bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in
Xenopus
oocytes
Authors: Niu Li , Vazquez Raymond W. , Nagel Georg , Friedrich Thomas , Bamberg Ernst , Oswald Robert E. , Hess George P.
Published: July 2002

Xenopus laevis
oocytes have been used extensively
during the past decade to express and study neurotransmitter receptors
of various origins and subunit composition and also to express and
study receptors altered by site-specific mutations. Interpretations of
the effects of structural differences on receptor mechanisms were,
however, hampered by a lack of rapid chemical reaction techniques
suitable for use with oocytes. Here we describe flow and photolysis
techniques, with 2-ms and 100-μs time resolution, respectively, for
studying neurotransmitter receptors in giant (≈20-μm diameter)
patches of oocyte membranes, using muscle and neuronal acetylcholine
receptors as examples. With these techniques, we find that the muscle
receptor in BC
3
H1 cells and the same receptor expressed in
oocytes have comparable kinetic properties. This finding is in contrast
to previous studies and raises questions regarding the interpretations
of the many studies of receptors expressed in oocytes in which an
insufficient time resolution was available. The results obtained
indicate that the rapid reaction techniques described here, in
conjunction with the oocyte expression system, will be useful in
answering many outstanding questions regarding the structure and
function of diverse neurotransmitter receptors.
Elimination of the
Chlamydomonas
gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
Authors: Khrebtukova Irina , Spreitzer Robert J.
Published: July 2002

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC
4.1.1.39
) is
the key photosynthetic enzyme that catalyzes the first step of
CO
2
fixation. The chloroplast-localized holoenzyme of
plants and green algae contains eight nuclear-encoded small subunits
and eight chloroplast-encoded large subunits. Although much has been
learned about the enzyme active site that resides within each large
subunit, it has been difficult to assess the role of eukaryotic small
subunits in holoenzyme function and expression. Small subunits are
coded by a family of genes, precluding genetic screening or nuclear
transformation approaches for the recovery of small-subunit mutants. In
this study, the two small-subunit genes of the green alga
Chlamydomonas reinhardtii
were eliminated during random
insertional mutagenesis. The photosynthesis-deficient deletion mutant
can be complemented with either of the two wild-type small-subunit
genes or with a chimeric gene that contains features of both. Thus,
either small subunit is sufficient for holoenzyme assembly and
function. In the absence of small subunits, expression of
chloroplast-encoded large subunits appears to be inhibited at the level
of translation.
An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.
Authors: Li B , Vilardell J , Warner J R
Published: July 2002

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.
Direct observation of the oceanic CO
2
increase revisited
Authors: Brewer Peter G. , Goyet Catherine , Friederich Gernot
Published: July 2002

We show, from recent data obtained at specimen North Pacific
stations, that the fossil fuel CO
2
signal is strongly
present in the upper 400 m, and that we may consider areal
extrapolations from geochemical surveys to determine the magnitude of
ocean fossil fuel CO
2
uptake. The debate surrounding this
topic is illustrated by contrasting reports which suggest, based upon
atmospheric observations and models, that the oceanic CO
2
sink is small at these latitudes; or that the oceanic CO
2
sink, based upon oceanic data and models, is large. The difference
between these two estimates is at least a factor of two. There are
contradictions arising from estimates based on surface partial
pressures of CO
2
alone, where the signal sought is small
compared with regional and seasonal variability; and estimates of the
accumulated subsurface burden, which correlates well other oceanic
tracers. Ocean surface waters today contain about 45
μmol⋅kg
−1
excess CO
2
compared with
those of the preindustrial era, and the signal is rising rapidly. What
limits should we place on such calculations? The answer lies in the
scientific questions to be asked. Recovery of the fossil fuel
CO
2
contamination signal from analysis of ocean water
masses is robust enough to permit reasonable budget estimates. However,
because we do not have sufficient data from the preindustrial ocean,
the estimation of the required Redfield oxidation ratio in the upper
several hundred meters is already blurred by the very fossil fuel
CO
2
signal we seek to resolve.
Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic
stripe
expression in the developing
Drosophila
epidermis
Authors: Vorbrüggen Gerd , Jäckle Herbert
Published: July 2002

