bluuebunny/crossref_metadata_2025 huggingface dataset:
Bradykinin inhibits M current via phospholipase C and Ca
2+
release from IP
3
-sensitive Ca
2+
stores in rat sympathetic neurons
Authors: Cruzblanca Humberto , Koh Duk-Su , Hille Bertil
Published: July 2002

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B
2
bradykinin and M
1
muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP
3
receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP
3
to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca
2+
]
i
rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca
2+
]
i
were blocked by depletion of Ca
2+
internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca
2+
from IP
3
-sensitive Ca
2+
stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.
Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix
Authors: Slack Barbara E.
Published: July 2002

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine–glycine–aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine–glycine–glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell’s interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.
Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons
Authors: Ito Koichi , Uno Makiko , Nakamura Yoshikazu
Published: July 2002

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG/UAA and UGA/UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of
Escherichia coli
and the viability of a chromosomal RF1/RF2 double knockout. In both
in vivo
and
in vitro
polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.
Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane
Authors: Khan Islam U. , Wallin Reidar , Gupta Radhey S. , Kammer Gary M.
Published: July 2002

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.
Effect of vitamin A supplementation on rhodopsin mutants threonine-17 → methionine and proline-347 → serine in transgenic mice and in cell cultures
Authors: Li Tiansen , Sandberg Michael A. , Pawlyk Basil S. , Rosner Bernard , Hayes K. C. , Dryja Thaddeus P. , Berson Eliot L.
Published: July 2002

A therapeutic effect of vitamin A supplementation on the course of photoreceptor degeneration, previously reported for patients with retinitis pigmentosa, was tested in two transgenic mouse models of this disease, each carrying a dominant rhodopsin mutation. The threonine-17 → methionine (T17M) mutation is a class II rhodopsin mutation, characterized by a thermal instability/folding defect and minimal regeneration with the chromophore. The proline-347 → serine (P347S) mutation belongs to class I, comprised of a smaller number of mutations that exhibit no recognized biochemical abnormality
in vitro
. In the present study, each of the two mouse models was fed a diet containing 2.5 mg of vitamin A palmitate (control) or 102.5 mg of vitamin A palmitate (high vitamin A) per kilogram of diet. Dark-adapted, full-field electroretinograms showed that the high vitamin A diet significantly reduced the rate of decline of a-wave and b-wave amplitudes in the T17M mice but had no significant effect on the decline of electroretinogram amplitude in the P347S mice. Correspondingly, histologic evaluation revealed that the treatment was associated with significantly longer photoreceptor inner and outer segments and a thicker outer nuclear layer in the T17M mice but had no effect on photoreceptor morphology in the P347S mice. In a separate series of experiments, the instability defect of the T17M mutant opsin expressed
in vitro
was partially alleviated by inclusion of 11-
cis
-retinal in the culture media. These results show that vitamin A supplementation slows the rate of photoreceptor degeneration caused by a class II rhodopsin mutation. Vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of the chromophore.
Identification of a human nuclear receptor defines a new signaling pathway for
CYP
3
A induction
Authors: Bertilsson Göran , Heidrich Jessica , Svensson Kristian , Åsman Michael , Jendeberg Lena , Sydow-Bäckman Mona , Ohlsson Rolf , Postlind Hans , Blomquist Patrik , Berkenstam Anders
Published: July 2002

Nuclear receptors regulate metabolic pathways in response to changes in the environment by appropriate alterations in gene expression of key metabolic enzymes. Here, a computational search approach based on iteratively built hidden Markov models of nuclear receptors was used to identify a human nuclear receptor, termed hPAR, that is expressed in liver and intestines. hPAR was found to be efficiently activated by pregnanes and by clinically used drugs including rifampicin, an antibiotic known to selectively induce human but not murine
CYP3A
expression. The
CYP3A
drug-metabolizing enzymes are expressed in gut and liver in response to environmental chemicals and clinically used drugs. Interestingly, hPAR is not activated by pregnenolone 16α-carbonitrile, which is a potent inducer of murine
CYP3A
genes and an activator of the mouse receptor PXR.1. Furthermore, hPAR was found to bind to and trans-activate through a conserved regulatory sequence present in human but not murine
CYP3A
genes. These results provide evidence that hPAR and PXR.1 may represent orthologous genes from different species that have evolved to regulate overlapping target genes in response to pharmacologically distinct
CYP3A
activators, and have potential implications for the
in vitro
identification of drug interactions important to humans.
The
Saccharomyces cerevisiae MLH
3
gene functions in MSH3-dependent suppression of frameshift mutations
Authors: Flores-Rozas Hernan , Kolodner Richard D.
Published: July 2002

