bluuebunny/crossref_metadata_2025 huggingface dataset:
Elemental propagation of calcium signals in response-specific patterns determined by environmental stimulus strength
Authors: Goddard Helen , Manison N. F. H. , Tomos D. , Brownlee C.
Published: July 2002

Plant cells can respond qualitatively and quantitatively to a wide range of environmental signals. Ca
2+
is used as an intracellular signal for volume regulation in response to external osmotic changes. We show here that the spatiotemporal patterns of hypo-osmotically induced Ca
2+
signals vary dramatically with stimulus strength in embryonic cells of the marine alga
Fucus
. Biphasic or multiphasic Ca
2+
signals reflect Ca
2+
elevations in distinct cellular domains. These propagate via elemental Ca
2+
release in nuclear or peripheral regions that are rich in endoplasmic reticulum. Cell volume regulation specifically requires Ca
2+
elevation in apical peripheral regions, whereas an altered cell division rate occurs only in response to stimuli that cause Ca
2+
elevation in nuclear regions.
Tyrosine phosphorylation of p62
dok
by p210
bcr-abl
inhibits RasGAP activity
Authors: Kashige Nobuhiro , Carpino Nick , Kobayashi Ryuji
Published: July 2002

The t(9;22) chromosomal translocation is found in almost all patients with chronic myelogenous leukemia. The resultant
Bcr-Abl
fusion gene expresses a chimeric fusion protein p210
bcr-abl
with increased tyrosine kinase activity. Hematopoietic progenitors isolated from chronic myelogenous leukemia patients in the chronic phase contain constitutively tyrosine-phosphorylated p62
dok
protein. p62
dok
associates with the Ras GTPase-activating protein (RasGAP), but only when p62
dok
is tyrosine phosphorylated. Here we have investigated the interaction between p62
dok
and RasGAP and the consequences of p62
dok
tyrosine phosphorylation on the activity of RasGAP. We have found that p62
dok
is directly tyrosine phosphorylated by p210
bcr-abl
, and the sites of phosphorylation are located in the C-terminal half of the p62
dok
molecule. We have identified five tyrosine residues that are involved in
in vitro
RasGAP binding and have found that tyrosine-phosphorylated p62
dok
inhibits RasGAP activity. Our results suggest that p210
bcr-abl
might lead to the activation of the Ras signaling pathway by inhibiting a key down-regulator of Ras signaling.
Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-
d
-erythritol 2-phosphate to 2C-methyl-
d
-erythritol 2,4-cyclodiphosphate
Authors: Herz Stefan , Wungsintaweekul Juraithip , Schuhr Christoph A. , Hecht Stefan , Lüttgen Holger , Sagner Sylvia , Fellermeier Monika , Eisenreich Wolfgang , Zenk Meinhart H. , Bacher Adelbert , Rohdich Felix
Published: July 2002

In many microorganisms, the putative orthologs of the
Escherichia coli
ygbB
gene are tightly
linked or fused to putative orthologs of
ygbP
,
which has been shown earlier to be involved in terpenoid biosynthesis.
The
ygbB
gene of
E. coli
was expressed in
a recombinant
E. coli
strain and was shown to direct the
synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was
found to convert 4-diphosphocytidyl-2C-methyl-
d
-erythritol
2-phosphate into 2C-methyl-
d
-erythritol
2,4-cyclodiphosphate and CMP. The structure of the reaction product was
established by NMR spectroscopy using
13
C-labeled substrate
samples. The enzyme-catalyzed reaction requires Mn
2+
or
Mg
2+
but no other cofactors. Radioactivity from
[2-
14
C]2C-methyl-
d
-erythritol
2,4-cyclodiphosphate was diverted efficiently to carotenoids by
isolated chromoplasts from
Capsicum annuum
and, thus,
was established as an intermediate in the deoxyxylulose phosphate
pathway of isoprenoid biosynthesis. YgbB protein also was found to
convert 4-diphosphocytidyl-2C-methyl-
d
-erythritol into
2C-methyl-
d
-erythritol 3,4-cyclophosphate. This compound
does not serve as substrate for the formation of carotenoids by
isolated chromoplasts and is assumed to be an
in vitro
product without metabolic relevance.
The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin
Authors: Frauenfelder Hans , McMahon Benjamin H. , Austin Robert H. , Chu Kelvin , Groves John T.
Published: July 2002

