, are also
in the cluster, showing that the non-homeotic Antp-class genes,
“accessory genes,” are an ancient feature of insect Hox clusters.
Identification of proton-active residues in a higher plant light-harvesting complex
Authors: Walters Robin G. , Ruban Alexander V. , Horton Peter
Published: July 2002

The thermal dissipation of absorbed light energy by the
light-harvesting apparatus of higher plants is important in protecting
the photosynthetic machinery from the effects of excess illumination. A
major mechanism for such photoprotection, known as trans-thylakoid
ΔpH-dependent chlorophyll fluorescence quenching (qE), is induced by
acidification of the lumen, is correlated with the interconversion of
xanthophyll pigments, and is manifested as quenching of chlorophyll
fluorescence. The mechanistic basis for qE remains unknown. The reagent
N
,
N
′-dicyclohexylcarbodiimide (DCCD)
specifically inhibits qE and covalently binds to two minor
light-harvesting pigment–protein complexes (LHCII), LHCIIa and LHCIIc.
It is shown that DCCD treatment of isolated LHCIIc complexes reverses
acid-induced chlorophyll fluorescence quenching in an
in
vitro
system. Fingerprinting of [
14
C]DCCD-labeled
LHCIIc demonstrates that there are two DCCD-sensitive amino acid
residues on this complex, and these are shown to be glutamate residues,
each of which is located near the lumen. In view of the effects of DCCD
on the pattern of proton release from photosystem II during
photosynthesis, we propose a model for the mechanism of the induction
of qE—that these residues form part of a proton pathway, the lumen pH
being sensed via its effects on the rate of proton release. One
possibility is that the resulting changes in the protonation state of
these carboxyl side chains may modulate the structural and energetic
organization of LHCII.
Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.
Authors: Botos I , Scapozza L , Zhang D , Liotta L A , Meyer E F
Published: July 2002

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.
Regulation of meiotic chromatin loop size by chromosomal position.
Authors: Heng H H , Chamberlain J W , Shi X M , Spyropoulos B , Tsui L C , Moens P B
Published: July 2002

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.
Variant antigens and endothelial receptor adhesion in Plasmodium falciparum.
Authors: Gardner J P , Pinches R A , Roberts D J , Newbold C I
Published: July 2002

Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface.
Molecular phylogeny of Archaea from soil
Authors: Bintrim Scott B. , Donohue Timothy J. , Handelsman Jo , Roberts Gary P. , Goodman Robert M.
Published: July 2002

Cultivation methods have contributed to our present knowledge about
the presence and diversity of microbes in naturally occurring
communities. However, it is well established that only a small fraction
of prokaryotes have been cultivated by standard methods and, therefore,
the prokaryotes that are cultivated may not reflect the composition and
diversity within those communities. Of the two prokaryotic phylogenetic
domains, Bacteria and Archaea, members of the former have been shown to
be ubiquitous in nature, with ample evidence of vast assemblages of
uncultured organisms. There is also now increasingly compelling
evidence that the Archaea, which were once thought to occupy a limited
number of environments, are also globally widespread. Here we report
the use of molecular phylogenetic techniques, which are independent of
microbial cultivation, to conduct an assessment of Archaea in a soil
microbial community. Small subunit ribosomal RNA genes of Archaea were
amplified from soil and cloned. Phylogenetic and nucleotide signature
analyses of these cloned small subunit ribosomal RNA gene sequences
revealed a cluster of Archaea from a soil microbial community that
diverge deeply from the crenarchaeotal line of descent and has the
closest affiliation to the lineage of planktonic Archaea. The
identification and phylogenetic classification of this archaeal lineage
from soil contributes to our understanding of the ecological
significance of Archaea as a component of microbial communities in
non-extreme environments.
The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in
Saccharomyces cerevisiae
Authors: de la Cruz Jesús , Iost Isabelle , Kressler Dieter , Linder Patrick
Published: July 2002

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5′ end of the mRNA. In
Saccharomyces cerevisiae
, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the
CAF20
gene. Here, we show a negative regulatory role for the p20 protein in translation initiation. Deletion of
CAF20
partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the
DED1
gene, which we have isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The
DED1
gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of
ded1
mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in
S. cerevisiae
.
Heme compounds in dinosaur trabecular bone
Authors: Schweitzer Mary H. , Marshall Mark , Carron Keith , Bohle D. Scott , Busse Scott C. , Arnold Ernst V. , Barnard Darlene , Horner J. R. , Starkey Jean R.
Published: July 2002

