bluuebunny/crossref_metadata_2025 huggingface dataset:
An essential role of the JAK-STAT pathway in ischemic preconditioning
Authors: Xuan Yu-Ting , Guo Yiru , Han Hui , Zhu Yanqing , Bolli Roberto
Published: July 2002

The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)–signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 ± 53%) and JAK2 (+238 ± 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 ± 61%) and pTyr-STAT3 (+253 ± 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 γ-IFN activation site DNA-binding activity (+606 ± 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac
iNOS
gene and the adaptation of the heart to ischemic stress.
Perforin-low memory CD8+cells are the predominant T cells in normal humans that synthesize the β-chemokine macrophage inflammatory protein-1β
Authors: Kamin-Lewis Roberta , Abdelwahab Sayed F. , Trang Christine , Baker Amanda , DeVico Anthony L. , Gallo Robert C. , Lewis George K.
Published: July 2002

The synthesis of antiviral β-chemokines has joined cytolysis as a potential mechanism for the control of HIV-1 infection by CD8+T cells. Recent evidence suggests that these two effector functions can diverge in some individuals infected with HIV-1; however, little is known about the CD8+T cell subsets in normal individuals that synthesize antiviral β-chemokines. In this report, we have used mutliparameter flow cytometry to characterize the T cell subsets that secrete the antiviral β-chemokine macrophage inflammatory protein (MIP)-1β. These studies have shown: (i) CD8+cells are the predominant T cell subset that synthesizes MIP-1β; (ii) MIP-1β and IFN-γ are synthesized congruently in most CD8+T cells; however, significant numbers of these cells synthesize only one of these effector molecules; (iii) approximately 60% of the CD8+T cells that synthesize MIP-1β lack perforin; (iv) MIP-1β is synthesized with approximately equal frequency by CD28+and CD28−subpopulations of CD8+T cells; (v) MIP-1β is synthesized by three distinct CD8+T cell subsets defined by the expression of CD45R0 and CD62L; and (vi) MIP-1β is not synthesized in short-term cultures of naive CD8+T cells. These results demonstrate substantial subset heterogeneity of MIP-1β synthesis among CD8+T cells and suggest that these subsets should be evaluated as correlates of protective immunity against HIV-1.
Apaf-1
deficiency and neural tube closure defects are found in
fog
mice
Authors: Honarpour Narimon , Gilbert Sandra L. , Lahn Bruce T. , Wang Xiaodong , Herz Joachim
Published: July 2002

The forebrain overgrowth mutation (
fog
) was
originally described as a spontaneous autosomal recessive mutation
mapping to mouse chromosome 10 that produces forebrain defects, facial
defects, and spina bifida. Although the
fog
mutant has
been characterized and available to investigators for several years,
the underlying mutation causing the pathology has not been known.
Because of its phenotypic resemblance to apoptotic protease
activating factor-1 (Apaf-1) knockout mice, we have investigated the
possibility that the
fog
mutation is in the
Apaf-1
gene. Allelic complementation, Western blot
analysis, and caspase activation assays indicate that
fog
mutant mice lack Apaf-1 activity. Northern blot and
reverse transcription-PCR analysis show that
Apaf-1
mRNA
is aberrantly processed, resulting in greatly reduced expression levels
of normal
Apaf-1
mRNA. These findings are strongly
suggestive of the
fog
mutation being a hypomorphic
Apaf-1
defect and implicate neural progenitor cell death
in the pathogenesis of spina bifida—a common human congenital
malformation. Because a complete deficiency in
Apaf-1
usually results in perinatal lethality and
fog
/
fog
mice more readily survive into
adulthood, these mutants serve as a valuable model with which
apoptotic cell death can be studied
in vivo
.
Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules
Authors: Vorobjev Ivan , Malikov Viacheslav , Rodionov Vladimir
Published: July 2002