The egr-type zinc-finger transcription factor encoded by the
Drosophila
gene
stripe
(
sr
) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that
sr
activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that
sr
encodes a transcriptional activator which acts as an autoregulated developmental switch gene.
sr
activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin.
sr
-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.
The protein product of the
het-s
heterokaryon incompatibility gene of the fungus
Podospora anserina
behaves as a prion analog
Authors: Coustou Virginie , Deleu Carol , Saupe Sven , Begueret Joel
Published: July 2002

The
het-s
locus of
Podospora anserina
is a heterokaryon incompatibility locus. The coexpression of the
antagonistic
het-s
and
het-S
alleles
triggers a lethal reaction that prevents the formation of viable
heterokaryons. Strains that contain the
het-s
allele can
display two different phenotypes, [Het-s] or [Het-s*], according to
their reactivity in incompatibility. The detection in these
phenotypically distinct strains of a protein expressed from the
het-s
gene indicates that the difference in reactivity
depends on a posttranslational difference between two forms of the
polypeptide encoded by the
het-s
gene. This
posttranslational modification does not affect the electrophoretic
mobility of the protein in SDS/PAGE. Several results suggest a
similarity of behavior between the protein encoded by the
het-s
gene and prions. The [Het-s] character can
propagate in [Het-s*] strains as an infectious agent, producing a
[Het-s*] → [Het-s] transition, independently of protein
synthesis. Expression of the [Het-s] character requires a functional
het-s
gene. The protein present in [Het-s] strains is
more resistant to proteinase K than that present in [Het-s*]
mycelium. Furthermore, overexpression of the
het-s
gene
increases the frequency of the transition from [Het-s*] to [Het-s].
We propose that this transition is the consequence of a
self-propagating conformational modification of the protein mediated by
the formation of complexes between the two different forms of the
polypeptide.
Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene
Authors: Ishikawa Masayuki , Díez Juana , Restrepo-Hartwig María , Ahlquist Paul
Published: July 2002

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants
mab1–1
,
mab2–1
, and
mab3–1
(for
ma
intenance of
B
MV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by
in vivo
transcription from DNA or by transfection of yeast with
in vitro
transcripts, confirming that cytoplasmic RNA replication steps were defective.
mab1–1
,
mab2–1
, and
mab3–1
slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects,
mab1–1
and
mab2–1
blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas
mab3–1
had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.
A sensory brain map for each behavior?
Authors: Metzner W. , Juranek J.
Published: July 2002

Multiple brain maps are commonly found in virtually every vertebrate sensory system. Although their functional significance is generally relatively little understood, they seem to specialize in processing distinct sensory parameters. Nevertheless, to yield the stimulus features that ultimately elicit the adaptive behavior, it appears that information streams have to be combined across maps. Results from current lesion experiments in the electrosensory system, however, suggest an alternative possibility. Inactivations of different maps of the first-order electrosensory nucleus in electric fish, the electrosensory lateral line lobe, resulted in markedly different behavioral deficits. The centromedial map is both necessary and sufficient for a particular electrolocation behavior, the jamming avoidance response, whereas it does not affect the communicative response to external electric signals. Conversely, the lateral map does not affect the jamming avoidance response but is necessary and sufficient to evoke communication behavior. Because the premotor pathways controlling the two behaviors in these fish appear to be separated as well, this system illustrates that sensory–motor control of different behaviors can occur in strictly segregated channels from the sensory input of the brain all through to its motor output. This might reflect an early evolutionary stage where multiplication of brain maps can satisfy the demand on processing a wider range of sensory signals ensuing from an enlarged behavioral repertoire, and bridging across maps is not yet required.
The Epstein–Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation
Authors: Izumi Kenneth M. , Kaye Kenneth M. , Kieff Elliott D.
Published: July 2002

Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185–211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1Δ185–211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1Δ185–211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.
Modulation by neurotransmitters of catecholamine secretion from sympathetic ganglion neurons detected by amperometry
Authors: Koh Duk-Su , Hille Bertil
Published: July 2002