Six independent lines of evidence point to the existence of heme-containing compounds and/or hemoglobin breakdown products in extracts of trabecular tissues of the large theropod dinosaur
Tyrannosaurus rex
. These include signatures from nuclear magnetic resonance and electron spin resonance that indicate the presence of a paramagnetic compound consistent with heme. In addition, UV/visible spectroscopy and high performance liquid chromatography data are consistent with the Soret absorbance characteristic of this molecule. Resonance Raman profiles are also consistent with a modified heme structure. Finally, when dinosaurian tissues were extracted for protein fragments and were used to immunize rats, the resulting antisera reacted positively with purified avian and mammalian hemoglobins. The most parsimonious explanation of this evidence is the presence of blood-derived hemoglobin compounds preserved in the dinosaurian tissues.
Relationships between enzymatic flux capacities and metabolic flux rates: Nonequilibrium reactions in muscle glycolysis
Authors: Suarez R. K. , Staples J. F. , Lighton J. R. B. , West T. G.
Published: July 2002

The rules that govern the relationships between enzymatic flux capacities (
V
max
) and maximum physiological flux rates (
v
) at enzyme-catalyzed steps in pathways are poorly understood. We relate
in vitro V
max
values with
in vivo
flux rates for glycogen phosphorylase, hexokinase, and phosphofructokinase, enzymes catalyzing nonequilibrium reactions, from a variety of muscle types in fishes, insects, birds, and mammals. Flux capacities are in large excess over physiological flux rates in low-flux muscles, resulting in low fractional velocities (%
V
max
=
v
/
V
max
× 100)
in vivo
. In high-flux muscles, close matches between flux capacities and flux rates (resulting in fractional velocities approaching 100%
in vivo
) are observed. These empirical observations are reconciled with current concepts concerning enzyme function and regulation. We suggest that in high-flux muscles, close matches between enzymatic flux capacities and metabolic flux rates (i.e., the lack of excess capacities) may result from space constraints in the sarcoplasm.
A maternal diet high in
n
− 6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring
Authors: Hilakivi-Clarke Leena , Clarke Robert , Onojafe Ighovie , Raygada Margarita , Cho Elizabeth , Lippman Marc
Published: July 2002

We hypothesized that feeding pregnant rats with a high-fat diet would increase both circulating 17β-estradiol (E2) levels in the dams and the risk of developing carcinogen-induced mammary tumors among their female offspring. Pregnant rats were fed isocaloric diets containing 12% or 16% (low fat) or 43% or 46% (high fat) of calories from corn oil, which primarily contains the
n
− 6 polyunsaturated fatty acid (PUFA) linoleic acid, throughout pregnancy. The plasma concentrations of E2 were significantly higher in pregnant females fed a high
n
− 6 PUFA diet. The female offspring of these rats were fed with a laboratory chow from birth onward, and when exposed to 7,12-dimethylbenz(a)anthracene had a significantly higher mammary tumor incidence (60% vs. 30%) and shorter latency for tumor appearance (11.4 ± 0.5 weeks vs. 14.2 ± 0.6 weeks) than the offspring of the low-fat mothers. The high-fat offspring also had puberty onset at a younger age, and their mammary glands contained significantly higher numbers of the epithelial structures that are the targets for malignant transformation. Comparable changes in puberty onset, mammary gland morphology, and tumor incidence were observed in the offspring of rats treated daily with 20 ng of E2 during pregnancy. These data, if extrapolated to humans, may explain the link among diet, early puberty onset, mammary parenchymal patterns, and breast cancer risk, and indicate that an
in utero
exposure to a diet high in
n
− 6 PUFA and/or estrogenic stimuli may be critical for affecting breast cancer risk.
The small GTP-binding protein Rho links G protein-coupled receptors and Gα
12
to the serum response element and to cellular transformation
Authors: Fromm Christian , Coso Omar A. , Montaner Silvia , Xu Ningzhi , Gutkind J. Silvio
Published: July 2002

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gα
s
and Gα
i2
families were found in human tumors; and members of the Gα
q
and Gα
12
families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-
fos
protooncogene. One of the c-
fos
promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα
12
family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ
1
γ
2
dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα
12
and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.
Minimum size limit for useful locomotion by free-swimming microbes
Authors: Dusenbery David B.
Published: July 2002