Polarized radial arrays of cytoplasmic microtubules (MTs) with minus ends clustered at the cell center define the organization of the cytoplasm through interaction with microtubule motors bound to membrane organelles or chromosomes. It is generally assumed that the radial organization results from nucleation of MTs at the centrosome. However, radial MT array can also be attained through self-organization that requires the activity of a minus-end-directed MT motor, cytoplasmic dynein. In this study we examine the role of cytoplasmic dynein in the self-organization of a radial MT array in cytoplasmic fragments of fish melanophores lacking the centrosome. After activation of dynein motors bound to membrane-bound organelles, pigment granules, the fragments rapidly form polarized radial arrays of MTs and position pigment aggregates at their centers. We show that rearrangement of MTs in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial pattern is generated by continuous disassembly and reassembly of MTs and concurrent minus-end-directed transport of pigment granules bearing the nucleation sites.
SOS mutator activity: Unequal mutagenesis on leading and lagging strands
Authors: Maliszewska-Tkaczyk Magdalena , Jonczyk Piotr , Bialoskorska Malgorzata , Schaaper Roel M. , Fijalkowska Iwona J.
Published: July 2002

A major pathway of mutagenesis in
Escherichia coli
is mediated by the inducible SOS response. Current models of SOS
mutagenesis invoke the interaction of RecA and UmuD′
2
C
proteins with a stalled DNA replication complex at sites of DNA lesions
or poorly extendable terminal mismatches, resulting in an (error-prone)
continuation of DNA synthesis. The precise mechanisms of SOS-mediated
lesion bypass or mismatch extension are not known. Here, we have
studied mutagenesis on the
E. coli
chromosome in
recA730
strains. In
recA730
strains, the
SOS system is expressed constitutively, resulting in a spontaneous
mutator effect (SOS mutator) because of reduced replication fidelity.
We investigated whether during SOS mutator activity replication
fidelity might be altered differentially in the leading and lagging
strand of replication. Pairs of
recA730
strains were
constructed differing in the orientation of the
lac
operon relative to the origin of replication. The strains were also
mismatch-repair defective (
mutL
) to facilitate scoring
of replication errors. Within each pair, a given
lac
sequence is replicated by the leading-strand machinery in one
orientation and by the lagging-strand machinery in the other
orientation. Measurements of defined
lac
mutant
frequencies in such pairs revealed large differences between the two
orientations. Furthermore, in all cases, the frequency bias was the
opposite of that seen in normal cells. We suggest that, for the
lacZ
target used in this study, SOS mutator activity
operates with very different efficiency in the two strands.
Specifically, the lagging strand of replication appears most
susceptible to the SOS mutator effect.
A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots
Authors: Haralampidis K. , Bryan G. , Qi X. , Papadopoulou K. , Bakht S. , Melton R. , Osbourn A.
Published: July 2002

Many plants synthesize antimicrobial secondary metabolites as part of their normal program of growth and development, often sequestering them in tissues where they may protect against microbial attack. These include glycosylated triterpenoids (saponins), natural products that are exploited by man for a variety of purposes including use as drugs [Hostettmann, K. & Marston, A. (1995)
Saponins
(Cambridge Univ. Press, Cambridge, U.K.)]. Very little is known about the genes required for the synthesis of this important family of secondary metabolites in plants. Here we show the novel oxidosqualene cyclase AsbAS1 catalyzes the first committed step in the synthesis of antifungal triterpenoid saponins that accumulate in oat roots. We also demonstrate that two sodium azide-generated saponin-deficient mutants of oat, which define the
Sad1
genetic complementation group, are defective in the gene encoding this enzyme and provide molecular genetic evidence indicating a direct link between AsbAS1, triterpenoid saponin biosynthesis, and disease resistance. Orthologs of
AsbAS1
are absent from modern cereals and may have been lost during selection, raising the possibility that this gene could be exploited to enhance disease resistance in crop plants.
A paradox resolved: Sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy
Authors: Freytag John K. , Girguis Peter R. , Bergquist Derk C. , Andras Jason P. , Childress James J. , Fisher Charles R.
Published: July 2002

Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites,
Lamellibrachia
cf.
luymesi
, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170–250 years.
L.
cf.
luymesi
can grow a posterior extension of its tube and tissue, termed a “root,” down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 μM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations. Respiration experiments, which exposed the root portions of
L.
cf.
luymesi
to sulfide concentrations between 51–561 μM, demonstrate that
L.
cf.
luymesi
use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 μmol⋅g
−1
⋅h
−1
. Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior (root) portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan
L.
cf.
luymesi
can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.
A likelihood ratio test for evolutionary rate shifts and functional divergence among proteins
Authors: Knudsen Bjarne , Miyamoto Michael M.
Published: July 2002

Changes in protein function can lead to changes in the selection
acting on specific residues. This can often be detected as
evolutionary rate changes at the sites in question. A
maximum-likelihood method for detecting evolutionary rate shifts at
specific protein positions is presented. The method determines
significance values of the rate differences to give a sound statistical
foundation for the conclusions drawn from the analyses. A statistical
test for detecting slowly evolving sites is also described. The methods
are applied to a set of Myc proteins for the identification of both
conserved sites and those with changing evolutionary rates. Those
positions with conserved and changing rates are related to the
structures and functions of their proteins. The results are compared
with an earlier Bayesian method, thereby highlighting the advantages of
the new likelihood ratio tests.
Ligand-induced signal transduction within heterodimeric GABA
B
receptor
Authors: Margeta-Mitrovic Marta , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

γ-aminobutyric acid type B (GABA
B
) receptors, G
protein-coupled receptors (GPCRs) for GABA, are obligate heterodimers
of two homologous subunits, GB1 and GB2. Typical for family C GPCRs,
the N termini of both GB1 and GB2 contain a domain with homology to
bacterial periplasmic amino acid-binding proteins (PBPs), but only the
GB1 PBP-like domain binds GABA. We found that both GB1 and GB2
extracellular N termini are required for normal coupling of
GABA
B
receptors to their physiological effectors,
G
i
and G protein-activated K
+
channels (GIRKs).
Receptors with two GB2 N termini did not respond to GABA, whereas
receptors with two GB1 N termini showed increased basal activity and
responded to GABA with inhibition, rather than activation, of GIRK
channels. This GABA-induced GIRK current inhibition depended on GABA
binding to the chimeric GB
1/2
subunit (the GB1 N-terminal
domain attached to the heptahelical domain of GB2), rather than the
wild-type GB1 subunit. Interestingly, receptors with reciprocal
exchange of N-terminal domains between the subunits were functionally
indistinguishable from wild-type receptors. We also found that peptide
linkers between GB1 and GB2 PBP-like domains and respective
heptahelical domains could be altered without affecting receptor
function. This finding suggests that other contacts between the
PBP-like and heptahelical domains underlie ligand-induced signal
transduction, a finding likely to be relevant for all family C
GPCRs.
Highly conserved glutamic acid in the extracellular IV–V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family
Authors: Terakita Akihisa , Yamashita Takahiro , Shichida Yoshinori
Published: July 2002

Retinochrome is a member of the rhodopsin family having a
chromophore retinal and functioning as a retinal photoisomerase in
squid photoreceptor cells. Unlike vertebrate rhodopsins, but like many
invertebrate rhodopsins, retinochrome does not have a glutamic acid at
position 113 to serve as a counterion for the protonated retinylidene
Schiff base. Here we investigated possible counterions in retinochrome
by site-specific mutagenesis. Our results showed that the counterion is
the glutamic acid at position 181, at which almost all the pigments in
the rhodopsin family, including vertebrate and invertebrate rhodopsins,
have a glutamic or aspartic acid. The remarkable exceptions are the
long-wavelength visual pigments that have a histidine that, together
with a nearby lysine, serves as a chloride-binding site. Replacement of
Glu-181 of bovine rhodopsin with Gln caused a 10-nm red-shift of
absorption maximum. Because the position at 181 is in the extracellular
loop connecting the transmembrane helices VI and V, these results
demonstrate the importance of this loop to function for spectral tuning
in the rhodopsin family.
Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut
Authors: Boudko Dmitri Y. , Moroz Leonid L. , Harvey William R. , Linser Paul J.
Published: July 2002