Many neuromodulators inhibit N-type Ca
2+
currents via G protein-coupled pathways in acutely isolated superior cervical ganglion (SCG) neurons. Less is known about which neuromodulators affect release of norepinephrine (NE) at varicosities and terminals of these neurons. To address this question, we used carbon fiber amperometry to measure catecholamine secretion evoked by electrical stimulation at presumed sites of high terminal density in cultures of SCG neurons. The pharmacological properties of action potential-evoked NE release paralleled those of N-type Ca
2+
channels: Release was completely blocked by Cd
2+
or ω-conotoxin GVIA, reduced 50% by 10 μM NE or 62% by 2 μM UK-14,304, an α
2
-adrenergic agonist, and reduced 63% by 10 μM oxotremorine M (Oxo-M), a muscarinic agonist. Consistent with action at M
2
or M
4
receptor subtypes, Oxo-M could be antagonized by 10 μM muscarinic antagonists methoctramine and tropicamide but not by pirenzepine. After overnight incubation with pertussis toxin, inhibition by UK-14,304 and Oxo-M was much reduced. Other neuromodulators known to inhibit Ca
2+
channels in these cells, including adenosine, prostaglandin E
2
, somatostatin, and secretin, also depressed secretion by 34–44%. In cultures treated with ω-conotoxin GVIA, secretion dependent on L-type Ca
2+
channels was evoked with long exposure to high K
+
Ringer’s solution. This secretion was not sensitive to UK-14,304 or Oxo-M. Evidently, many neuromodulators act on the secretory terminals of SCG neurons, and the depression of NE release at terminals closely parallels the membrane-delimited inhibition of N-type Ca
2+
currents in the soma.
Working underground: Respiratory adaptations in the blind mole rat
Authors: Widmer Hans R. , Hoppeler Hans , Nevo Eviatar , Taylor C. Richard , Weibel Ewald R.
Published: July 2002

Mole rats (
Spalax ehrenbergi
superspecies) perform the heavy work of digging their subterranean burrows in Israel under highly hypoxic/hypercapnic conditions. Unlike most other mammals, they can achieve high levels of metabolic rate under these conditions, while their metabolic rate at low work rates is depressed. We explored, by comparing mole rats with white rats, whether and how this is related to adaptations in the design of the respiratory system, which determines the transfer of O
2
from the lung to muscle mitochondria. At the same body mass, mole rats were found to have a significantly smaller total skeletal muscle mass than ordinary white rats (−22%). In contrast, the fractional volume of muscle mitochondria was larger by 46%. As a consequence, both species had the same total amount of mitochondria and achieved, under normoxia, the same
V̇

O
2

max
. Whereas the O
2
transport capacity of the blood was not different, we found a larger capillary density (+31%) in the mole rat muscle, resulting in a reduced diffusion distance to mitochondria. The structural pulmonary diffusing capacity for O
2
was greater in the mole rat (+44%), thus facilitating O
2
uptake in hypoxia. We conclude that structural adaptations in lung and muscle tissue improve O
2
diffusion conditions and serve to maintain high metabolic rates in hypoxia but have no consequences for achieving
V̇

O
2

max
under normoxic conditions.
Receptor-mediated activation of a plant Ca
2+
-permeable ion channel involved in pathogen defense
Authors: Zimmermann Sabine , Nürnberger Thorsten , Frachisse Jean-Marie , Wirtz Wolfgang , Guern Jean , Hedrich Rainer , Scheel Dierk
Published: July 2002

Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca
2+
across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca
2+
-permeable, La
3+
-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl
2
). At an extracellular Ca
2+
concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for
l
arge conductance
e
licitor-
a
ctivated ion
c
hannel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of
Phytophthora sojae
. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.
The
Caenorhabditis elegans
gene
unc-76
and its human homologs define a new gene family involved in axonal outgrowth and fasciculation
Authors: Bloom Laird , Horvitz H. Robert
Published: July 2002

The gene
unc-76
(
unc
, uncoordinated) is necessary for normal axonal bundling and elongation within axon bundles in the nematode
Caenorhabditis elegans
. The UNC-76 protein and two human homologs identified as expressed sequence tags are not similar to previously characterized proteins and thus represent a new protein family. At least one of these human homologs can function in
C. elegans
, suggesting that it, like UNC-76, acts in axonal outgrowth. We propose that the UNC-76 protein, which is found in cell bodies and processes of all neurons throughout development, either has a structural role in the formation and maintenance of axonal bundles or transduces signals to the intracellular machinery that regulates axonal extension and adhesion.
Arsenic-induced PML targeting onto nuclear bodies: Implications for the treatment of acute promyelocytic leukemia
Authors: Zhu Jun , Koken Marcel H. M. , Quignon Frédérique , Chelbi-Alix Mounira K. , Degos Laurent , Wang Zhen Yi , Chen Zhu , de Thé Hugues
Published: July 2002

