bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues
Authors: Fei Hong , Okano Hirotaka J. , Li Cai , Lee Gwo-Hwa , Zhao Connie , Darnell Robert , Friedman Jeffrey M.
Published: July 2002

Leptin’s effects are mediated by interactions with a receptor that is alternatively spliced, resulting in at least five different murine forms: Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, and Ob-Re. A mutation in one splice form, Ob-Rb, results in obesity in mice. Northern blots, RNase protection assays, and PCR indicate that Ob-Rb is expressed at a relatively high level in hypothalamus and low level in several other tissues. Ob-Ra is expressed ubiquitously, whereas Ob-Rc, -Rd, and -Re RNAs are only detectable using PCR. In hypothalamus, Ob-Rb is present in the arcuate, ventromedial, dorsomedial, and lateral hypothalamic nuclei but is not detectable in other brain regions. These nuclei are known to regulate food intake and body weight. The level of Ob-Rb in hypothalamus is reduced in mice rendered obese by gold thioglucose (GTG), which causes hypothalamic lesions. The obesity in GTG-treated mice is likely to be caused by ablation of Ob-Rb-expressing neurons, which results in leptin resistance.
BRCA1 is a cell cycle-regulated nuclear phosphoprotein
Authors: Ruffner Heinz , Verma Inder M.
Published: July 2002

We have characterized the
BRCA1
gene product by using four polyclonal antibodies raised against peptides from four different regions of the protein. The antibodies specifically recognize an ≈220-kDa BRCA1 protein that is predominantly expressed in the nucleus of both normal and neoplastic breast cancer cells. It is a serine phosphoprotein that undergoes hyperphosphorylation during late G
1
and S phases of the cell cycle and is transiently dephosphorylated early after M phase. We propose that BRCA1 is a phosphoprotein that alters in a qualitative and quantitative manner during cell cycle progression.
The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro
Authors: Pratt K. P. , Côté H. C. F. , Chung D. W. , Stenkamp R. E. , Davie E. W.
Published: July 2002

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.
Silent chromatin determines target preference of the
Saccharomyces
retrotransposon Ty5
Authors: Zou Sige , Voytas Daniel F.
Published: July 2002

The
HML
and
HMR
mating loci of
Saccharomyces cerevisiae
are bound in silent chromatin, which is assembled at the flanking E and I transcriptional silencers. The retrotransposon Ty5 preferentially integrates into regions of silent chromatin, and Ty5 insertions near the
HMR-E
silencer account for ≈2% of total transposition events. Most Ty5 insertions occur within 800 bp on either side of the autonomously replicating consensus sequence within
HMR-E
. Ty5 target preference is determined by silent chromatin, because integration near
HMR-E
is abolished in strains with silencer mutations that alleviate transcriptional repression. The recognition of specific DNA sequences
per se
does not direct integration, rather, it is the protein complex assembled at the silencers. As demonstrated here for Ty5, recognition of specific chromatin domains may be a general mechanism by which retrotransposons and retroviruses determine integration sites.
Striking sequence similarity in inter- and intra-specific comparisons of class I
SLG
alleles from
Brassica oleracea
and
Brassica campestris
: Implications for the evolution and recognition mechanism
Authors: Kusaba Makoto , Nishio Takeshi , Satta Yoko , Hinata Kokichi , Ockendon David
Published: July 2002

Self-incompatibility in
Brassica
is controlled by a single multi-allelic locus (
S
locus), which contains at least two highly polymorphic genes expressed in the stigma: an
S
glycoprotein gene (
SLG
) and an
S
receptor kinase gene (
SRK
). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel
SLG
sequences of
Brassica oleracea
and
Brassica campestris
. Sequence comparisons of a large number of
SLG
alleles and
SLG
-related genes revealed the following points. (
i
) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between
SLG

14

of
B. oleracea
and
SLG

25

of
B. campestris
in deduced amino acid sequence) suggests that
SLG
diversification predates speciation. (
ii
) A perfect match of the sequences in hypervariable regions, which are thought to determine
S
specificity in an intra-specific comparison (
SLG

8

and
SLG

46

of
B. campestris
) and the observation that the hypervariable regions of SLG and SRK of the same
S
haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine
S
specificity. (
iii
) Comparison of the hypervariable regions of
SLG
alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in
SLG
alleles. Models for the evolution of
SLG
/
SRK
are presented.
Synergistic and promoter-selective activation of transcription by recruitment of transcription factors TFIID and TFIIB
Authors: Gonzalez-Couto Eduardo , Klages Natacha , Strubin Michel
Published: July 2002