The midgut of mosquito larvae maintains a specific lumen
alkalinization profile with large longitudinal gradients (pH
≈ 3 units⋅mm
−1
) in which an extremely alkaline
(pH ≈ 11) anterior midgut lies between near-neutral posterior
midgut and gastric cecum (pH 7–8). A plasma membrane
H
+
V-ATPase energizes this alkalinization but the ion
carriers involved are unknown. Capillary zone electrophoresis of body
samples with outlet conductivity detection showed a specific
transepithelial distribution of chloride and bicarbonate/carbonate
ions, with high concentrations of both anions in the midgut tissue:
68.3 ± 5.64 and 50.8 ± 4.21 mM, respectively. Chloride was
higher in the hemolymph, 57.6 ± 7.84, than in the lumen,
3.51 ± 2.58, whereas bicarbonate was higher in the lumen,
58.1 ± 7.34, than the hemolymph, 3.96 ± 2.89.
Time-lapse video assays of pH profiles
in vivo
revealed that ingestion of the carbonic anhydrase inhibitor
acetazolamide and the ion exchange inhibitor DIDS
(4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), at
10
−4
M eliminates lumen alkalinization. Basal application
of these inhibitors
in situ
also reduced gradients
recorded with self-referencing pH-sensitive microelectrodes near the
basal membrane by ≈65% and 85% respectively. Self-referencing
chloride-selective microelectrodes revealed a specific spatial profile
of transepithelial chloride transport with an efflux maximum in
anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes.
These data suggest that an H
+
V-ATPase-energized anion
exchange occurs across the apical membrane of the epithelial cells and
implicate an electrophoretic Cl
−
/HCO

\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\setlength{\oddsidemargin}{-69pt}
\begin{document}
\begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}

exchanger and carbonic anhydrase as crucial components of the
steady-state alkalinization in anterior midgut of mosquito larvae.
Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding
Authors: Hattendorf Douglas A. , Lindquist Susan L.
Published: July 2002

Hsp104 from
Saccharomyces cerevisiae
is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the
K
d
and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.
Order-sensitive plasticity in adult primary auditory cortex
Authors: Kilgard Michael P. , Merzenich Michael M.
Published: July 2002

The neural response to a stimulus presented as part of a rapid sequence is often quite different from the response to the same stimulus presented in isolation. In primary auditory cortex (A1), although the most common effect of preceding stimuli is inhibitory, most neurons can also exhibit response facilitation if the appropriate spectral and temporal separation of sequence elements is presented. In this study, we investigated whether A1 neurons in adult animals can develop context-dependent facilitation to a novel acoustic sequence. After repeatedly pairing electrical stimulation of the basal forebrain with a three-element sequence (high frequency tone–low frequency tone– noise burst), 25% of A1 neurons exhibited facilitation to the low tone when preceded by the high tone, compared with only 5% in controls. In contrast, there was no increase in the percent of sites that showed facilitation for the reversed tone order (low preceding high). Nearly 60% of sites exhibited a facilitated response to the noise burst when preceded by the two tones. Although facilitation was greatest in response to the paired sequence, facilitation also generalized to related sequences that were either temporally distorted or missing one of the tones. Pairing basal forebrain stimulation with the acoustic sequence also caused a decrease in the time to peak response and an increase in population discharge synchrony, which was not seen after pairing simple tones, tone trains, or broadband stimuli. These results indicate that context-dependent facilitation and response synchronization can be substantially altered in an experience-dependent fashion and provide a potential mechanism for learning spectrotemporal patterns.
Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.
Authors: Azad A K , Sirakova T D , Rogers L M , Kolattukudy P E
Published: July 2002

A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.
CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes.
Authors: Stokes D G , Tartof K D , Perry R P
Published: July 2002