The
Saccharomyces cerevisiae
genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in
MLH3
increased the rate of reversion of the
hom3–10
allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in
MLH3
and
MSH6
caused a synergistic increase in the
hom3–10
reversion rate, whereas the
hom3–10
reversion rate in an
mlh3 msh3
double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the
LYS2
gene was analyzed, although mutation of
MLH3
did not cause the same extent of affect at every
LYS2
frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.
Overexpression of DNA polymerase β in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs
Authors: Canitrot Yvan , Cazaux Christophe , Fréchet Mathilde , Bouayadi Khalil , Lesca Claire , Salles Bernard , Hoffmann Jean-Sébastien
Published: July 2002

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested
in vitro
. Here, we show that cells overexpressing
pol
β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.
Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays
Authors: Steger David J. , Eberharter Anton , John Sam , Grant Patrick A. , Workman Jerry L.
Published: July 2002

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription
in vitro
. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.
Explosive invasion of plant mitochondria by a group I intron
Authors: Cho Yangrae , Qiu Yin-Long , Kuhlman Peter , Palmer Jeffrey D.
Published: July 2002

Group I introns are mobile, self-splicing genetic elements found principally in organellar genomes and nuclear rRNA genes. The only group I intron known from mitochondrial genomes of vascular plants is located in the
cox1
gene of
Peperomia
, where it is thought to have been recently acquired by lateral transfer from a fungal donor. Southern-blot surveys of 335 diverse genera of land plants now show that this intron is in fact widespread among angiosperm
cox1
genes, but with an exceptionally patchy phylogenetic distribution. Four lines of evidence—the intron’s highly disjunct distribution, many incongruencies between intron and organismal phylogenies, and two sources of evidence from exonic coconversion tracts—lead us to conclude that the 48 angiosperm genera found to contain this
cox1
intron acquired it by 32 separate horizontal transfer events. Extrapolating to the over 13,500 genera of angiosperms, we estimate that this intron has invaded
cox1
genes by cross-species horizontal transfer over 1,000 times during angiosperm evolution. This massive wave of lateral transfers is of entirely recent occurrence, perhaps triggered by some key shift in the intron’s invasiveness within angiosperms.
The cancer growth suppressor gene
mda-
7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice
Authors: Su Zao-zhong , Madireddi Malavi T. , Lin Jiao Jiao , Young Charles S. H. , Kitada Shinichi , Reed John C. , Goldstein Neil I. , Fisher Paul B.
Published: July 2002

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (
mda-
7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of
mda-
7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which
mda-
7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of
mda-
7 on human breast tumor formation
in vivo
in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing
mda-
7, Ad.
mda-
7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.
mda-
7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of
mda-
7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that
mda-
7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects,
mda-
7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.
Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting
Authors: Gonzalez-Cadavid Nestor F. , Taylor Wayne E. , Yarasheski Kevin , Sinha-Hikim Indrani , Ma Kun , Ezzat Shereen , Shen Ruoqing , Lalani Rukhsana , Asa Sylvia , Mamita Mohamad , Nair Gouri , Arver Stefan , Bhasin Shalender
Published: July 2002

Myostatin, a member of the transforming growth factor-β superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene’s expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.
Electric field-induced critical demixing in lipid bilayer membranes
Authors: Groves Jay T. , Boxer Steven G. , McConnell Harden M.
Published: July 2002

Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75°K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.
The yeast nascent polypeptide-associated complex initiates protein targeting to mitochondria
in vivo
Authors: George Rebecca , Beddoe Travis , Landl Karina , Lithgow Trevor
Published: July 2002

The yeast nascent polypeptide-associated complex (NAC) is encoded by two genes,
EGD1
and
EGD2
, and is associated with cytoplasmic ribosomes. Yeast mutants lacking NAC (Δ
egd2
) are viable but suffer slight defects in the targeting of nascent polypeptides to several locations including the endoplasmic reticulum and mitochondria. If both NAC and Mft52p are missing from yeast cells, inefficient targeting of mitochondrial precursor proteins leads to defects in both mitochondrial function and morphology. We suggest that NAC provides a ribosomal environment for nascent mitochondrial targeting sequences to achieve secondary structure, thereby enhancing the efficiency of protein targeting.
Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator
Authors: Schwiebert Erik M. , Morales Marcelo M. , Devidas Sreenivas , Egan Marie E. , Guggino William B.
Published: July 2002

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl
−
) channel and a regulator of outwardly rectifying Cl
−
channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl
−
channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3–1) or by injection of
in vitro
transcribed complementary RNAs (cRNAs) into
Xenopus
oocytes. Two-electrode voltage clamp recordings,
36
Cl
−
efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl
−
channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted α-helices 5 and 6, forms an essential part of the Cl
−
channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl
−
channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.
Differing roles for zinc fingers in DNA recognition: Structure of a six-finger transcription factor IIIA complex
Authors: Nolte Robert T. , Conlin Rachel M. , Harrison Stephen C. , Brown Raymond S.
Published: July 2002

The crystal structure of the six NH
2
-terminal zinc fingers of
Xenopus laevis
transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 Å resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.
The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice
Authors: Yang Xiao , Li Cuiling , Xu Xiaoling , Deng Chuxia
Published: July 2002

Members of the transforming growth factor (TGF)-β superfamily have been shown to play a variety of important roles in embryogenesis, including dorsal and ventral mesoderm induction. The tumor suppressor SMAD4, also known as DPC4, is believed to be an essential factor that mediates TGF-β signals. To explore functions of SMAD4 in development, we have mutated it by truncating its functional C-domain. We show that
Smad4
is expressed ubiquitously during murine embryogenesis. Mice heterozygous for the
Smad4
ex8/+
mutation are developmentally normal, whereas homozygotes die between embryonic day 6.5 (E6.5) and 8.5. All
Smad4
ex8/ex8
mutants are developmentally delayed at E6 and show little or no elongation in the extraembryonic portion of late egg cylinder stage embryos. Consistent with this, cultured
Smad4
ex8/ex8
blastocyst outgrowths suffer cellular proliferation defects and fail to undergo endoderm differentiation. Although a portion of mutant embryos at E8.5 show an increase in the embryonic ectoderm and endoderm, morphological and molecular analyses indicate that they do not form mesoderm. Altogether, these data demonstrate that SMAD4-mediated signals are required for epiblast proliferation, egg cylinder formation, and mesoderm induction.
Cooperation between the activin and Wnt pathways in the spatial control of organizer gene expression
Authors: Crease Devanand J. , Dyson Steven , Gurdon J. B.
Published: July 2002

The normal expression pattern of the Wnt responsive homeobox gene
Siamois
is restricted to the dorso-vegetal region of the
Xenopus
embryo. Because the Wnt signaling pathway (via β-catenin) is active on the entire dorsal side of the early embryo, we have asked why
Siamois
expression is not seen in the dorsal ectoderm. Only Wnt signaling, via activation of β-catenin, can induce directly
Siamois
, and signaling via the SMAD1 (BMP2/4) or SMAD2 (activin/Vg-1) pathways cannot. We now directly show that the SMAD2 pathway can cooperate with the Wnt pathway to induce expression of
Siamois
much more strongly than the Wnt pathway alone, in normal embryos. We demonstrate the significance of this cooperation in normal embryos by blocking the SMAD2 signaling pathway with a dominant negative activin receptor. The activin dominant negative receptor blocks this cooperative effect and reduces the expression of
Siamois
by threefold in early embryos. Furthermore, we find that this cooperative relationship between the SMAD2 and Wnt pathways is reciprocal. Thus, in normal embryos, the Wnt pathway can enhance induction, by the SMAD 2 pathway, of the organizer genes
Gsc
and
Chd
but not the pan-mesodermal marker genes
Xbra
and
Eomes
. We conclude that the Wnt and SMAD2 signaling pathways cooperate to induce the expression of Spemann-organizer specific genes and so help to localize their spatial expression.
Bipolar localization of
Bacillus subtilis
topoisomerase IV, an enzyme required for chromosome segregation
Authors: Huang Wai Mun , Libbey Jane L. , van der Hoeven Petra , Yu Shelly Xiaohong
Published: July 2002