Eukaryotic transcriptional activators may function by stimulating formation of RNA polymerase II preinitiation complexes at the core promoter of genes. In this case, their mode of action will intrinsically depend on how these complexes assemble on promoters in living cells, an issue that remains largely unexplored. Here we show that in yeast the basal transcription machinery is brought to the promoter in the form of at least two subcomplexes, TFIID and a complex comprising TFIIB and other essential components. Individual recruitment of either complex by artificial contact with a transcriptionally inactive, sequence-specific DNA-binding protein suffices to trigger transcriptional activation from a wild-type core promoter bearing the appropriate binding site. In contrast, activation from a promoter containing a weakened TATA element is only observed upon recruitment of TFIID. Tethering TFIIB on that promoter remains without effect, but the simultaneous recruitment of both components leads to strong synergistic activation. These findings suggest a simple mechanism whereby two activators that contact distinct subcomplexes of the basal machinery may stimulate transcription synergistically and differentially depending on the nature of the promoter.
Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new
myc
collaborating oncogene
Authors: Stewart M. , Terry A. , Hu M. , O’Hara M. , Blyth K. , Baxter E. , Cameron E. , Onions D. E. , Neil J. C.
Published: July 2002

The
til
-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-
myc
transgenic mice. We now show that viral insertions at
til
-1 lead to transcriptional activation of PEBP2αA (CBFA1), a transcription factor related to the
Drosophila
segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2αA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated
til
-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2αA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.
A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection
Authors: Adema Coen M. , Hertel Lynn A. , Miller Robert D. , Loker Eric S.
Published: July 2002

After infection with the digenetic trematode
Echinostoma paraensei
, hemolymph of the snail
Biomphalaria glabrata
contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by
E. paraensei
intramolluscan larvae. Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members.
B. glabrata
has at least five FREP genes, three of which are expressed at increased levels after infection. Elucidation of components of the defense system of
B. glabrata
is relevant because this snail is an intermediate host for
Schistosoma mansoni
, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.
Estimating the relative roles of top-down and bottom-up forces on insect herbivore populations: A classic study revisited
Authors: Hunter Mark D. , Varley George C. , Gradwell George R.
Published: July 2002

Although most ecologists agree that both top-down and bottom-up forces (predation and resource limitation, respectively) act in concert to influence populations of herbivores, it has proven difficult to estimate the relative contributions of such forces in terrestrial systems. Using a combination of time–series analysis of population counts recorded over 16 years and experimental data, we present the first estimates of the relative roles of top-down and bottom-up forces on the population dynamics of two terrestrial insect herbivores on the English oak (Quercus robur). Data suggest that temporal variation in winter moth,Operophtera brumata, density is dominated by time-lagged effects of pupal predators. By comparison, spatial variation inO. brumatadensity is dominated by host–plant quality. Overall, top-down forces explain 34.2% of population variance, bottom-up forces explain 17.2% of population variance, and 48.6% remains unexplained. In contrast, populations of the green oak tortrix,Tortrix viridana, appear dominated by bottom-up forces. Resource limitation, expressed as intraspecific competition among larvae for oak leaves, explains 29.4% of population variance. Host quality effects explain an additional 5.7% of population variance. We detected no major top-down effects onT. viridanapopulations. An unknown factor causing a linear decline inT. viridanapopulations over the 16-year study period accounts for most of the remaining unexplained variance. We discuss the observed differences between the insect species and the utility of time–series analysis as a tool in assessing the relative importance of top-down and bottom-up forces on herbivore populations.
Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains
Authors: Goldshleger Rivka , Karlish Steven J. D.
Published: July 2002

Incubation of Na/K-ATPase with ascorbate plus H
2
O
2
produces specific cleavage of the α subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The β subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe
2+
ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe
3+
-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe
2+
but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH⋅ radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E
2
) or media containing Rb ions [E
2
(Rb)], cleavage is much faster than in high ionic strength media (E
1
) or media containing Na ions (E
1
Na). N-terminal fragments and two C-terminal fragments (N-terminals E
214
and V
712
) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe
2+
(or Fe
3+
) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe
2+
(or Fe
3+
) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the α subunit, within the minor and major cytoplasmic loops, interact in the E
2
or E
2
(Rb) conformations but move apart in the E
1
or E
1
Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.
Character displacement in someCnemidophoruslizards revisited: A phylogenetic analysis
Authors: Radtkey Ray. R. , Fallon Sylvia M. , Case Ted J.
Published: July 2002