The grail of protein science is the connection between structure
and function. For myoglobin (Mb) this goal is close. Described as only
a passive dioxygen storage protein in texts, we argue here that Mb is
actually an allosteric enzyme that can catalyze reactions among small
molecules. Studies of the structural, spectroscopic, and kinetic
properties of Mb lead to a model that relates structure, energy
landscape, dynamics, and function. Mb functions as a miniature chemical
reactor, concentrating and orienting diatomic molecules such as NO, CO,
O
2
, and H
2
O
2
in highly conserved
internal cavities. Reactions can be controlled because Mb exists in
distinct taxonomic substates with different catalytic properties and
connectivities of internal cavities.
Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin
Authors: Robert-Nicoud Maya , Flahaut Marjorie , Elalouf Jean-Marc , Nicod Marie , Salinas Miguel , Bens Marcelle , Doucet Alain , Wincker Patrick , Artiguenave François , Horisberger Jean-Daniel , Vandewalle Alain , Rossier Bernard C. , Firsov Dmitri
Published: July 2002

Aldosterone and vasopressin are responsible for the final
adjustment of sodium and water reabsorption in the kidney. In principal
cells of the kidney cortical collecting duct (CCD), the integral
response to aldosterone and the long-term functional effects of
vasopressin depend on transcription. In this study, we analyzed the
transcriptome of a highly differentiated mouse clonal CCD principal
cell line (mpkCCD
cl4
) and the changes in the transcriptome
induced by aldosterone and vasopressin. Serial analysis of gene
expression (SAGE) was performed on untreated cells and on cells treated
with either aldosterone or vasopressin for 4 h. The transcriptomes
in these three experimental conditions were determined by sequencing
169,721 transcript tags from the corresponding SAGE libraries. Limiting
the analysis to tags that occurred twice or more in the data set,
14,654 different transcripts were identified, 3,642 of which do not
match known mouse sequences. Statistical comparison (at
P
< 0.05 level) of the three SAGE libraries
revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs
(aldosterone-repressed transcripts), 48 VITs (vasopressin-induced
transcripts) and 11 VRTs (vasopressin-repressed transcripts). A
selection of the differentially-expressed, hormone-specific transcripts
(5 VITs, 2 AITs and 1 ART) has been validated in the
mpkCCD
cl4
cell line either by Northern blot hybridization
or reverse transcription–PCR
.
The hepatocyte nuclear
transcription factor HNF-3-α (VIT39), the receptor activity modifying
protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper
protein (GILZ) (AIT28) are candidate proteins playing a role in
physiological responses of this cell line to vasopressin and
aldosterone.
Structure and function of the C-terminal PABC domain of human poly(A)-binding protein
Authors: Kozlov Guennadi , Trempe Jean-François , Khaleghpour Kianoush , Kahvejian Avak , Ekiel Irena , Gehring Kalle
Published: July 2002

We have determined the solution structure of the C-terminal quarter
of human poly(A)-binding protein (hPABP). The protein fragment contains
a protein domain, PABC [for poly(A)-binding protein C-terminal
domain], which is also found associated with the HECT family of
ubiquitin ligases. By using peptides derived from PABP interacting
protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a
peptide binding domain. We use chemical shift perturbation analysis to
identify the peptide binding site in PABC and the major elements
involved in peptide recognition. From comparative sequence analysis of
PABC-binding peptides, we formulate a preliminary PABC consensus
sequence and identify human ataxin-2, the protein responsible for type
2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.
Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase
Authors: Nomanbhoy Tyzoon K. , Schimmel Paul R.
Published: July 2002

Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNA
Ile
) at its active site. The misactivated amino acid is then translocated to an editing site located >25 Å away. The role of the misactivated amino acid in determining the rate of translocation is not known. Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, α-aminobutyrate, and cysteine. In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, α-aminobutyrate, and cysteine. Although their rates of misactivation are clearly distinct, their rates of translocation are similar. Thus, the rate of translocation is independent of the nature of the misactivated amino acid. This result suggests that the misactivated amino acid
per se
has little or no role in directing translocation.
Segregation distortion induced by wild-type RanGAP in
Drosophila
Authors: Kusano Ayumi , Staber Cynthia , Ganetzky Barry
Published: July 2002