Acute promyelocytic leukemia (APL) is associated with the t(15;17) translocation, which generates a PML/RARα fusion protein between PML, a growth suppressor localized on nuclear matrix-associated bodies, and RARα, a nuclear receptor for retinoic acid (RA). PML/RARα was proposed to block myeloid differentiation through inhibition of nuclear receptor response, as does a dominant negative RARα mutant. In addition, in APL cells, PML/RARα displaces PML and other nuclear body (NB) antigens onto nuclear microspeckles, likely resulting in the loss of PML and/or NB functions. RA leads to clinical remissions through induction of terminal differentiation, for which the respective contributions of RARα (or PML/RARα) activation, PML/RARα degradation, and restoration of NB antigens localization are poorly determined. Arsenic trioxide also leads to remissions in APL patients, presumably through induction of apoptosis. We demonstrate that in non-APL cells, arsenic recruits the nucleoplasmic form of several NB antigens onto NB, but induces the degradation of PML only, identifying a powerful tool to approach NB function. In APL cells, arsenic targets PML and PML/RARα onto NB and induces their degradation. Thus, RA and arsenic target RARα and PML, respectively, but both induce the degradation of the PML/RARα fusion protein, which should contribute to their therapeutic effects. The difference in the cellular events triggered by these two agents likely stems from RA-induced transcriptional activation and arsenic effects on NB proteins.
Evidence for a common sex determination mechanism for pistil abortion in maize and in its wild relative
Tripsacum
Authors: Li Dadong , Blakey C. Ann , Dewald Chet , Dellaporta Stephen L.
Published: July 2002

Cultivated maize (
Zea mays
) and several other members of the Tribe Andropogoneae produce unisexual florets. In maize, the formation of two staminate florets in each spikelet on the tassel and a single pistillate floret in each spikelet on the ear includes a pistil abortion process that requires the action of the
TASSELSEED2
gene. In Eastern gamagrass (
Tripsacum dactyloides
) the
GYNOMONOECIOUS SEX FORM1
gene appears to perform a similar role in pistil abortion. These genes were shown to be homeologs by restriction fragment length polymorphism mapping and by the failure of the
gsf1
and
ts2
alleles to complement one another in intergeneric hybrids. Molecular analysis of the
gsf1
allele shows that it is caused by a 1.4-kb deletion mutation. Both
TASSELSEED2
and
GYNOMONOECIOUS SEX FORM1
show similar expression patterns in subepidermal cells of pistils just before abortion. These results suggest that the formation of staminate florets in the Andropogoneae represents a monophyletic trait.
DNA looping by Ku and the DNA-dependent protein kinase
Authors: Cary Robert B. , Peterson Scott R. , Wang Jinting , Bear David G. , Bradbury E. Morton , Chen David J.
Published: July 2002

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an ≈460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein–nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein–nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.
The role of charged residues mediating low affinity protein–protein recognition at the cell surface by CD2
Authors: Davis Simon J. , Davies Elizabeth A. , Tucknott Michael G. , Jones E. Yvonne , van der Merwe P. Anton
Published: July 2002

Insights into the structural basis of protein–protein recognition have come principally from the analysis of proteins such as antibodies, hormone receptors, and proteases that bind their ligands with relatively high affinity (
K
a
≈ 10
9
M
−1
). In contrast, few studies have been done on the very low affinity interactions mediating cell adhesion and cell–cell recognition. As a site of protein–protein recognition, the ligand binding face of the T lymphocyte cell–cell recognition molecule, CD2, which binds its ligands 10
4
- to 10
5
-fold more weakly than do antibodies and proteases, is unusual in being both very flat and highly charged. An analysis of the effect of mutations and ionic strength on CD2 binding to its ligand, CD48, indicates that these charged residues contribute little, if any, binding energy to this interaction. However, the loss of these charged residues is shown to markedly reduce ligand-binding specificity. Thus, the charged residues increase the specificity of CD2 binding without increasing the affinity. This phenomenon is likely to result from a requirement for electrostatic complementarity between charged binding surfaces to compensate for the removal, upon binding, of water interacting with the charged residues. It is proposed that this mode of recognition is highly suited to biological interactions requiring a low affinity because it uncouples increases in specificity from increases in affinity.
Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily
Authors: Pellequer Jean-Luc , Wager-Smith Karen A. , Kay Steve A. , Getzoff Elizabeth D.
Published: July 2002