Previously, we reported on the discovery and characterization of a mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), with features that led us to suspect that it might have an important role in the modification of chromatin structure. We now report on the characterization of the Drosophila melanogaster CHD1 homologue (dCHD1) and its localization on polytene chromosomes. A set of overlapping cDNAs encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. When the chromo and ATPase/helicase domain sequences in various CHD1 homologues were compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains were revealed. The dCHD1 gene was mapped to position 23C-24A on chromosome 2L. Western blot analyses with antibodies raised against a dCHD1 fusion protein specifically recognized an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies revealed that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression.
The extraordinarily rapid disappearance of entopic images.
Authors: Coppola D , Purves D
Published: July 2002

It has been known for more than 40 years that images fade from perception when they are kept at the same position on the retina by abrogating eye movements. Although aspects of this phenomenon were described earlier, the use of close-fitting contact lenses in the 1950s made possible a series of detailed observations on eye movements and visual continuity. In the intervening decades, many investigators have studied the role of image motion on visual perception. Although several controversies remain, it is clear that images deteriorate and in some cases disappear following stabilization; eye movements are, therefore, essential to sustained exoptic vision. The time course of image degradation has generally been reported to be a few seconds to a minute or more, depending upon the conditions. Here we show that images of entoptic vascular shadows can disappear in less than 80 msec. The rapid vanishing of these images implies an active mechanism of image erasure and creation as the basis of normal visual processing.
Discrimination among pheromone component blends by interneurons in male antennal lobes of two populations of the turnip moth, Agrotis segetum.
Authors: Wu W , Anton S , Löfstedt C , Hansson B S
Published: July 2002

A difference in female pheromone production and male behavioral response has previously been found in two populations of the turnip moth, Agrotis segetum, originating from Sweden and Zimbabwe, respectively. In this study, we investigated the pheromone response of antennal lobe interneurons of males of the two populations by intracellular recordings, stimulating with single pheromone components and various inter- and intra-populational pheromone blends. Three major physiological types of antennal lobe neurons were established in the two populations according to their responses to different stimuli. One type responded broadly to almost all the stimuli tested. The second type responded selectively to some of the single components and blends. The third type did not respond to any single components but did respond to certain blends. Furthermore, some neurons of the second and third type recognized strain specific differences in ratios between pheromone components. Both projection neurons and local interneurons were found among these three types. Two pheromone responding bilateral projection neurons are reported for the first time in this paper.
I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction.
Authors: Rothe M , Xiong J , Shu H B , Williamson K , Goddard A , Goeddel D V
Published: July 2002

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40- and TNF-mediated activation of the transcription factor NF-kappa B. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-kappa B activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state.
Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.
Authors: Akaogi K , Okabe Y , Sato J , Nagashima Y , Yasumitsu H , Sugahara K , Miyazaki K
Published: July 2002

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.
Identification and characterization of differentially expressed cDNAs of the vector mosquito,
Anopheles gambiae
Authors: Dimopoulos George , Richman Adam , della Torre Alessandra , Kafatos Fotis C. , Louis Christos
Published: July 2002

The isolation and study of
Anopheles gambiae
genes
that are differentially expressed in development, notably in tissues
associated with the maturation and transmission of the malaria
parasite, is important for the elucidation of basic molecular
mechanisms underlying vector–parasite interactions. We have used the
differential display technique to screen for mRNAs specifically
expressed in adult males, females, and midgut tissues of blood-fed and
unfed females. We also screened for mRNAs specifically induced upon
bacterial infection of larval stage mosquitoes. We have characterized
19 distinct cDNAs, most of which show developmentally regulated
expression specificity during the mosquito life cycle. The most
interesting are six new sequences that are midgut-specific in the
adult, three of which are also modulated by blood-feeding. The
gut-specific sequences encode a maltase, a V-ATPase subunit, a GTP
binding protein, two different lectins, and a nontrypsin serine
protease. The latter sequence is also induced in larvae subjected to
bacterial challenge. With the exception of a mitochondrial DNA
fragment, the other 18 sequences constitute expressed genomic sequence
tags, 4 of which have been mapped cytogenetically.
Suppression of HIV replication by lymphoid tissue CD8+cells correlates with the clinical state of HIV-infected individuals
Authors: Blackbourn David J. , Mackewicz Carl E. , Barker Edward , Hunt Thomas K. , Herndier Brian , Haase Ashley T. , Levy Jay A.
Published: July 2002