Segregation Distorter (
SD
) is a meiotic drive system in
Drosophila
that causes preferential transmission of the
SD
chromosome from
SD
/
SD
+
males owing to the induced dysfunction of
SD
+
spermatids. The key distorter locus,
Sd
, is a dominant neomorphic allele encoding a truncated, but enzymatically active, RanGAP (RanGTPase-activating protein) whose nuclear mislocalization underlies distortion by disrupting the Ran signaling pathway. Here, we show that even wild-type RanGAP can cause segregation distortion when it is overexpressed in the male germ line or when the gene dosage of a particular modifier locus is increased. Both manipulations result in substantial nuclear accumulation of RanGAP. Distortion can be suppressed by overexpression of Ran or Ran guanine nucleotide exchange factor (RanGEF) in the male germ line, indicating that the primary consequence of nuclear mislocalization of RanGAP is reduction of intranuclear RanGTP levels. These results prove that segregation distortion does not depend on any unique properties of the mutant RanGAP encoded by
Sd
and provide a unifying explanation for the occurrence of distortion in a variety of experimental situations.
A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions
Authors: Joung J. Keith , Ramm Elizabeth I. , Pabo Carl O.
Published: July 2002

We have developed a bacterial “two-hybrid” system that readily allows selection from libraries larger than 10
8
in size. Our bacterial system may be used to study either protein–DNA or protein—protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein–DNA and protein–protein interactions.
An expanded view of bacterial DNA replication
Authors: Noirot-Gros Marie-Françoise , Dervyn Etienne , Wu Ling Juan , Mervelet Peggy , Errington Jeffery , Ehrlich S. Dusko , Noirot Philippe
Published: July 2002

A protein-interaction network centered on the replication machinery of
Bacillus subtilis
was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.
Direct biochemical evidence for type III secretion-dependent translocation of the AvrBs2 effector protein into plant cells
Authors: Casper-Lindley Catharina , Dahlbeck Douglas , Clark Eszter T. , Staskawicz Brian J.
Published: July 2002

The calmodulin-dependent adenylate cyclase domain (Cya) of the
Bordetella pertussis
cyclolysin was used as a reporter protein to study the direct translocation of the
Xanthomonas
effector protein, AvrBs2, into the plant host cell. Adenylate cyclase activity (production of cAMP) depends on the presence of eukaryotic plant calmodulin and is only active after translocation from the prokaryotic cell into the eukaryotic plant cell. Here, we show that infection of pepper plants by
Xanthomonas campestris
pv.
vesicatoria
strains expressing the AvrBs2:Cya fusion protein results in detectable increases of cAMP levels in plant cells as early as 3 h after inoculation. Adenylate cyclase activity was shown to be type III secretion-dependent as the
Xanthomonas hrp
mutations,
hrcV
or
hrpF
, failed to produce detectable levels of cAMP in infected pepper plants. Furthermore, the N-terminal secretion and translocation signals of AvrBs2 were shown to be required for activity of the fusion protein in the plant. A single genomic copy of the
avrBs2
:
cya
fusion gene expressed under the control of the wild-type
avrBs2
promoter was used to compare the effect of a susceptible and resistant plant interaction on the kinetics of effector protein delivery. Implications of these results and additional applications of this reporter construct are discussed.
Globin-coupled sensors: A class of heme-containing sensors in Archaea and Bacteria
Authors: Hou Shaobin , Freitas Tracey , Larsen Randy W. , Piatibratov Mikhail , Sivozhelezov Victor , Yamamoto Amy , Meleshkevitch Ella A. , Zimmer Mike , Ordal George W. , Alam Maqsudul
Published: July 2002

The recently discovered prokaryotic signal transducer HemAT, which has been described in both
Archaea
and
Bacteria
, mediates aerotactic responses. The N-terminal regions of HemAT from the archaeon
Halobacterium salinarum
(HemAT-
Hs
) and from the Gram-positive bacterium
Bacillus subtilis
(HemAT-
Bs
) contain a myoglobin-like motif, display characteristic heme–protein absorption spectra, and bind oxygen reversibly. Recombinant HemAT-
Hs
and HemAT-
Bs
shorter than 195 and 176 residues, respectively, do not bind heme effectively. Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species. The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold. We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers. Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes. We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them. Both bind heme and show myoglobin-like spectra. This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.
A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity
Authors: Aman A. T. , Fraser S. , Merritt E. A. , Rodigherio C. , Kenny M. , Ahn M. , Hol W. G. J. , Williams N. A. , Lencer W. I. , Hirst T. R.
Published: July 2002