Ecological studies have demonstrated the role of competition in structuring communities; however, the importance of competition as a vehicle for evolution by natural selection and speciation remains unresolved. Study systems of insular faunas have provided several well known cases where ecological character displacement, coevolution of competitors leading to increased morphological separation, is thought to have occurred (e.g., anoline lizards and geospizine finches). Whiptail lizards (genusCnemidophorus) from the islands of the Sea of Cortez and the surrounding mainland demonstrate a biogeographic pattern of morphological variation suggestive of character displacement. Two species ofCnemidophorusoccur on the Baja peninsula, one relatively large (Cnemidophorus tigris) and one smaller (Cnemidophorus hyperythrus). Oceanic islands in the Sea of Cortez contain only single species, five of six having sizes intermediate to both species found on the Baja peninsula. On mainland MexicoC. hyperythrusis absent, whereasC. tigrisis the smaller species in whiptail guilds. Here we construct a phylogeny using nucleotide sequences of the cytochromebgene to infer the evolutionary history of body size change and historical patterns of colonization in theCnemidophorussystem. The phylogenetic analysis indicates that (i) oceanic islands have been founded at least five times from mainland sources by relatives of eitherC. tigrisorC. hyperythrus, (ii) there have been two separate instances of character relaxation on oceanic islands forC. tigris, and (iii) there has been colonization of the oceanic island Cerralvo with retention of ancestral size forCnemidophorus ceralbensis, a relative ofC. hyperythrus. Finally, the phylogenetic analysis reveals potential cryptic species within mainland populations ofC. tigris.
Hemato-lymphoidin vivoreconstitution potential of subpopulations derived fromin vitrodifferentiated embryonic stem cells
Authors: Potocnik Alexandre J. , Kohler Hubertus , Eichmann Klaus
Published: July 2002

During differentiationin vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+population generated a single transient wave of IgM+IgD+B cells but failed to generate T cells. In contrast, transfer of the B220−fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated duringin vitrodifferentiation of ES cells and that both subsets can undergo complete final maturationin vivo.
Experimental evidence for a behavior-mediated trophic cascade in a terrestrial food chain
Authors: Beckerman Andrew P. , Uriarte Maria , Schmitz Oswald J.
Published: July 2002

Predators of herbivorous animals can affect plant populations by altering herbivore density, behavior, or both. To test whether the indirect effect of predators on plants arises from density or behavioral responses in a herbivore population, we experimentally examined the dynamics of terrestrial food chains comprised of old field plants, leaf-chewing grasshoppers, and spider predators in Northeast Connecticut. To separate the effects of predators on herbivore density from the effects on herbivore behavior, we created two classes of spiders: (i) risk spiders that had their feeding mouth parts glued to render them incapable of killing prey and (ii) predator spiders that remained unmanipulated. We found that the effect of predators on plants resulted from predator-induced changes in herbivore behavior (shifts in activity time and diet selection) rather than from predator-induced changes in grasshopper density. Neither predator nor risk spiders had a significant effect on grasshopper density relative to a control. This demonstrates that the behavioral response of prey to predators can have a strong impact on the dynamics of terrestrial food chains. The results make a compelling case to examine behavioral as well as density effects in theoretical and empirical research on food chain dynamics.
Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly
Authors: Feder Jeffrey L. , Roethele Joseph B. , Wlazlo Brian , Berlocher Stewart H.
Published: July 2002

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (
Malus pumila
)- and hawthorn (
Crataegus
spp.)-infesting races of
Rhagoletis pomonella
(Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect’s life cycle with the phenology of its host plant.
The role of
Wolbachia
bacteria in reproductive incompatibilities and hybrid zones of
Diabrotica
beetles and
Gryllus
crickets
Authors: Giordano Rosanna , Jackson Jan J. , Robertson Hugh M.
Published: July 2002