PAS domains are found in diverse proteins throughout all three
kingdoms of life, where they apparently function in sensing and signal
transduction. Although a wealth of useful sequence and functional
information has become recently available, these data have not been
integrated into a three-dimensional (3D) framework. The very early
evolutionary development and diverse functions of PAS domains have made
sequence analysis and modeling of this protein superfamily challenging.
Limited sequence similarities between the ∼50-residue PAS repeats and
one region of the bacterial blue-light photosensor photoactive yellow
protein (PYP), for which ground-state and light-activated
crystallographic structures have been determined to high resolution,
originally were identified in sequence searches using consensus
sequence probes from PAS-containing proteins. Here, we found that by
changing a few residues particular to PYP function, the modified PYP
sequence probe also could select PAS protein sequences. By mapping a
typical ∼150-residue PAS domain sequence onto the entire
crystallographic structure of PYP, we show that the PAS sequence
similarities and differences are consistent with a shared 3D fold (the
PAS/PYP module) with obvious potential for a ligand-binding cavity.
Thus, PYP appears to prototypically exhibit all the major structural
and functional features characteristic of the PAS domain superfamily:
the shared PAS/PYP modular domain fold of ∼125–150 residues, a
sensor function often linked to ligand or cofactor (chromophore)
binding, and signal transduction capability governed by heterodimeric
assembly (to the downstream partner of PYP). This 3D PAS/PYP module
provides a structural model to guide experimental testing of hypotheses
regarding ligand-binding, dimerization, and signal transduction.
Fission yeast
orb
6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle
Authors: Verde Fulvia , Wiley David J. , Nurse Paul
Published: July 2002

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The
orb6
gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/
cot1
/
warts
family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34
cdc2
mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis.
orb6
interacts genetically with
orb2
, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.
Identification of a large Myc-binding protein that contains RCC1-like repeats
Authors: Guo Qingbin , Xie Jingwu , Dang Chi V. , Liu Edison T. , Bishop J. Michael
Published: July 2002

The protooncogene
MYC
plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors.
MYC
and the closely related
MYCN
encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. We have identified a large and highly conserved nuclear protein that interacts directly with the transcriptional activating domain of Myc (designated “protein associated with Myc” or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. The gene encoding Pam (
PAM
) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt’s lymphomas.
PAM
is located within a 300-kb region on chromosome 13q22.
Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/ glyceraldehyde-3-phosphate dehydrogenase complex dissociation
Authors: Wedel Norbert , Soll Jürgen
Published: July 2002

For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC
2.7.1.19
) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC
1.2.1.13
), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12. Enzymatic activity depends on complex dissociation, mediated by NADPH. The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of
Chlamydomonas
and
Synechocystis
suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes.
In vitro
complex reconstitution assays with heterologously expressed
Synechocystis
wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex. Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H). These fusion events occurred at least twice in evolution. First was the fusion of the duplicated genes for CP12 and the ORF4 protein of
Anabaena variabilis
to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N
2
fixation. A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.
Defect in IgV gene somatic hypermutation in Common Variable Immuno-Deficiency syndrome
Authors: Levy Yves , Gupta Neetu , Le Deist Françoise , Garcia Corinne , Fischer Alain , Weill Jean-Claude , Reynaud Claude-Agnès
Published: July 2002

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40–75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.
Mobility of cytochrome P450 in the endoplasmic reticulum membrane
Authors: Szczesna-Skorupa Elzbieta , Chen Ci-Di , Rogers Steven , Kemper Byron
Published: July 2002

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 × 10
−10
cm
2
/s. A coefficient only slightly larger (7.1 × 10
−10
cm
2
/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.
Metabolism to a response pathway selective retinoid ligand during axial pattern formation
Authors: Pijnappel W. W. M. , Folkers G. E. , de Jonge W. J. , Verdegem P. J. E. , de Laat S. W. , Lugtenburg J. , Hendriks H. F. J. , van der Saag P. T. , Durston A. J.
Published: July 2002