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit ofEscherichia coliheat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.
The role of cdc2 in the expression of herpes simplex virus genes
Authors: Advani Sunil J. , Weichselbaum Ralph R. , Roizman Bernard
Published: July 2002

Earlier reports have shown that cdc2 kinase is activated in
cells infected with herpes simplex virus 1 and that the activation is
mediated principally by two viral proteins, the infected cell protein
22 (ICP22) and the protein kinase encoded by U
L
13. The same
proteins are required for optimal expression of a subset of late
(γ
2
) genes exemplified by U
S
11. In this
study, we used a dominant-negative cdc2 protein to determine the role
of cdc2 in viral gene expression. We report the following.
(
i
) The cdc2 dominant-negative protein had no effect in
the synthesis and accumulation of at least two α-regulatory proteins
(ICP4 and ICP0), two β-proteins (ribonucleotide reductase major
subunit and single-stranded DNA-binding protein), and two
γ
1
-proteins (glycoprotein D and viral protease).
U
S
11, a γ
2
-protein, accumulated only in cells
in which cdc2 dominant-negative protein could not be detected or was
made in very small amounts. (
ii
) The sequence of amino
acids predicted to be phosphorylated by cdc2 is present in at least 27
viral proteins inclusive of the regulatory proteins ICP4, ICP0, and
ICP22. In
in vitro
assays, we demonstrated that cdc2
specifically phosphorylated a polypeptide consisting of the second exon
of ICP0 but not a polypeptide containing the sequence of the third exon
as would be predicted from the sequence analysis. We conclude that cdc2
is required for optimal expression of a subset of
γ
2
-proteins whose expression is also regulated by the
viral proteins (ICP22 and U
L
13) that mediate the activation
of cdc2 kinase.
Two distinct signaling pathways in hair cycle induction: Stat3-dependent and -independent pathways
Authors: Sano Shigetoshi , Kira Masahiro , Takagi Satoshi , Yoshikawa Kunihiko , Takeda Junji , Itami Satoshi
Published: July 2002

The hair follicle is an epidermal derivative that undergoes cycles
of growth, involution, and rest. The hair cycle has well-orchestrated
kinetics regulated by interactions between mesenchymal and epithelial
cells, although the intracellular signals remain unclear. We previously
established keratinocyte-specific Stat3-disrupted mice, by which we
demonstrated that signal transducer and activator of transcription 3
(Stat3) is required for wound healing and anagen progression in the
hair cycle. Growth factor-dependent migration of Stat3-disrupted
keratinocytes was severely impaired, suggesting that not only wound
healing but also telogen-to-anagen progression required organized
keratinocyte migration in response to mesenchymal stimuli. In the
present study, to examine whether Stat3 activation in keratinocytes is
a prerequisite for hair cycle progression, we applied methods for
experimental anagen induction to Stat3-disrupted mice. It was
demonstrated that anagen was successfully induced in Stat3-disrupted as
well as wild-type mice by chemical or mechanical stimulation, i.e., by
topical application of phorbol 12-myristate 13-acetate (PMA) or by hair
plucking, respectively. This result indicated that anagen in these
methods occurred in the absence of Stat3. Furthermore, PMA stimulated
the migration of Stat3-disrupted keratinocytes
in vitro
,
supporting a hypothesis that the protein kinase C (PKC) and Stat3
pathways occur independently in the postnatal anagen induction. Both
Stat3-dependent and -independent migration of keratinocytes was
inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin.
Therefore, we infer that entry into anagen is mediated by at least two
distinct signaling pathways: Stat3-dependent pathway for spontaneous
hair cycling and Stat3-independent (probably PKC-dependent) pathway for
exogenously induced hair cycling, whereas both pathways require PI3K
activation.
Complement activation in factor D-deficient mice
Authors: Xu Yuanyuan , Ma Minghe , Ippolito Gregory C. , Schroeder Harry W. , Carroll Michael C. , Volanakis John E.
Published: July 2002

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balancein vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activationin vitroby CVF demonstrated that in factor D-deficient serum the α chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg2+, whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization ofStreptococcus pneumoniaeby C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.
Sequence and expression of the murine Hoxd-3 homeobox gene.
Authors: Tan D P , Shao X , Pu L , Guo V , Nirenberg M
Published: July 2002

