bluuebunny/crossref_metadata_2025 huggingface dataset:
Coherent addition of AlGaAs lasers using microlenses and diffractive coupling
Authors: Leger James R. , Scott Miles L. , Veldkamp Wilfrid B.
Published: July 2002

A near single-lobed far-field pattern was obtained from coherent operation of a nonevanescently coupled AlGaAs laser diode array. A diffractive microlens array collimated the individual beams to approximate a plane wave, and diffractive coupling from an external cavity mirror provided mutual coherence. A diffraction-limited far-field pattern was observed with 82% of the power contained in the central lobe. The method is directly applicable to two-dimensional laser arrays and can be implemented as a single thin optical element.
Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site
Authors: Loo Tip W. , Clarke David M.
Published: July 2002

The human multidrug resistance P-glycoprotein uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. In this study, we used cysteine-scanning mutagenesis and cross-linking studies to identify residues that are exposed to the drug-binding site upon vanadate trapping. In the absence of nucleotides, C222(TM4) was cross-linked to C868(TM10) and C872(TM10); C306(TM5) was cross-linked to C868(TM10), C872(TM10), C945(TM11), C982(TM12), and C984(TM12); and C339(TM6) was cross-linked to C868(TM10), C872(TM10), C942(TM11), C982(TM12), and C985(TM12). These cysteines are in the middle of the predicted transmembrane (TM) segments and form the drug-binding site. Cross-linking between 332C(TM6) and cysteines introduced at the extracellular side of other TM segments was also done. In the absence of nucleotides, residues 332C and 856C on the extracellular side of TMs 6 and 10, respectively, were cross-linked with a 13-Å cross-linker (M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate). ATP plus vanadate inhibited cross-linking between 332C(TM6) and 856C(TM10) as well as those in the drug-binding site. Instead, vanadate trapping promoted cross-linking between 332C(TM6) and 976C(TM12) with a 10-Å cross-linker (M6M, 1,6-hexanediyl bismethanethiosulfonate). When ATP hydrolysis was allowed to proceed, then 332C(TM12) could form a disulfide bond with 975C(TM12). The cross-linking pattern of 332C(TM6) with residues in TM10 and TM12 indicates that the drug-binding site undergoes dynamic and relatively large conformational changes, and that different residues are exposed to the drug-binding site during the resting phase, upon vanadate trapping and at the completion of the catalytic cycle.
Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment
Authors: Muchowski Paul J. , Ning Ke , D'Souza-Schorey Crislyn , Fields Stanley
Published: July 2002

Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein. The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein. However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear. Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a
Saccharomyces cerevisiae
model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1. Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.
Blue light-induced kinetics of H
+
and Ca
2+
fluxes in etiolated wild-type and phototropin-mutant
Arabidopsis
seedlings
Authors: Babourina Olga , Newman Ian , Shabala Sergey
Published: July 2002

Ion flux kinetics associated with blue light (BL) treatment of two wild types (WTs) and the
phot1
,
phot2
and
phot1/phot2
mutants of
Arabidopsis
were studied by using the MIFE noninvasive ion-selective microelectrode technique. BL induced significant changes in activity of H
+
and Ca
2+
transporters within the first 10 min of BL onset, peaking between 3 and 5 min. In all WT plants and in
phot2
mutants, BL induced immediate Ca
2+
influx. In
phot1
and
phot1/phot2
mutants, net Ca
2+
flux remained steady. It is suggested that PHOT1 regulates Ca
2+
uptake into the cytoplasm from the apoplast. Changes in ion fluxes were measured from cotyledons of intact seedlings and from the cut top of the hypocotyl of decapitated seedlings. Thus the photoreceptors mediating BL-induced Ca
2+
and H
+
fluxes are present in the rest of the decapitated seedling and probably in the cotyledons as well. The H
+
and Ca
2+
flux responses to BL appear not to be linked because, (
i
) when changes were observed for both ions, Ca
2+
flux changed almost immediately, whereas H
+
flux lagged by about 1.5 min; (
ii
) in the Wassilewskija ecotype, changes in H
+
fluxes were small. Finally, wave-like changes in Ca
2+
and H
+
concentrations were observed along the cotyledon–hook axis regardless of its orientation to the light.
Target-induced formation of neuraminidase inhibitors from
in vitro
virtual combinatorial libraries
Authors: Hochgürtel Matthias , Kroth Heiko , Piecha Dorothea , Hofmann Michael W. , Nicolau Claude , Krause Sonja , Schaaf Otmar , Sonnenmoser Gabriele , Eliseev Alexey V.
Published: July 2002