We report identification of 9-
cis
-4-oxo-retinoic acid (9-
cis
-4-oxo-RA) as an
in
vivo
retinoid metabolite in
Xenopus
embryos. 9-
Cis
-4-oxo-RA bound receptors (RARs) α, β, and γ as well as retinoid X receptors (RXRs) α, β, and γ
in vitro
. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-
Cis
-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-
cis
-4-oxo-RA displayed high potency for modulating axial pattern formation in
Xenopus
, metabolism to 9-
cis
-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.
The antigen-binding characteristics of mAbs derived fromin vivopriming of avian B cells
Authors: Michael Nancy , Accavitti Mary Ann , Masteller Emma , Thompson Craig B.
Published: July 2002

In most vertebrates, a primary antibody repertoire is created through the recombination of a diverse set of Ig variable (V), diversity (D), and joining (J) gene segments. In contrast, an avian immune repertoire is generated by gene conversion of rearranged Ig genes during B cell development within the bursa of Fabricius, a lymphoid organ unique to birds. To investigate the properties of antigen-specific Igs created through the process of gene conversion, we have developed a system for the production of avian-derived mAbs. This system was used to produce multiple antibodies after a single immunization with a conserved peptide from the human cystic fibrosis transmembrane conductance regulator gene. Each antibody isolated was found to have arisen independently through a distinct series of gene conversion events. These primary antibodies displayed evidence of diversity in all of the complementarity determining regions of both heavy and light chains, and both the heavy and the light chains contributed to antigen specificity. In the light chains, diversity could be attributed to gene conversion events. The measured affinity constants of two of the antibodies were between 108and 109M−1, and the antibodies were functional in quantitative ELISA as well as immunohistochemical studies of cystic fibrosis transmembrane conductance regulator expression. These data demonstrate that antigen-specific antibodies produced by Ig gene conversion display both high affinity and specificity. In addition, the methods developed here provide the description of a system for the production of mAbs derived from a nonmammalian species.
Evidence that the lipid moiety of oxidized low density lipoprotein plays a role in its interaction with macrophage receptors
Authors: Terpstra Valeska , Bird David A. , Steinberg Daniel
Published: July 2002

The binding of oxidatively damaged red blood cells (OxRBCs) to resident mouse peritoneal macrophages correlates with an increase in phosphatidylserine on the external leaflet of the plasma membrane. Liposomes rich in phosphatidylserine can inhibit this binding and also the binding of certain apoptotic cells. We have shown previously that oxidized low density lipoproteins (OxLDL) also can inhibit the binding of OxRBCs to resident mouse peritoneal macrophages. The present studies show that microemulsions prepared from the lipids extracted from OxLDL are very effective in inhibiting the binding of OxRBCs and also, to a lesser extent, of apoptotic thymocytes to macrophages. OxRBC binding was also inhibited by cholesterol phospholipid liposomes containing oxidized 1-stearoyl-2-linoleoyl-phosphatidylcholine. The binding and uptake of125I-labeled OxLDL were also strongly inhibited by microemulsions of the lipids extracted from OxLDL and by cholesterol phospholipid liposomes containing oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine. Earlier studies have shown that the delipidated protein moiety of OxLDL can competitively inhibit macrophage binding of intact OxLDL, implicating the protein moiety as an effective receptor-binding domain of OxLDL with respect to some macrophage scavenger receptors. The present studies suggest that the lipid moiety of OxLDL may also play a role.
Caenorhabditis elegansorthologs of the aryl hydrocarbon receptor and its heterodimerization partner the aryl hydrocarbon receptor nuclear translocator
Authors: Powell-Coffman Jo Anne , Bradfield Christopher A. , Wood William B.
Published: July 2002

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematodeCaenorhabditis elegans, encoded by the genesahr-1andaha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate thatC. elegansAHR-1, like vertebrate AHR, requires some form of post-translational activation. Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor:C. elegansAHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by β-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators.
Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules
Authors: Markiewicz Mary A. , Girao Cristina , Opferman Joseph T. , Sun Jiling , Hu Qinghui , Agulnik Alexander A. , Bishop Colin E. , Thompson Craig B. , Ashton-Rickardt Philip G.
Published: July 2002