A rickettsial bacterium in the genus
Wolbachia
is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm,
Diabrotica virgifera virgifera
, and the Mexican corn rootworm,
D. v. zeae
. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of
D. v. virgifera
, with the exception of two populations in southern Arizona, are infected with a strain of
Wolbachia
. Populations of
D. v. zeae
are not infected. Treatment of
D. v. virgifera
with tetracycline eliminated the
Wolbachia
and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus
Gryllus. Gryllus assimilis
,
G. integer
,
G. ovisopis
,
G. pennsylvanicus
, and
G. rubens
are infected with
Wolbachia
whereas
G. firmus
is usually not. Populations of
G. rubens
and
G. ovisopis
carry the same
Wolbachia
strain, which is distinct from that of
G. integer. G. pennsylvanicus
is infected with two
Wolbachia
strains, that found in
G. rubens
and one unique to
G. pennsylvanicus
. Moreover, a proportion of
G. pennsylvanicus
individuals harbors both strains.
Wolbachia
may have influenced speciation in some members of the genus
Gryllus
by affecting the degree of hybridization between species. Given that
Wolbachia
infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with
Wolbachia
.
Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization
Authors: Finnemann Silvia C. , Bonilha Vera L. , Marmorstein Alan D. , Rodriguez-Boulan Enrique
Published: July 2002

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localizedin vivoto the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assessin vitrouptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.
Multiple pathways for SOS-induced mutagenesis in
Escherichia coli
: An overexpression of
dinB
/
dinP
results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA
Authors: Kim Su-Ryang , Maenhaut-Michel Geneviéve , Yamada Masami , Yamamoto Yoshihiro , Matsui Keiko , Sofuni Toshio , Nohmi Takehiko , Ohmori Haruo
Published: July 2002

dinP
is an
Escherichia coli
gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the
E. coli
UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to
dinB
, an SOS gene previously localized near the
lac
locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed
dinP
null mutant exhibited the same defect for λUTM as observed previously with a
dinB
::Mu mutant, and the defect was complemented by plasmids carrying
dinP
as the only intact bacterial gene. Furthermore, merely increasing the
dinP
gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′
lac
plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on
recA
,
uvrA
, or
umuDC
. Thus, our results establish that
E. coli
has at least two distinct pathways for SOS-induced mutagenesis: one dependent on
umuDC
and the other on
dinB
/
P
.
Drosophila
Myc is oncogenic in mammalian cells and plays a role in the
diminutive
phenotype
Authors: Schreiber-Agus Nicole , Stein David , Chen Ken , Goltz Jason S. , Stevens Leslie , DePinho Ronald A.
Published: July 2002

Biochemical and biological activities of Myc oncoproteins are highly dependent upon their association with another basic region helix–loop–helix/leucine zipper (bHLH/LZ) protein, Max. Our previous observation that the DNA-binding/dimerization region of Max is absolutely conserved throughout vertebrate evolution provided the basis for a yeast two-hybrid interaction screen that led to the isolation of the
Drosophila
Myc (dMyc1) protein. Structural conservation in regions of known functional significance is consistent with the ability of dMyc1 to interact with vertebrate Max, to transactivate gene expression in yeast cells, and to cooperate with activated H-RAS to effect the malignant transformation of primary mammalian cells. The ability of
P
-element-mediated ectopic expression of
dmyc1
to reverse a subset of the phenotypic alterations associated with the
diminutive
mutation suggests that
diminutive
may correspond to
dmyc1
. This finding, along with the localization of
dmyc1
expression to zones of high proliferative activity in the embryo, implicates dMyc1 as an integral regulator of
Drosophila
growth and development.
Gα
o
is necessary for muscarinic regulation of Ca
2+
channels in mouse heart
Authors: Valenzuela D. , Han X. , Mende U. , Fankhauser C. , Mashimo H. , Huang P. , Pfeffer J. , Neer E. J. , Fishman M. C.
Published: July 2002

Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The Gα
o
protein is the most abundant Gα subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca
2+
channels in GH
3
cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of Gα
o
, we have generated mice lacking both isoforms of Gα
o
by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that Gα
o
makes up 0.2–0.5% of brain particulate protein and 10% of the growth cone membrane. The Gα
o
−/− mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, Gα
o
−/− mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K
+
channels is normal, as is the electrocardiogram. The levels of other Gα subunits (Gα
s
, Gα
q
, and Gα
i
) are unchanged in the hearts of Gα
o
−/− mice, but the amount of Gβγ is decreased. Whichever subunit, Gα
o
or Gβγ, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca
2+
channels requires coupling of the muscarinic receptor to Gα
o
. Other cardiac Gα subunits cannot substitute.
The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo
Authors: Lim Lorena , Zhou Heping , Costa Robert H.
Published: July 2002