Neuraminidase, a key enzyme responsible for influenza virus propagation, has been used as a template for selective synthesis of small subsets of its own inhibitors from theoretically highly diverse dynamic combinatorial libraries. We show that the library building blocks, aldehydes and amines, form significant amounts of the library components resulting from their coupling by reductive amination
only
in the presence of the enzyme. The target amplifies the best hits at least 120-fold. The dynamic libraries synthesized and screened in such an
in vitro
virtual mode form the components that possess high inhibitory activity, as confirmed by enzyme assays with independently synthesized individual compounds.
The role of distinct p185
neu
extracellular subdomains for dimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling
Authors: Kumagai Toru , Davis James G. , Horie Takeo , O'Rourke Donald M. , Greene Mark I.
Published: July 2002

The extracellular domain of p185
c-neu
can be viewed as
a complex structure of four subdomains, two of which are cysteine-rich
subdomains. We have investigated the contribution of these distinct
p185
c-neu
extracellular subdomains to p185/epidermal
growth factor receptor (EGFR) heteromer formation and EGF-induced
heteromeric signaling. Our studies indicate that at least two separate
p185 subdomains, a region spanning subdomains I and II and subdomain IV
are involved in association of p185 with the EGFR. We also demonstrated
that subdomain IV reduced the heteromeric signaling and transforming
activities induced by EGF after associating with EGFR. When 126 aa were
deleted from subdomain IV, this small subdomain IV-derived fragment
could still lead to heterodimers with EGFR and suppress EGF-induced
mitogen-activated protein kinase activation and subsequent
transformation abilities. These data provide information about
trans-inhibitory mechanisms of mutant p185 species and also indicate
that both the entire and a part of subdomain IV may represent a
therapeutic target for erbB-overexpressing tumors. Finally, these
studies define a basic feature of receptor-receptor associations that
are determined by cystine-knot containing subdomains.
Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches
Authors: Heaton Richard J. , Peterson Alexander W. , Georgiadis Rosina M.
Published: July 2002

We demonstrate that
in situ
optical surface plasmon
resonance spectroscopy can be used to monitor hybridization kinetics
for unlabeled DNA in tethered monolayer nucleic acid films on gold in
the presence of an applied electrostatic field. The dc field can
enhance or retard hybridization and can also denature
surface-immobilized DNA duplexes. Discrimination between matched and
mismatched hybrids is achieved by simple adjustment of the electrode
potential. Although the electric field at the interface is extremely
large, the tethered single-stranded DNA thiol probes remain bound and
can be reused for subsequent hybridization reactions without loss of
efficiency. Only capacitive charging currents are drawn; redox
reactions are avoided by maintaining the gold electrode potential
within the ideally polarizable region. Because of potential-induced
changes in the shape of the surface plasmon resonance curve, we account
for the full curve rather than simply the shift in the resonance
minimum.
Human antibodies against spores of the genus
Bacillus
: A model study for detection of and protection against anthrax and the bioterrorist threat
Authors: Zhou Bin , Wirsching Peter , Janda Kim D.
Published: July 2002

A naïve, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of
Bacillus subtilis
IFO 3336 suspended in solution. A direct
in vitro
panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of
B. subtilis
IFO 3336 vs. a panel of spores from 11 other
Bacillus
species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with
B. licheniformis
5A24. The Fab-phage clone 52 was improved compared with 5B and was comparable to 7E in binding
B. subtilis
IFO 3336 vs.
B. licheniformis
5A24, yet showed a distinctive crossreactivity pattern with other spores. We also developed a method to directly detect individual spores by using fluorescently labeled antibody-phage. Finally, a variety of “powders” that might be used in deploying spores of
B. anthracis
were examined for antibody-phage binding. The strategies described provide a foundation to discover human antibodies specific for native spores of
B. anthracis
that can be developed as diagnostic and therapeutic reagents.
A crystallographic map of the transition from B-DNA to A-DNA
Authors: Vargason Jeffrey M. , Henderson Keith , Ho P. Shing
Published: July 2002