How memory T cells are maintained
in vivo
is poorly understood. To address this problem, a male-specific peptide (H-Y) was identified and used to activate female anti-H-Y T cells
in vitro.
Anti-H-Y T cells survived
in vivo
for at least 70 days in the absence of antigen. This persistence was not because of the intrinsic ability of memory T cells to survive
in vivo
. Instead, the survival and function of adoptively transferred memory cells was found to require transporter of antigen protein 1-dependent expression of self-peptide/major histocompatibility complex class I molecules in recipient animals. Therefore, it appears that the level of T cell receptor engagement provided by transporter of antigen protein 1-dependent, self-peptide/major histocompatibility complexes is sufficient to maintain the long-term survival and functional phenotype of memory cells in the absence of persistent antigen. These data suggest that positive selection plays a role not only in T cell development but also in the maintenance of T cell memory.
Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice
Authors: Surwit Richard S. , Wang Shiying , Petro Ann E. , Sanchis Daniel , Raimbault Serge , Ricquier Daniel , Collins Sheila
Published: July 2002

Uncoupling protein 2 (UCP2) maps to a region on distal mouse chromosome 7 that has been linked to the phenotypes of obesity and type II diabetes. We recently reported that UCP2 expression is increased by high fat feeding in adipose tissue of the A/J strain of mice, which is resistant to the development of dietary obesity. More recently, a third UCP (UCP3) was identified, which is expressed largely in skeletal muscle and brown adipose tissue. The UCP2 and UCP3 genes are located adjacent to one another on mouse chromosome 7. Thus, the roles of these UCPs in both metabolic efficiency and the linkage to obesity and diabetes syndromes is unclear. For this reason, we examined the expression of UCP2 and UCP3 in white adipose tissue and interscapular brown adipose tissue and in gastrocnemius/soleus muscle preparations from the obesity-resistant A/J and C57BL/KsJ (KsJ) strains and the obesity-prone C57BL/6J (B6) mouse strain. In both KsJ and A/J mice, UCP2 expression in white fat was increased ≈2-fold in response to 2 weeks of a high fat diet, but there was no effect of diet on UCP2 levels in B6 mice. In skeletal muscle and in brown fat, neither UCP2 nor UCP3 expression was affected by diet in A/J, B6, or KsJ mice. However, in brown fat, we observed a 2–3-fold increase in the expression of UCP1 in response to dietary fat challenge, which may be related to diet-induced elevations in plasma leptin levels. Together, these results indicate that the consumption of a high fat diet selectively regulates UCP2 expression in white fat and UCP1 expression in brown fat and that resistance to obesity is correlated with this early, selective induction of UCP1 and UCP2 and is not associated with changes in expression of UCP3.
A common-sense climate index: Is climate changing noticeably?
Authors: Hansen James , Sato Makiko , Glascoe Jay , Ruedy Reto
Published: July 2002

We propose an index of climate change based on practical climate indicators such as heating degree days and the frequency of intense precipitation. We find that in most regions the index is positive, the sense predicted to accompany global warming. In a few regions, especially in Asia and western North America, the index indicates that climate change should be apparent already, but in most places climate trends are too small to stand out above year-to-year variability. The climate index is strongly correlated with global surface temperature, which has increased as rapidly as projected by climate models in the 1980s. We argue that the global area with obvious climate change will increase notably in the next few years. But we show that the growth rate of greenhouse gas climate forcing has declined in recent years, and thus there is an opportunity to keep climate change in the 21st century less than “business-as-usual” scenarios.
Magnetars
Authors: Kouveliotou Chryssa
Published: July 2002

Recent x-ray observations have shown that a
substantial fraction of newly born neutron stars have magnetic fields
of several 10
14
G. They reveal themselves as soft gamma
repeaters and anomalous x-ray pulsars and may account for the missing
radio pulsars in young supernova remnants.
From pre-Hispanic to future conservation alternatives: Lessons from Mexico
Authors: Gómez-Pompa Arturo , Kaus Andrea
Published: July 2002

In this paper, we review some past and present trends in
biodiversity conservation in Mexico and explore possible explanations
of why, in spite of this long history of depredation and ineffective
conservation policies, the ecosystems have been able to cope with and
retain most of their biological components. We suggest a hypothesis
based on the persistence of a complex mosaic of past and present
traditional land uses as a possible explanation for this resilience. We
propose an agenda for the scope of future conservation research and
policy, particularly the need to take the socioeconomic context of
environmental degradation into account. We put forth a series of
questions that we think need to be investigated if the conservation
research community is to participate in developing solutions for the
future welfare of the human species and of biodiversity on earth.