Formulas are derived for the effect of size on a free-swimming microbe’s ability to follow chemical, light, or temperature stimuli or to disperse in random directions. The four main assumptions are as follows: (
i
) the organisms can be modeled as spheres, (
ii
) the power available to the organism for swimming is proportional to its volume, (
iii
) the noise in measuring a signal limits determination of the direction of a stimulus, and (
iv
) the time available to determine stimulus direction or to swim a straight path is limited by rotational diffusion caused by Brownian motion. In all cases, it is found that there is a sharp size limit below which locomotion has no apparent benefit. This size limit is estimated to most probably be about 0.6 μm diameter and is relatively insensitive to assumed values of the other parameters. A review of existing descriptions of free-floating bacteria reveals that the smallest of 97 motile genera has a mean length of 0.8 μm, whereas 18 of 94 nonmotile genera are smaller. Similar calculations have led to the conclusion that a minimum size also exists for use of pheromones in mate location, although this size limit is about three orders of magnitude larger. In both cases, the application of well-established physical laws and biological generalities has demonstrated that a common feature of animal behavior is of no use to small free-swimming organisms.
In vivo
functions of the
Saccharomyces cerevisiae
Hsp90 chaperone
Authors: Nathan Debra F. , Vos Melissa Harju , Lindquist Susan
Published: July 2002

In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear.
In vivo
, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins
in vitro
, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in
Saccharomyces cerevisiae
, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions
in vivo
. The data suggest that Hsp90 is not required for the
de novo
folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its
in vivo
functions are highly restricted.
Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization
Authors: Puech Anne , Saint-Jore Bruno , Funke Birgit , Gilbert Debra J. , Sirotkin Howard , Copeland Neal G. , Jenkins Nancy A. , Kucherlapati Raju , Morrow Bernice , Skoultchi Arthur I.
Published: July 2002

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.
Combined transgenic expression of α-galactosidase and α1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galα(1,3)Gal
Authors: Osman Narin , McKenzie Ian F. C. , Ostenried Karen , Ioannou Yiannis A. , Desnick Robert J. , Sandrin Mauro S.
Published: July 2002

Hyperacute rejection of pig organs by humans involves the interaction of Galα(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galα(1,3)Gal were investigated by overexpression of human lysosomal α-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human α-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galα(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human α-galactosidase showed only a 15–25% reduction in binding to natural human anti-Galα(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galα(1,3)Gal and degalactosylation results in the exposure ofN-acetyllactosamine residues and potential new xenoepitopes, using α-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human α1,2-fucosyltransferase as a transgene had ≈90% reduced Galα(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing α-galactosidase and α1,2-fucosyltransferase on Galα(1,3)Gal levels. Galα(1,3)Gal-positive COS cells expressing α1,3-galactosyltransferase, α1,2-fucosyltransferase, and α-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, α1,2-fucosyltransferase and α-galactosidase effectively reduced the expression of Galα(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galα(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.
Strong density- and diversity-related effects help to maintain tree species diversity in a neotropical forest
Authors: Wills Christopher , Condit Richard , Foster Robin B. , Hubbell Stephen P.
Published: July 2002

Intraspecific density-dependent effects in the Barro Colorado Island (Panama) study area are far stronger, and involve far more species, than previously had been suspected. Significant effects on recruitment, many extremely strong, are seen for 67 out of the 84 most common species in the plot, including the 10 most common. Significant effects on the intrinsic rate of increase are seen in 54 of the 84 species. These effects are far more common than interspecific effects, and are predominantly of the type that should maintain tree diversity. As a result, the more diverse an area in the forest is, the higher is the overall rate of increase of the trees in that area, although sheer crowding has by itself a negative effect. These findings are consistent with, but do not prove, an important role for host–pathogen interactions (defined broadly) in the maintenance of diversity. Ways are suggested by which to test host–pathogen models and competing models.
Bipolar localization of a chromosome partition protein in
Bacillus subtilis
Authors: Lin Daniel Chi-Hong , Levin Petra Anne , Grossman Alan D.
Published: July 2002

We have determined the subcellular localization of the chromosome partition protein Spo0J of
Bacillus subtilis
by immunofluorescence microscopy and visualizing fluorescence of a Spo0J–GFP fusion protein. Spo0J was associated with a region of the nucleoid proximal to the cell pole, both in growing cells dividing symmetrically and in sporulating cells dividing asymmetrically. Additional experiments indicated that Spo0J was bound to sites in the origin-proximal third of the chromosome. These results show that the replicating chromosomes are oriented in a specific manner during the division cycle, with the Spo0J binding region positioned toward the cell poles. Experiments characterizing cells at different stages of the cell cycle showed that chromosome orientation is established prior to the initiation of cell division. Our results indicate that there is a mechanism for orienting the chromosomes and that the chromosome partition protein Spo0J might be part of a bacterial mitotic-like apparatus.
Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides
Authors: Hiraiwa Masao , Taylor Eve M. , Campana W. Marie , Darin Sam J. , O’Brien John S.
Published: July 2002