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.
The notion of the Cambrian pananimalia genome.
Authors: Ohno S
Published: July 2002

The toil by photosynthesizing cyanobacteria and blue-green algae of nearly three billion years appeared to have finally resulted in the sufficient accumulation of molecular oxygen. So, the stage was set for the emergence, at the ocean bottom, of diverse animals that were consumers of molecular oxygen. It now appears that this Cambrian explosion, during which nearly all the extant animal phyla have emerged, was of an astonishingly short duration, lasting only 6-10 million years. Inasmuch as only a 1% DNA base sequence change is expected in 10 million years under the standard spontaneous mutation rate, I propose that all those diverse animals of the early Cambrian period, some 550 million years ago, were endowed with nearly identical genomes, with differential usage of the same set of genes accounting for the extreme diversities of body forms. Some of the more pertinent genes that are thought to be included in the Cambrian pananimalia genome are as follows. (i) A gene for lysyloxidase that, in the presence of molecular oxygen, crosslinked collagen triple helices to produce ligaments and tendons, thus contributing to the stout bodies of the Cambrian animals. (ii) Genes for hemoglobin; these internal transporters of molecular oxygen are today seen sporadically in members of diverse animal phyla. (iii) The Pax-6 gene for eye formation; the eyes of a ribbon worm to a human are organized by this gene. In animals without eyes, the same gene organizes other sensory systems and organs. (iv) A series of Hox genes for the anterior-posterior (cranio-caudal) body plans: these genes are also present in all phyla of the kingdom Animalia.
Potential use of additivity of mutational effects in simplifying protein engineering.
Authors: Skinner M M , Terwilliger T C
Published: July 2002

The problem of rationally engineering protein molecules can be simplified where effects of mutations on protein function are additive. Crystal structures of single and double mutants in the hydrophobic core of gene V protein indicate that structural and functional effects of core mutations are additive when the regions structurally influenced by the mutations do not substantially overlap. These regions of influence can provide a simple basis for identifying sets of mutations that will show additive effects.
Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans.
Authors: Grenache D G , Caldicott I , Albert P S , Riddle D L , Politz S M
Published: July 2002

Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.
Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.
Authors: Carter K L , Roizman B
Published: July 2002

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.
Stability of nonrelativistic quantum mechanical matter coupled to the (ultraviolet cutoff) radiation field
Authors: Fefferman Charles , Fröhlich Jürg , Graf Gian Michele
Published: July 2002

We announce a proof of
H
-stability for the quantized
radiation field, with ultraviolet cutoff, coupled to arbitrarily many
non-relativistic quantized electrons and static nuclei. Our result
holds for arbitrary atomic numbers and fine structure constant. We also
announce bounds for the energy of many electrons and nuclei in a
classical vector potential and for the eigenvalue sum of a one-electron
Pauli Hamiltonian with magnetic field.
Differential functions of the two Src homology 2 domains in protein tyrosine phosphatase SH-PTP1.
Authors: Pei D , Wang J , Walsh C T
Published: July 2002

SH-PTP1 (also known as PTP1C, HCP, and SHP) is a non-transmembrane protein tyrosine phosphatase (PTPase) containing two tandem Src homology 2 (SH2) domains. We show here that the two SH2 (N-SH2 and C-SH2) domains in SH-PTP1 have different functions in regulation of the PTPase domain and thereby signal transduction. While the N-terminal SH2 domain is both necessary and sufficient for autoinhibition through an intramolecular association with the PTPase domain, truncation of the C-SH2 domain [SH-PTP1 (delta CSH2) construct] has little effect on SH-PTP1 activity. A synthetic phosphotyrosine residue (pY) peptide derived from the erythropoietin receptor (EpoR pY429) binds to the N-SH2 domain and activates both wild-type SH-PTP1 and SH-PTP1 (delta CSH2) 60- to 80-fold. Another pY peptide corresponding to a phosphorylation site on the IgG Fc receptor (Fc gamma RIIB1 pY309) associates with both the C-SH2 domain (Kd = 2.8 microM and the N-SH2 domain (Kd = 15.0 microM) and also activates SH-PTP1 12-fold. By analysis of the effect of the Fc gamma RIIB1 pY309 peptide on SH-PTP1 (delta CSH2), SH-PTP1 (R30K/R33E), SH-PTP1 (R30K/R136K), and SH-PTP1 (R136K) mutants in which the function of either the N- or C-SH2 domain has been impaired, we have determined that both synthetic pY peptides stimulate SH-PTP1 by binding to its N-SH2 domain; binding of pY ligand to the C-SH2 domain has no effect on SH-PTP1 activity. We propose that the N-terminal SH2 domain serves both as a regulatory domain and as a recruiting unit, whereas the C-terminal SH2 domain acts merely as a recruiting unit.
Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.
Authors: Bamford R N , Battiata A P , Burton J D , Sharma H , Waldmann T A
Published: July 2002