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+cell ratio of 0.1. The CD8+cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+cell ratio of 0.25; the subject’s peripheral blood CD8+cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.
The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex
Authors: Lauble H. , Kennedy M. C. , Emptage M. H. , Beinert H. , Stout C. D.
Published: July 2002

It has been known for many years that fluoroacetate and
fluorocitrate when metabolized are highly toxic, and that at least one
effect of fluorocitrate is to inactivate aconitase. In this paper we
present evidence supporting the hypothesis that the
(−)-
erythro
diastereomer of 2-fluorocitrate acts as a
mechanism based inhibitor of aconitase by first being converted to
fluoro-
cis
-aconitate, followed by addition of hydroxide
and with loss of fluoride to form
4-hydroxy-
trans
-aconitate (HTn), which binds very
tightly, but not covalently, to the enzyme. Formation of HTn by these
reactions is in accord with the working model for the enzyme mechanism.
That HTn is the product of fluorocitrate inhibition is supported by the
crystal structure of the enzyme-inhibitor complex at 2.05-Å
resolution, release of fluoride stoichiometric with total enzyme when
(−)-
erythro
-2-fluorocitrate is added, HPLC analysis of
the product, slow displacement of HTn by 10
6
-fold excess of
isocitrate, and previously published Mössbauer experiments. When
(+)-
erythro
-2-fluorocitrate is added to aconitase, the
release of fluoride is stoichiometric with total substrate added, and
HPLC analysis of the products indicates the formation of
oxalosuccinate, and its derivative α-ketoglutarate. This is
consistent with the proposed mechanism, as is the formation of HTn from
(−)-
erythro
-2-fluorocitrate. The structure of the
inhibited complex reveals that HTn binds like the inhibitor
trans
-aconitate while providing all the interactions of
the natural substrate, isocitrate. The structure exhibits four hydrogen
bonds <2.7 Å in length involving HTn, H
2
O bound to the
[4Fe–4S] cluster, Asp-165 and His-167, as well as low temperature
factors for these moieties, consistent with the observed very tight
binding of the inhibitor.
Phosphohistidyl active sites in polyphosphate kinase of
Escherichia coli
Authors: Kumble Krishnanand D. , Ahn Kyunghye , Kornberg Arthur
Published: July 2002

In the synthesis of inorganic polyphosphate (polyP) from ATP by
polyphosphate kinase (PPK; EC
2.7.4.1
) of
Escherichia
coli
, an N—P-linked phosphoenzyme was previously identified
as the intermediate. The phosphate is presumed to be linked to N3 of
the histidine residue because of its chemical stabilities and its
resemblance to other enzymes known to contain
N
3
-phosphohistidine. Tryptic digests of
[
32
P]PPK contain a predominant
32
P-labeled
peptide that includes His-441. Of the 16 histidine residues in PPK of
E. coli
, 4 are conserved among several bacterial
species. Mutagenesis of these 4 histidines shows that two (His-430 and
His-598) are unaffected in function when mutated to glutamine, whereas
two others (His-441 and His-460) mutated to glutamine or alanine fail
to be phosphorylated, show no enzymatic activities, and fail to support
polyP accumulation in cells bearing these mutant enzymes.
Mouse cytoplasmic polyadenylylation element binding protein: An evolutionarily conserved protein that interacts with the cytoplasmic polyadenylylation elements of c-
mos
mRNA
Authors: Gebauer Fátima , Richter Joel D.
Published: July 2002