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor. Herein prosaposin was found to increase sulfatide concentrations in primary and transformed Schwann cells (iSC) and oligodendrocytes (differentiated CG4 cells). Of the four mature saposins, only saposin C was found to increase sulfatide concentrations in these cell types. A similar result was obtained by using peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain. Dose–response curves demonstrated maximal enhancement by saposin C and prosaptides at low nanomolar concentrations (5–10 nM). The increase in sulfatide concentration by a 14-mer prosaptide, TX14(A), in CG4 oligodendrocytes was about 3-fold greater than in primary Schwann cells. A mutant prosaptide with a single amino acid replacement of Asn → Asp was inactive. Prosaptides did not induce cell proliferation of primary Schwann cells, iSC cells, or CG4 oligodendrocytes but nanomolar concentrations of prosaptides prevented cell death of iSC cells and CG4 oligodendrocytes. Immunoblot analysis demonstrated that phosphorylation of both mitogen-activated protein kinase p-42 and p-44 isoforms were enhanced 3- to 5-fold after 5 min of treatment with prosaptides at concentrations of 1–5 nM. These findings suggest that prosaposin and prosaptides bind to a receptor that initiates signal transduction to promote myelin lipid synthesis and prolong cell survival in both Schwann cells and oligodendrocytes. Prosaposin may function as a myelinotrophic factor
in vivo
during development and repair of myelinated nerves explaining the deficiency of myelin observed in prosaposin-deficient mice and humans.
Drosophila
NURF-55, a WD repeat protein involved in histone metabolism
Authors: Martínez-Balbás Marian A. , Tsukiyama Toshio , Gdula David , Wu Carl
Published: July 2002

The
Drosophila
nucleosome remodeling factor (NURF) is a protein complex of four distinct subunits that assists transcription factor-mediated chromatin remodeling. One NURF subunit, ISWI, is related to the transcriptional regulators
Drosophila
brahma and yeast SWI2/SNF2. We have determined peptide sequences and isolated cDNA clones for a second NURF component (the 55-kDa subunit). Immunological studies show that p55 is an integral subunit of NURF and is generally associated with polytene chromosomes. The predicted sequence of p55 reveals a WD repeat protein that is identical with the 55-kDa subunit of the
Drosophila
chromatin assembly factor (CAF-1). Given that WD repeat proteins related to p55 are associated with histone deacetylase and histone acetyltransferase, our findings suggest that p55 and its homologs may function as a common platform for the assembly of protein complexes involved in chromatin metabolism.
Human α-endosulfine, a possible regulator of sulfonylurea-sensitive K
ATP
channel: Molecular cloning, expression and biological properties
Authors: Heron Lisa , Virsolvy Anne , Peyrollier Karine , Gribble Fiona M. , Le Cam Alphonse , Ashcroft Frances M. , Bataille Dominique
Published: July 2002

Sulfonylureas are a class of drugs commonly used in the management of non-insulin-dependent diabetes mellitus. Their therapeutic action results primarily from their ability to inhibit ATP-sensitive potassium (K
ATP
) channels in the plasma membrane of pancreatic β cells and thereby stimulate insulin release. A key question is whether an endogenous ligand for the K
ATP
channel exists that is able to mimic the inhibitory effects of sulfonylureas. We describe here the cloning of the cDNA encoding human α-endosulfine, a 13-kDa peptide that is a putative candidate for such a role. α-Endosulfine is expressed in a wide range of tissues including muscle, brain, and endocrine tissues. The recombinant protein displaces binding of the sulfonylurea [
3
H]glibenclamide to β cell membranes, inhibits cloned K
ATP
channel currents, and stimulates insulin secretion. We propose that endosulfine is an endogenous regulator of the K
ATP
channel, which has a key role in the control of insulin release and, more generally, couples cell metabolism to electrical activity.
BCR/ABL-mediated leukemogenesis requires the activity of the small GTP-binding protein Rac
Authors: Skorski Tomasz , Wlodarski Pawel , Daheron Laurence , Salomoni Paolo , Nieborowska-Skorska Malgorzata , Majewski Miroslaw , Wasik Mariusz , Calabretta Bruno
Published: July 2002

The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2. We show here that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retained the resistance to growth factor deprivation-induced apoptosis but showed a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants were capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants was markedly prolonged as compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis.
Complex gangliosides are essential in spermatogenesis of mice: Possible roles in the transport of testosterone
Authors: Takamiya Kogo , Yamamoto Akihito , Furukawa Keiko , Zhao Jinmin , Fukumoto Satoshi , Yamashiro Shuji , Okada Masahiko , Haraguchi Masashi , Shin Masashi , Kishikawa Masao , Shiku Hiroshi , Aizawa Shinichi , Furukawa Koichi
Published: July 2002

Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC
2.4.1.94
) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides
in vivo
.
An autonomous folding unit mediates the assembly of two-stranded coiled coils
Authors: Kammerer Richard A. , Schulthess Therese , Landwehr Ruth , Lustig Ariel , Engel Jürgen , Aebi Ueli , Steinmetz Michel O.
Published: July 2002

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the
Dictyostelium discoideum
actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil “trigger” sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein
de novo
design.
ESA1 is a histone acetyltransferase that is essential for growth in yeast
Authors: Smith Edwin R. , Eisen Arri , Gu Weigang , Sattah Martin , Pannuti Antonio , Zhou Jianxin , Cook Richard G. , Lucchesi John C. , Allis C. David
Published: July 2002

Posttranslational acetylation of core histone amino termini has long been associated with transcriptionally active chromatin. Recent reports have demonstrated histone acetyltransferase activity in a small group of conserved transcriptional regulators directly linked to gene activation. In addition, the presence of a putative acetyltransferase domain has been discovered in a group of proteins known as the MYST family (for its founding members MOZ, YBF2/SAS3, SAS2, and Tip60). Members of this family are implicated in acute myeloid leukemia (MOZ), transcriptional silencing in yeast (SAS2 and YBF2/SAS3), HIV Tat interaction in humans (Tip60), and dosage compensation in
Drosophila
(MOF). In this report, we express a yeast ORF with homology to MYST family members and show it possesses histone acetyltransferase activity. Unlike the other MYST family members in
Saccharomyces cerevisiae
this gene is essential for growth.
CpABC, a
Cryptosporidium parvum
ATP-binding cassette protein at the host–parasite boundary in intracellular stages
Authors: Perkins Margaret E. , Riojas Ynolde A. , Wu Teresa W. , Le Blancq Sylvie M.
Published: July 2002

The intracellular parasite
Cryptosporidium parvum
develops inside a vacuole at the apex of its epithelial host cell. The developing parasite is separated from the host cell cytoplasm by a zone of attachment that consists of an extensively folded membranous structure known as the feeder organelle. It has been proposed that the feeder organelle is the site of regulation of transport of nutrients and drugs into the parasite. In this report, we localize an ≈200-kDa integral membrane protein, CpABC, from
Cryptosporidium parvum
to the host–parasite boundary, possibly the feeder organelle. The predicted amino acid sequence of CpABC has significant structural similarity with the cystic fibrosis conductance regulator and the multidrug resistance protein subfamily of ATP-binding cassette proteins. This is an example of a parasite-encoded transport protein localized at the parasite–host interface of an intracellular protozoan.
Relationship between presynaptic calcium transients and postsynaptic currents at single γ-aminobutyric acid (GABA)ergic boutons
Authors: Kirischuk S. , Veselovsky N. , Grantyn R.
Published: July 2002

Postsynaptic responses to stereotyped activation of single axons are known to fluctuate, but the origin of synaptic variability in the vertebrate central nervous system is still unclear. To test the hypothesis that fluctuations of inhibitory postsynaptic currents reflect variations in presynaptic Ca
2+
concentration, we examined single GABAergic axodendritic contacts in low-density cultures. Collicular neurons from rat embryos were loaded with the Ca
2+
indicator Oregon Green 488 BAPTA-1. Presynaptic axon terminals were visualized by staining with the styryl dye RH414. Under the condition of action potential block, RH414-labeled boutons were activated selectively by current pulses applied through a fine-tipped glass pipette. Short (1- to 3-ms) depolarization of isolated boutons resulted in stimulus-locked changes of presynaptic Ca
2+
concentration ([Ca
2+
]
pre
) and in evoked inhibitory postsynaptic currents (eIPSCs). Varying the strength of the stimulating currents produced a wide amplitude range of both presynaptic fluorescence transients (up to 220% of the resting value) and postsynaptic conductance changes (up to 2–3 nS). It was found that average eIPSCs displayed an approximately third-power dependency on [Ca
2+
]
pre
. Transmitter release retained its probabilistic character throughout the range of observed [Ca
2+
]
pre
values. In any tested single bouton, maximal eIPSCs occurred in association with the largest [Ca
2+
]
pre
transients, but failures were present at any [Ca
2+
]
pre
. The increase of maximal eIPSC amplitudes in connection with higher [Ca
2+
]
pre
supports the hypothesis that GABAergic boutons have the capacity to regulate synaptic strength by changing the number of simultaneously released vesicles.