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.
Effect of estrogen upon cyclic ADP ribose metabolism: β-Estradiol stimulates ADP ribosyl cyclase in rat uterus
Authors: Chini Eduardo N. , de Toledo Frederico G. S. , Thompson Michael A. , Dousa Thomas P.
Published: July 2002

Cyclic ADP ribose (cADPR) has been shown to trigger Ca
2+
release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all-
trans-
retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of β-estradiol (E
2
), an archetypal steroid superfamily hormone, upon its major target organ, uterus,
in vivo
. Administration of E
2
to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an ≈Δ + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E
2
-stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E
2
was due to the increase of
V
MAX
without change in
K
m
. Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E
2
(0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E
2
-treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E
2
, via action to its nuclear receptors
in vivo
, increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca
2+
release pathway.
Vacuolar H
+
-ATPase in ocular ciliary epithelium
Authors: Wax Martin B. , Saito Isao , Tenkova Tatyana , Krupin Theodore , Becker Bernard , Nelson Nathan , Brown Dennis , Gluck Stephen L.
Published: July 2002

The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H
+
-ATPase (V-ATPase) in the ocular ciliary epithelium is a key component of this process. In intracellular pH (pH
i
) measurements of isolated ciliary epithelium performed with 2′,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A
1
slowed the recovery of pH
i
in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A
1
produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A
1
reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.
Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein
Authors: Takemaru Ken-ichi , Li Feng-Qian , Ueda Hitoshi , Hirose Susumu
Published: July 2002

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the
Drosophila melanogaster
nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm
Bombyx mori
whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the
fushi tarazu
gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.
A protein encoded by a group I intron in
Aspergillus nidulans
directly assists RNA splicing and is a DNA endonuclease
Authors: Ho Yugong , Kim Sea-Jin , Waring Richard B.
Published: July 2002

Some group I introns self-splice
in vitro
, but almost all are thought to be assisted by proteins
in vivo
. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from
Aspergillus nidulans
directly facilitates splicing
in vitro
. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.
Comparison of fusion phage libraries displaying V
H
or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules
Authors: Cai Xiaohong , Garen Alan
Published: July 2002

A single-chain Fv (scFv) fusion phage library derived from random combinations of V
H
and V
L
(variable heavy and light chains) domains in the antibody repertoire of a vaccinated melanoma patient was previously used to isolate clones that bind specifically to melanoma cells. An unexpected finding was that one of the clones encoded a truncated scFv molecule with most of the V
L
domain deleted, indicating that a V
H
domain alone can exhibit tumor-specific binding. In this report a V
H
fusion phage library containing V
H
domains unassociated with V
L
domains was compared with a scFv fusion phage library as a source of melanoma-specific clones; both libraries contained the same V
H
domains from the vaccinated melanoma patient. The results demonstrate that the clones can be isolated from both libraries, and that both libraries should be used to optimize the chance of isolating clones binding to different epitopes. Although this strategy has been tested only for melanoma, it is also applicable to other cancers. Because of their small size, human origin and specificity for cell surface tumor antigens, the V
H
and scFv molecules have significant advantages as tumor-targeting molecules for diagnostic and therapeutic procedures and can also serve as probes for identifying the cognate tumor antigens.
Firefly “femmes fatales” acquire defensive steroids (lucibufagins) from their firefly prey
Authors: Eisner Thomas , Goetz Michael A. , Hill David E. , Smedley Scott R. , Meinwald Jerrold
Published: July 2002