The transition between B- and A-DNA was first observed nearly 50 years ago. We have now mapped this transformation through a set of single-crystal structures of the sequence d(GGCGCC)
2
, with various intermediates being trapped by methylating or brominating the cytosine bases. The resulting pathway progresses through 13 conformational steps, with a composite structure that pairs A-nucleotides with complementary B-nucleotides serving as a distinct transition intermediate. The details of each step in the conversion of B- to A-DNA are thus revealed at the atomic level, placing intermediates for this and other sequences in the context of a common pathway.
Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers
Authors: Sant'Angelo Derek B. , Cresswell Peter , Janeway Charles A. , Denzin Lisa K.
Published: July 2002

T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the β chain and, more commonly, for the α chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This “functional allelic exclusion” is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.
FLIP switches Fas-mediated glucose signaling in human pancreatic β cells from apoptosis to cell replication
Authors: Maedler Kathrin , Fontana Adriano , Ris Frédéric , Sergeev Pavel , Toso Christian , Oberholzer José , Lehmann Roger , Bachmann Felix , Tasinato Andrea , Spinas Giatgen A. , Halban Philippe A. , Donath Marc Y.
Published: July 2002

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic β cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of β cell turnover. In human islets, elevated glucose concentrations impair β cell proliferation and induce β cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients.
In vitro
exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive β cells; FLIP was no longer detectable in such TUNEL-positive β cells. Up-regulation of FLIP, by incubation with transforming growth factor β or by transfection with an expression vector coding for FLIP, protected β cells from glucose-induced apoptosis, restored β cell proliferation, and improved β cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human β cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.
Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in
Escherichia coli
Authors: Moreno-Bruna Beatriz , Baroja-Fernández Edurne , Muñoz Francisco José , Bastarrica-Berasategui Ainara , Zandueta-Criado Aitor , Rodríguez-López Milagros , Lasa Iñigo , Akazawa Takashi , Pozueta-Romero Javier
Published: July 2002

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC
3.6.1.21
) has been characterized by using
Escherichia coli
. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene,
aspP
, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria.
aspP
was cloned into an expression vector and introduced into
E. coli
. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the
aspP
-overexpressing and
aspP
-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in
E. coli
.
V(D)J recombination is not activated by demethylation of the kappa locus
Authors: Cherry Sara R. , Beard Caroline , Jaenisch Rudolf , Baltimore David
Published: July 2002

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation,
Dnmt1
, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.
Mutation of a BRCT domain selectively disrupts DNA single-strand break repair in noncycling Chinese hamster ovary cells
Authors: Moore David J. , Taylor Richard M. , Clements Paula , Caldecott Keith W.
Published: July 2002

The DNA single-strand break repair protein XRCC1 contains a BRCT
domain that binds and stabilizes intracellular DNA ligase III protein.
We recently demonstrated that this domain is largely dispensable for
single-strand break repair and cellular resistance to DNA base damage
in cycling cells. Here, we report that the BRCT domain is required for
single-strand break repair in noncycling cells. Mutations that disrupt
the BRCT domain and prevent DNA ligase III interaction abolished
XRCC1-dependent repair in serum-starved Chinese hamster ovary cells,
and reentry into cell cycle induced by readdition of serum restored
repair. Elevating DNA ligase III levels in XRCC1 mutant cells using
proteosome inhibitors or by expressing XRCC1 protein in which the BRCT
domain is disrupted but can still bind DNA ligase III failed to restore
repair in noncycling cells. The requirement for the BRCT domain for DNA
strand break repair is thus for more than simply binding and
stabilizing DNA ligase III. These data provide evidence in support of a
selective role for a DNA repair protein or protein domain in noncycling
cells. We propose that the XRCC1 C-terminal BRCT domain may be
important for genetic stability in postmitotic cells
in
vivo
.
Distribution of spontaneous plant hybrids.
Authors: Ellstrand N C , Whitkus R , Rieseberg L H
Published: July 2002