Mammalian hepatocyte nuclear factor-3 (HNF-3) and the
Drosophila
homeotic gene
fork head
proteins are prototypes of an extensive family of cell-specific
transcription factors that share homology in the winged helix
DNA-binding domain. One of these mammalian family members,
HNF-3/
fork head
homolog-4 (HFH-4), was isolated by PCR
amplification of rodent brain cDNA and exhibits abundant expression in
the adult bronchiolar epithelium. In this study, we performed
in
situ
hybridization of stage-specific mouse embryos and report
on a novel expression pattern of the HFH-4 gene in both the presumptive
and differentiated choroid plexus epithelium, which is responsible for
the synthesis and secretion of cerebrospinal fluid (CSF) proteins. We
also showed that HFH-4 is a potent transcriptional activator in
cotransfection assays and defined several protein sequences important
for HFH-4 transcriptional activity. We used
in vitro
DNA-binding site selection with recombinant HFH-4 protein and
determined that the HFH-4 protein recognizes the DNA consensus
sequences HWDTGTTTGTTTA or KTTTGTTGTTKTW (where H is not G, W is A or
T, D is not C, and K is G or T). We used this HFH-4 consensus to
identify potential HFH-4 target genes in the choroid plexus epithelium
and demonstrated that these promoter sequences bind to recombinant
HFH-4 protein in electrophoretic mobility shift assays. Recombinant
HFH-4 formed specific protein–DNA complexes with the promoter regions
of the human prothrombin, beta amyloid precursor protein,
α1-antichymotrypsin, cystic fibrosis transmembrane
conductance regulator and rodent α2-macroglobulin, growth hormone
receptors, and insulin-like growth factor II genes. Furthermore, we
identified putative HFH-4 target genes in the bronchiolar epithelium
including the clara cell secretory protein gene and the HNF-3α gene,
a winged helix family member involved in the transcriptional regulation
of genes in the bronchiolar epithelium. In support of these binding
studies, cotransfection assays show that HFH-4 potentiates expression
of the HNF-3α and clara cell secretory protein promoter regions.
The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase
Authors: Huibregtse Jon M. , Yang Joyce C. , Beaudenon Sylvie L.
Published: July 2002

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential
hect
(homologous to E6-AP carboxyl terminus) E3 of
Saccharomyces cerevisiae
. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1)
in vitro
. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of
RSP5
expression
in vivo
led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for
hect
E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal
hect
domain catalyzes ubiquitination of bound substrates.
Dissection of a circadian oscillation into discrete domains
Authors: Merrow Martha W. , Garceau Norman Y. , Dunlap Jay C.
Published: July 2002

The circadian oscillator in
Neurospora
is a negative feedback loop involving as principal players the products of the
frequency
(
frq
) locus.
frq
encodes multiple forms of its protein product FRQ, which act to depress the amounts of
frq
transcript. In this scheme there are two discrete and separable steps to the circadian cycle, negative feedback itself (repression) in which FRQ acts to decrease the levels of its own transcript, and recovery from repression (derepression) in which
frq
transcript levels return to peak amounts. By introducing an exogenously regulatable
frq
transgene into a
frq
loss-of-function strain (
frq
9
), we created an artificial system in which the two separate steps in the circadian cycle can be initiated and followed separately for purposes of observing their kinetics. Under these conditions the
frq
-FRQ cycle occupies the time scale of a full circadian cycle. During this time, the process of negative feedback of FRQ on
frq
transcript levels is rapid and efficient; it requires only 3 to 6 h and can be mediated by on the order of 10 molecules of FRQ per nucleus, a level even less than that seen in the normal oscillation. In contrast, recovery from negative feedback requires 14 to 18 h, most of the circadian cycle, during which time
de novo
FRQ synthesis has stopped, and existing FRQ is progressively posttranslationally modified. Altogether the time required to complete both of these steps is in good agreement with the 22-h observed period length of the normal circadian cycle.
Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage Φ6
Authors: Qiao Xueying , Qiao Jian , Mindich Leonard
Published: July 2002

A model that explains the stoichiometric packaging of the chromosomes of Φ6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.
A role for heterodimerization in nuclear localization of a homeodomain protein
Authors: Spit Anthony , Hyland Robert H. , Mellor E. Jane C. , Casselton Lorna A.
Published: July 2002