Cytoplasmic polyadenylylation is an essential process that controls
the translation of maternal mRNAs during early development and depends
on two cis elements in the 3′ untranslated region: the
polyadenylylation hexanucleotide AAUAAA and a U-rich cytoplasmic
polyadenylylation element (CPE). In searching for factors that could
mediate cytoplasmic polyadenylylation of mouse c-
mos
mRNA, which encodes a serine/threonine kinase necessary for oocyte
maturation, we have isolated the mouse homolog of CPEB, a protein that
binds to the CPEs of a number of mRNAs in
Xenopus
oocytes and is required for their polyadenylylation. Mouse CPEB (mCPEB)
is a 62-kDa protein that binds to the CPEs of c-
mos
mRNA. mCPEB mRNA is present in the ovary, testis, and kidney; within
the ovary, this RNA is restricted to oocytes. mCPEB shows 80% overall
identity with its
Xenopus
counterpart, with a higher
homology in the carboxyl-terminal portion, which contains two RNA
recognition motifs and a cysteine/histidine repeat. Proteins from
arthropods and nematodes are also similar to this region, suggesting an
ancient and widely used mechanism to control polyadenylylation and
translation.
Induction of HSP70 and polyubiquitin expression associated with plant virus replication
Authors: Aranda Miguel A. , Escaler Margarita , Wang Daowen , Maule Andrew J.
Published: July 2002

By examining the front of virus invasion in immature pea embryos
infected with pea seed-borne mosaic virus (PSbMV), the selective
control of different host genes has been observed. From our
observations, the early responses to PSbMV replication can be grouped
into three classes, inhibited host gene expression, induced host gene
expression, and no effect on a normal host function. The expression of
two heat-inducible genes encoding HSP70 and polyubiquitin was induced
coordinately with the onset of virus replication and the
down-regulation of two other genes encoding lipoxygenase and heat shock
cognate protein. The down-regulation was part of a general suppression
of host gene expression that may be achieved through the degradation of
host transcripts. We discuss the possibilities of whether the induction
of HSP70 and polyubiquitin genes represents a requirement for the
respective protein products by the virus or is merely a consequence of
the depletion of other host transcripts. The former is feasible, as the
induction of both genes does result in increased HSP70 and ubiquitin
accumulation. This also indicates that, in contrast to some animal
virus infections, there is not a general inhibition of translation of
host mRNAs following PSbMV infection. This selective control of host
gene expression was observed in all cell types of the embryo and
identifies mechanisms of cellular disruption that could act as triggers
for symptom expression.
Tiggers and DNA transposon fossils in the human genome.
Authors: Smit A F , Riggs A D
Published: July 2002

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.
Foldons, protein structural modules, and exons.
Authors: Panchenko A R , Luthey-Schulten Z , Wolynes P G
Published: July 2002

Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For gammaII-crystallin, myoglobin, barnase, alpha-lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.
Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.
Authors: Rubinstein M , Mogil J S , Japón M , Chan E C , Allen R G , Low M J
Published: July 2002

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.
The three-dimensional structure of an H-2Ld-peptide complex explains the unique interaction of Ldwith beta-2 microglobulin and peptide
Authors: Balendiran Ganesaratnam K. , Solheim Joyce C. , Young Aideen C. M. , Hansen Ted H. , Nathenson Stanley G. , Sacchettini James C.
Published: July 2002

Solution at 2.5-Å resolution of the three-dimensional structure of H-2Ldwith a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (β2m) and peptide. Consistent with the biological data that show an unusually weak association of Ldwith β2m, a novel orientation of the α1/α2 domains of Ldrelative to β2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ldantigen-binding cleft determine the unique motif of peptides that it binds. Ldhas no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ldand its peptide antigen. The unique structural interactions of Ldwith β2m and peptide could make Ldmore suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.
The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling
Authors: Cheng Alec M. , Negishi Izumi , Anderson Steven J. , Chan Andrew C. , Bolen Joseph , Loh Dennis Y. , Pawson Tony
Published: July 2002

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4−CD8−double negative (DN) cells to CD4+CD8+double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.
Characterization of the chicken β-globin insulator
Authors: Chung Jay H. , Bell Adam C. , Felsenfeld Gary
Published: July 2002

Insulators, first identified in
Drosophila
, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5′ end of the chicken β-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse β-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5′HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.
Proteins unfold in steps
Authors: Zocchi G.
Published: July 2002

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1–100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein’s structure.