Natural hybridization is a relatively common feature of vascular plant species and has been demonstrated to have played an important role in their evolution. Nonetheless, it is not clear whether spontaneous hybridization occurs as a general feature of all plant families and genera or whether certain groups are especially prone to spontaneous hybridization. Therefore, we inspected five modern biosystematic floras to survey the frequency and taxonomic distribution of spontaneous hybrids. We found spontaneous hybridization to be nonrandomly distributed among taxa, concentrated in certain families and certain genera, often at a frequency out of proportion to the size of the family or genus. Most of these groups were primarily outcrossing perennials with reproductive modes that stabilized hybridity such as agamospermy, vegetative spread, or permanent odd polyploidy. These data suggest that certain phylogenetic groups are biologically predisposed for the formation and maintenance of hybrids.
An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.
Authors: Baumann P , Jackson S P
Published: July 2002

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.
Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.
Authors: Li M , Dyda F , Benhar I , Pastan I , Davies D R
Published: July 2002

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.
Starburst amacrine cells change from spiking to nonspiking neurons during retinal development.
Authors: Zhou Z J , Fain G L
Published: July 2002

The membrane excitability of cholinergic (starburst) amacrine cells was studied in the rabbit retina during postnatal development. Whole-cell patch-clamp recordings were made from 110 displaced starburst cells in a thin retina] slice preparation of rabbits between postnatal days P1 and P56 old. We report that displaced starburst cells undergo a dramatic transition from spiking to nonspiking, caused by a loss of voltage-gated Na currents. This change in membrane excitability occurred just after eye opening (P10), such that all of the starburst cells tested before eye opening had conspicuous tetrodotoxin-sensitive Na currents and action potentials, but none tested after the first 3 postnatal weeks had detectable Na currents or spikes. Our results suggest that starburst cells use action potentials transiently during development and probably play a functional role in visual development. These cells then cease to spike as the retina matures, presumably consistent with their role in visual processing in the mature retina.
Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.
Authors: Pilch D S , Poklar N , Gelfand C A , Law S M , Breslauer K J , Baird E E , Dervan P B
Published: July 2002

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.
Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.
Authors: Gdula D A , Gerasimova T I , Corces V G
Published: July 2002

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.
Sodium: a male moth's gift to its offspring.
Authors: Smedley S R , Eisner T
Published: July 2002

Males of the moth Gluphisia septentrionis acquire sodium by drinking from mud puddles. Analyses of male and female bodies indicate that such "puddling" behavior enables the male to provide his mate with a nuptial gift of sodium, presumably via the spermatophore. This gift (about 10 microg), amounting to more than half of a puddler male's total body sodium, is in large measure apportioned by the female to her eggs. Puddler-sired eggs contain 2 to 4 times more sodium than those control-sired; this difference is already apparent in eggs laid the night after mating. Paternal endowment of offspring with sodium had not previously been demonstrated for an insect to our knowledge. The potential adaptive significance of such chemical bestowal is evident, given that the foliar diet of G. septentrionis larvae is extremely low in sodium content.
Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from
Xenopus
oocytes
Authors: Guo Zhong , Liliom Károly , Fischer David J. , Bathurst Ian C. , Tomei L. David , Kiefer Michael C. , Tigyi Gábor
Published: July 2002

Lysophosphatidic acid
(1-acyl-2-lyso-
sn
-glycero-3-phosphate, LPA) is a
multifunctional lipid mediator found in a variety of organisms that
span the phylogenetic tree from humans to plants. Although its
physiological function is not clearly understood, LPA is a potent
regulator of mammalian cell proliferation; it is one of the major
mitogens found in blood serum. In
Xenopus laevis
oocytes, LPA elicits oscillatory Cl
−
currents. This
current, like other effects of LPA, is consistent with a plasma
membrane receptor-mediated activation of G protein-linked signal
transduction pathways. Herein we report the identification of a
complementary DNA from
Xenopus
that encodes a functional
high-affinity LPA receptor. The predicted structure of this protein of
372 amino acids contains features common to members of the seven
transmembrane receptor superfamily with a predicted extracellular amino
and intracellular carboxyl terminus. An antisense oligonucleotide
derived from the first 5–11 predicted amino acids, selectively
inhibited the expression of the endogenous high-affinity
LPA receptors in
Xenopus
oocytes, whereas the same
oligonucleotide did not affect the low-affinity LPA receptor.
Expression of the full-length cRNA in oocytes led to an increase in
maximal Cl
−
current due to increased expression of the
high-affinity LPA receptor, but activation of the low-affinity receptor
was, again, unaffected. Oocytes expressing cRNA prepared from this
clone showed no response to other lipid mediators including
prostaglandins, leukotrienes, sphingosine 1-phosphate,
sphingosylphosphorylcholine, and platelet-activating factor, suggesting
that the receptor is highly selective for LPA.
Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis.
Authors: Miyata T , Taneda S , Kawai R , Ueda Y , Horiuchi S , Hara M , Maeda K , Monnier V M
Published: July 2002