TheAmating type genes of the mushroomCoprinus cinereusencode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterialuidA(β-glucuronidase) gene and showing that β-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.
Cerebral cortical hyperactivation in response to mental stress in patients with coronary artery disease
Authors: Soufer Robert , Bremner J. Douglas , Arrighi James A. , Cohen Ira , Zaret Barry L. , Burg Matthew M. , Goldman-Rakic Patricia
Published: July 2002

The central nervous system (CNS) effects of mental stress in patients with coronary artery disease (CAD) are unexplored. The present study used positron emission tomography (PET) to measure brain correlates of mental stress induced by an arithmetic serial subtraction task in CAD and healthy subjects. Mental stress resulted in hyperactivation in CAD patients compared with healthy subjects in several brain areas including the left parietal cortex [angular gyrus/parallel sulcus (area 39)], left anterior cingulate (area 32), right visual association cortex (area 18), left fusiform gyrus, and cerebellum. These same regions were activated within the CAD patient group during mental stress versus control conditions. In the group of healthy subjects, activation was significant only in the left inferior frontal gyrus during mental stress compared with counting control. Decreases in blood flow also were produced by mental stress in CAD versus healthy subjects in right thalamus (lateral dorsal, lateral posterior), right superior frontal gyrus (areas 32, 24, and 10), and right middle temporal gyrus (area 21) (in the region of the auditory association cortex). Of particular interest, a subgroup of CAD patients that developed painless myocardial ischemia during mental stress had hyperactivation in the left hippocampus and inferior parietal lobule (area 40), left middle (area 10) and superior frontal gyrus (area 8), temporal pole, and visual association cortex (area 18), and a concomitant decrease in activation observed in the anterior cingulate bilaterally, right middle and superior frontal gyri, and right visual association cortex (area 18) compared with CAD patients without myocardial ischemia. These findings demonstrate an exaggerated cerebral cortical response and exaggerated asymmetry to mental stress in individuals with CAD.
Interleukin 10 secretion in relation to human IL-10 locus haplotypes
Authors: Eskdale Joyce , Gallagher Grant , Verweij Cor L. , Keijsers Vivian , Westendorp Rudi G. J. , Huizinga Tom W. J.
Published: July 2002

Stimulation of human blood cultures with bacterial lipopolysaccharide (LPS) shows large inter-individual variation in interleukin 10 (IL-10) secretion, which has been shown to have a genetic component of over 70%. Alleles at two microsatellite loci in the 4 kb immediately upstream of the human IL-10 transcription initiation site in 132 individuals from 56 Dutch families were defined and assigned as haplotypes. LPS-induced IL-10 secretion was measured by ELISA and related to the IL-10 promoter haplotypes present in 78 unrelated individuals obtained from these families. Analysis showed that LPS-induced IL-10 secretion from unrelated individuals varied with IL-10 promoter haplotypes (P= 0.024; Kruskal-Wallis test). Two observations were made in relation to secreted IL-10 levels and promoter haplotypes; first, those haplotypes containing the allele IL10.R3 were associated with lower IL-10 secretion than haplotypes containing any other IL10.R allele. Second, the haplotype IL10.R2/IL10.G14 was associated with highest IL-10 secretion overall, whereas the haplotype IL10.R3/IL10.G7 was associated with lowest IL-10 secretion. These data demonstrate that the ability to secrete IL-10 can vary in man according to the genetic composition of the IL-10 locus.
Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant
Authors: Williamson Toni L. , Bruijn Lucie I. , Zhu Qinzhang , Anderson Karen L. , Anderson Scott D. , Julien Jean-Pierre , Cleveland Don W.
Published: July 2002

Mutations in superoxide dismutase 1 (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), provoke disease through an unidentified toxic property. Neurofilament aggregates are pathologic hallmarks of both sporadic and SOD1-mediated familial ALS. By deleting NF-L, the major neurofilament subunit required for filament assembly, onset and progression of disease caused by familial ALS-linked SOD1 mutant G85R are significantly slowed, while selectivity of mutant-mediated toxicity for motor neurons is reduced. In NF-L-deleted animals, levels of the two remaining neurofilament subunits, NF-M and NF-H, are markedly reduced in axons but are elevated in motor neuron cell bodies. Thus, while neither perikaryal nor axonal neurofilaments are essential for SOD1-mediated disease, the absence of assembled neurofilaments both diminishes selective vulnerability and slows SOD1
G85R
mutant-mediated toxicity to motor neurons.
Restorer genes for different forms of
Brassica
cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several mitochondrial genes
Authors: Li Xiu-Qing , Jean Martine , Landry Benoit S. , Brown Gregory G.
Published: July 2002