Female fireflies of the genusPhoturis, the so-called firefly “femmes fatales,” prey on male fireflies of the genusPhotinus. The females are able to entrap the males by faking the flash signal characteristics of thePhotinusfemale. We found that by feeding onPhotinusmales,Photurisfemales gain more than nutrients. They also acquire defensive steroidal pyrones called lucibufagins, which are contained inPhotinusbut whichPhoturisfireflies are unable to produce on their own.Photurisfemales that eatPhotinusmales or lucibufagin are rejected byPhidippusjumping spiders. Lucibufagin itself proved to be a deterrent to such spiders. Field-collectedPhoturisfemales contain lucibufagin in varying amounts. The more lucibufagin they contain the more unacceptable they are toPhidippus.
Context-sensitive synaptic plasticity and temporal-to-spatial transformations in hippocampal slices
Authors: Buonomano Dean V. , Hickmott Peter W. , Merzenich Michael M.
Published: July 2002

Hippocampal slices are used to show that, as a temporal input pattern of activity flows through a neuronal layer, a temporal-to-spatial transformation takes place. That is, neurons can respond selectively to the first or second of a pair of input pulses, thus transforming different temporal patterns of activity into the activity of different neurons. This is demonstrated using associative long-term potentiation of polysynaptic CA1 responses as an activity-dependent marker: by depolarizing a postsynaptic CA1 neuron exclusively with the first or second of a pair of pulses from the dentate gyrus, it is possible to “tag” different subpopulations of CA3 neurons. This technique allows sampling of a population of neurons without recording simultaneously from multiple neurons. Furthermore, it reflects a biologically plausible mechanism by which single neurons may develop selective responses to time-varying stimuli and permits the induction of context-sensitive synaptic plasticity. These experimental results support the view that networks of neurons are intrinsically able to process temporal information and that it is not necessary to invoke the existence of internal clocks or delay lines for temporal processing on the time scale of tens to hundreds of milliseconds.
Broken symmetry and critical transport properties of random metals
Authors: Phillips J. C.
Published: July 2002

Recent experimental data on the conductivity σ
+
(
T
),
T
→ 0, on the metallic side of the metal–insulator transition in ideally random (neutron transmutation-doped)
70
Ge:Ga have shown that σ
+
(0) ∝ (
N
−
N
c
)
μ
with μ = ½, confirming earlier ultra-low-temperature results for Si:P. This value is inconsistent with theoretical predictions based on diffusive classical scaling models, but it can be understood by a quantum-directed percolative filamentary amplitude model in which electronic basis states exist which have a well-defined momentum parallel but not normal to the applied electric field. The model, which is based on a new kind of broken symmetry, also explains the anomalous sign reversal of the derivative of the temperature dependence in the critical regime.
The structure of Selmer groups
Authors: Greenberg Ralph
Published: July 2002

The purpose of this article is to describe certain results and
conjectures concerning the structure of Galois cohomology groups and
Selmer groups, especially for abelian varieties. These results are
analogues of a classical theorem of Iwasawa. We formulate a very
general version of the Weak Leopoldt Conjecture. One consequence of
this conjecture is the nonexistence of proper Λ-submodules of finite
index in a certain Galois cohomology group. Under certain hypotheses,
one can prove the nonexistence of proper Λ-submodules of finite index
in Selmer groups. An example shows that some hypotheses are needed.
The
Caenorhabditis elegans
seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells
Authors: Zhang Yinong , Chou Joseph H. , Bradley Jonathan , Bargmann Cornelia I. , Zinn Kai
Published: July 2002

The nematode
Caenorhabditis elegans
exhibits behavioral responses to many volatile odorants. Chemotaxis toward one such odorant, diacetyl (butanedione), requires the function of a seven-transmembrane receptor protein encoded by the
odr-10
gene. To determine directly whether ODR-10 protein is an odorant receptor, it is necessary to express the protein in a heterologous system and show that it responds to diacetyl by activation of a G protein signaling pathway. Here we demonstrate that human cells expressing ODR-10 on their surfaces exhibit a transient elevation in intracellular Ca
2+
levels after diacetyl application. Volatile compounds that differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone) are not ODR-10 agonists. Behavioral responses to these compounds are not dependent on
odr-10
function, so ODR-10 specificity in human cells resembles
in vivo
specificity. The apparent affinity of ODR-10 for diacetyl observed in human cells is consistent with the diacetyl concentration ranges that allow efficient nematode chemotaxis. ODR-10 expressed in human cells also responds to two anionic compounds, pyruvate and citrate, which are metabolic precursors used for diacetyl production by certain bacterial species. Ca
2+
elevation in response to ODR-10 activation is due to release from intracellular stores.