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.
Dated co-occurrence of Homo erectus and Gigantopithecus from Tham Khuyen Cave, Vietnam.
Authors: Ciochon R , Long V T , Larick R , González L , Grün R , de Vos J , Yonge C , Taylor L , Yoshida H , Reagan M
Published: July 2002

Tham Khuyen Cave (Lang Son Province, northern Vietnam) is one of the more significant sites to yield fossil vertebrates in east Asia. During the mid-1960s, excavation in a suite of deposits produced important hominoid dental remains of middle Pleistocene age. We undertake more rigorous analyses of these sediments to understand the fluvial dynamics of Pleistocene cave infilling as they determine how skeletal elements accumulate within Tham Khuyen and other east Asian sites. Uranium/thorium series analysis of speleothems brackets the Pleistocene chronology for breaching, infilling, and exhuming the regional paleokarst. Clast analysis indicates sedimentary constituents, including hominoid teeth and cranial fragments accumulated from very short distances and under low fluvial energy. Electron spin resonance analysis of vertebrate tooth enamel and sediments shows that the main fossil-bearing suite (S1-S3) was deposited about 475 thousand years ago. Among the hominoid teeth excavated from S1-S3, some represent Homo erectus and Gigantopithecus blacki. Criteria are defined to differentiate these teeth from more numerous Pongo pygmaeus elements. The dated co-occurrence of Homo erectus and Gigantopithecus blacki at Tham Khuyen helps to establish the long co-existence of these two species throughout east Asia during the Early and Middle Pleistocene.
Penelope
, a new family of transposable elements and its possible role in hybrid dysgenesis in
Drosophila virilis
Authors: Evgen’ev Michael B. , Zelentsova Helena , Shostak Natalia , Kozitsina Marina , Barskyi Victor , Lankenau Dirk-Henner , Corces Victor G.
Published: July 2002

A hybrid dysgenesis syndrome occurs in
Drosophila virilis
when males from an established laboratory strain are crossed to females obtained from the wild, causing the simultaneous mobilization of several different transposable elements. The insertion sequence responsible for the mutant phenotype of a dysgenic
yellow
allele has been characterized and named
Penelope. In situ
hybridization and Southern analyses reveal the presence of more than 30 copies of this element in the P-like parental strain, whereas
Penelope
is absent in all M-like strains tested.
Penelope
contains one 2.5-kb-long ORF that could encode products with homology to integrase and reverse transcriptase. Northern analysis and whole-mount
in situ
hybridization show strong induction of a 2.6-kb RNA in the ovaries of dysgenic females that is expressed at very low levels in the parental strains or in the progeny from the reciprocal cross. Injection of
Penelope
-containing plasmids into preblastoderm embryos of an M-like strain results in mutant progeny caused by insertion of
Ulysses
and perhaps other transposons, suggesting that
Penelope
expression might be responsible for the observed dysgenesis syndrome and the simultaneous mobilization of other transposable elements.
Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from
Trypanosoma brucei
Authors: Hofer Anders , Schmidt Peter P. , Gräslund Astrid , Thelander Lars
Published: July 2002