The oilseed rape plant,
Brassica napus
, possesses two endogenous male sterile cytoplasms,
nap
and
pol
. Previous studies have shown that nuclear restoration of
pol
cytoplasmic male sterility (CMS) is conditioned by a gene,
Rfp
, that is also involved in modifying transcripts of the
pol
CMS-associated
orf224/atp6
mtDNA region. We now find that the
nap
nuclear restorer gene
Rfn
apparently is identical to
Mmt
, a gene that conditions the modification of transcripts from several different mtDNA regions, including one that is associated with
nap
CMS and contains
orf222
, a chimeric gene related to
orf224. Mmt
, in turn, is found to be allelic to
Rfp
, suggesting that restorer genes for the two cytoplasms represent different alleles or haplotypes of a single nuclear locus. This view is supported by restriction fragment length polymorphism mapping studies that indicate that
Rfn
and
Rfp
map to the same chromosomal position. Thus, in contrast to CMS in other species, different forms of Brassica CMS are restored by alleles of a single nuclear locus, and the restoration properties of these alleles reflect their involvement in the modification of transcripts of corresponding CMS-associated mtDNA regions. A survey of 51 varieties from 8
Brassica
and
Sinapis
species failed to find evidence of
Rfn(Mmt)
in other than fertility-restored,
nap
cytoplasm
B. napus.
This suggests that
Rfn(Mmt)
arose in Brassica with
nap
cytoplasm and that the necessity for fertility restoration may have provided the selective pressure for its origin and maintenance.
Morphine-activated opioid receptors elude desensitization by β-arrestin
Authors: Whistler Jennifer L. , von Zastrow Mark
Published: July 2002

μ opioid receptors are targets of native opioid peptides and addictive analgesic drugs. A major clinical liability of opiate drugs is their ability to cause physiological tolerance. Individual opiates, such as morphine and etorphine, differ both in their ability to promote physiological tolerance and in their effects on receptor regulation by endocytosis. Here, we demonstrate that arrestins play a fundamental role in mediating this agonist-selective regulation and that morphine-activated μ receptors fail to undergo arrestin-dependent uncoupling from cognate G proteins. Thus, highly addictive opiate drugs elude a fundamental mode of physiological regulation that is mediated by agonist-specific interaction of opioid receptors with arrestins.
Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases
Authors: Sengupta Tapas K. , Talbot Erika S. , Scherle Peggy A. , Ivashkiv Lionel B.
Published: July 2002

Gene activation and cellular differentiation induced by interleukin-6 (IL-6) and transcription factor Stat3 are suppressed by several factors, including ionomycin, granulocyte/macrophage-colony-stimulating factor, and phorbol 12-myristate 13-acetate (PMA), that block IL-6-induced Stat3 activation. These inhibitory agents activate mitogen activated protein kinases (MAPKs), and thus the role of MAPKs in the mechanism of inhibition of Stat3 activation was investigated. Inhibition of IL-6-induced Stat3 activation by PMA and ionomycin was rapid (within 5 min) and did not require new RNA or protein synthesis. Inhibition of Stat3 DNA-binding activity and tyrosine phosphorylation by PMA, ionomycin, and granulocyte/macrophage-colony-stimulating factor was reversed when activation of the extracellular signal-regulated kinase (ERK) group of MAPKs was blocked by using specific kinase inhibitors. Expression of constitutively active MEK1, the kinase that activates ERKs, or overexpression of ERK2, but not JNK1, inhibited Stat3 activation. Inhibition of Stat3 correlated with suppression of IL-6-induction of a signal transducer and activator of transcription (STAT)-dependent reporter gene. In contrast to IL-6, activation of Stat3 by interferon-α was not inhibited. MEKs and ERKs inhibited IL-6 activation of Stat3 harboring a mutation at serine-727, the major site for serine phosphorylation, similar to inhibition of wild-type Stat3, and inhibited Janus kinases Jak1 and Jak2 upstream of Stat3 in the Jak-STAT-signaling pathway. These results demonstrate an ERK-mediated mechanism for inhibiting IL-6-induced Jak-STAT signaling that is rapid and inducible, and thus differs from previously described mechanisms for downmodulation of the Jak-STAT pathway. This inhibitory pathway provides a molecular mechanism for the antagonism of Stat3-mediated IL-6 activity by factors that activate ERKs.