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the
de novo
synthesis of deoxyribonucleotides by directly reducing ribonucleotides to the corresponding deoxyribonucleotides. To keep balanced pools of deoxyribonucleotides, all nonviral RNRs studied so far are allosterically regulated. Most eukaryotes contain a class I RNR, which is a heterodimer of two nonidentical subunits called proteins R1 and R2. We have isolated cDNAs encoding the R1 and R2 proteins from
Trypanosoma brucei
. The amino acid sequence identities with the mouse R1 and R2 subunits are 58% and 63%, respectively. Recombinant active trypanosome R1 and R2 proteins were expressed in
Escherichia coli
and purified. The R2 protein contains an iron–tyrosyl free radical center verified by EPR spectroscopy and iron analyses. Measurement of cytidine 5′-diphosphate reduction by the trypanosome RNR in the presence of various allosteric effectors showed that the activity is highest with dTTP, dGTP, or dATP and considerably lower with ATP. The effect of dGTP is either activating (alone) or inhibitory (in the presence of ATP). Filter binding studies indicated that there are two classes of allosteric effector binding sites that bind ATP or dATP (low-affinity dATP site) and ATP, dATP, dGTP, or dTTP (high-affinity dATP site), respectively. Therefore, the structural organization of the allosteric sites is very similar to the mammalian RNRs, whereas the allosteric regulation of cytidine 5′-diphosphate reduction is unique. Hopefully, this difference can be used to target the trypanosome RNR for therapeutic purposes.
Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon,
Pyrococcus furiosus
, functions as a CoA-dependent pyruvate decarboxylase
Authors: Ma Kesen , Hutchins Andrea , Sung Shi-Jean S. , Adams Michael W. W.
Published: July 2002

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon,
Pyrococcus furiosus
, an organism that grows optimally at 100°C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO
2
and reduces
P. furiosus
ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent
K
m
values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90°C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80°C (pH 8.0), the apparent
V
m
value for pyruvate decarboxylation was about 40% of the apparent
V
m
value for pyruvate oxidation rate (using
P. furiosus
ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases
in vivo
are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.
Effects of extracellular Ca
2+
concentration on hair-bundle stiffness and gating-spring integrity in hair cells
Authors: Marquis Robert E. , Hudspeth A. J.
Published: July 2002

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca
2+
is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca
2+
chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca
2+
also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca
2+
concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca
2+
concentration; the maximal stiffness of ≈1200 μN⋅m
−
1
occurred when bundles were bathed in solutions containing 250 μM Ca
2+
, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca
2+
concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m
−
1
and no longer exhibited Ca
2+
-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca
2+
concentration.
Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein
Authors: Goletz Theresa J. , Klimpel Kurt R. , Arora Naveen , Leppla Stephen H. , Keith Jerry M. , Berzofsky Jay A.
Published: July 2002

A challenge for subunit vaccines whose goal is to elicit CD8+cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.
CDC
45 is required in conjunction with
CDC
7/
DBF
4 to trigger the initiation of DNA replication
Authors: Owens Julia C. , Detweiler Corrella S. , Li Joachim J.
Published: July 2002

The initiation of DNA replication in
Saccharomyces cerevisiae
requires the protein product of the
CDC45
gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G
1
, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for
CDC45
are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G
1
/S phase transition, are dependent on one another for function. These data indicate that
CDC45
functions in late G
1
phase in concert with
CDC7/DBF4
to trigger initiation at replication origins after the assembly of the prereplicative complexes.
The mechanism of Golgi segregation during mitosis is cell type-specific
Authors: Stanley Harold , Botas Juan , Malhotra Vivek
Published: July 2002

Golgi membranes in
Drosophila
embryos and tissue culture cells are found as discrete units dispersed in the cytoplasm. We provide evidence that Golgi membranes do not undergo any dramatic change in their organization during the rapid mitotic divisions of the nuclei in the syncitial embryo or during cell division postcellularization. By contrast, in
Drosophila
tissue culture cells, the Golgi membranes undergo complete fragmentation during mitosis. Our studies show that the mechanism of Golgi partitioning during cell division is cell type-specific.
Germ-line knockout heterokaryons of an essential α-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in
Tetrahymena thermophila
Authors: Hai Bing , Gorovsky Martin A.
Published: July 2002

The haploid
Tetrahymena thermophila
genome contains a single α-tubulin (
ATU
) gene. Using biolistic transformation, we disrupted one of the two copies of the
ATU
gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of the
ATU
gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any α-tubulin genes. However, when these conjugants were transformed with a functional marked
ATU
gene, viable transformants were obtained that contained the transforming
ATU
gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per μg of DNA) with a wild-type
ATU
gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into
Tetrahymena
, providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.
Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant
Authors: Subramaniam Sriram , Faruqi A. R. , Oesterhelt Dieter , Henderson Richard
Published: July 2002

We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 → Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 → Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-
trans
state. Projection Fourier difference maps show that upon illumination of the Leu-93 → Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (
i
) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (
ii
) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.