In
Bacillus subtilis
,
parE
and
parC
were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.
Collaborative role of E2F transcriptional activity and G
1
cyclindependent kinase activity in the induction of S phase
Authors: Leone Gustavo , DeGregori James , Jakoi Laszlo , Cook Jeanette G. , Nevins Joseph R.
Published: July 2002

A considerable body of evidence points to a role for both cyclin E/cyclin-dependent kinase (cdk)2 activity and E2F transcription activity in the induction of S phase. We show that overexpression of cyclin E/cdk2 in quiescent cells induces S phase, that this coincides with an induction of E2F activity, and that coexpression of E2F enhances the cyclin E/cdk2-mediated induction of S phase. Likewise, E2F overexpression can induce S phase and does so in the apparent absence of cyclin E/cdk2 activity. In addition, although the inhibition of cyclin E/cdk2 activity blocks the induction of S phase after growth stimulation of normal mouse embryo fibroblasts, inhibition of cyclin E/cdk2 does not block S phase induction in Rb−/− cells where E2F activity is deregulated. These results point to the important roles for E2F and cyclin E/cdk2 in the induction of S phase. Moreover, the nature of the E2F targets and the suspected targets for cyclin E/cdk2 suggests a potential molecular mechanism for the collaborative action of cyclin E/cdk2 and E2F in the induction of S phase.
The kinetoplast structure-specific endonuclease I is related to the 5′ exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the kinetoplast topoisomerase II and DNA polymerase β during replication
Authors: Engel Michele L. , Ray Dan S.
Published: July 2002

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid
Crithidia fasciculata
has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5′ exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase β at antipodal sites flanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase β [Johnson, C. E. & Englund, P. T. (1998)
J. Cell Biol.
143, 911–919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during kinetoplast replication. Together, these results suggest a point of control for kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.
Evolution of a light-harvesting protein by addition of new subunits and rearrangement of conserved elements: Crystal structure of a cryptophyte phycoerythrin at 1.63-Å resolution
Authors: Wilk Krystyna E. , Harrop Stephen J. , Jankova Lucy , Edler Diana , Keenan Gary , Sharples Francis , Hiller Roger G. , Curmi Paul M. G.
Published: July 2002

Cryptophytes are unicellular photosynthetic algae that use a lumenally located light-harvesting system, which is distinct from the phycobilisome structure found in cyanobacteria and red algae. One of the key components of this system is water-soluble phycoerythrin (PE) 545 whose expression is enhanced by low light levels. The crystal structure of the heterodimeric α
1
α
2
ββ PE 545 from the marine cryptophyte
Rhodomonas CS24
has been determined at 1.63-Å resolution. Although the β-chain structure is similar to the α and β chains of other known phycobiliproteins, the overall structure of PE 545 is novel with the α chains forming a simple extended fold with an antiparallel β-ribbon followed by an α-helix. The two doubly linked β50/β61 chromophores (one on each β subunit) are in van der Waals contact, suggesting that exciton-coupling mechanisms may alter their spectral properties. Each α subunit carries a covalently linked 15,16-dihydrobiliverdin chromophore that is likely to be the final energy acceptor. The architecture of the heterodimer suggests that PE 545 may dock to an acceptor protein via a deep cleft and that energy may be transferred via this intermediary protein to the reaction center.
c-
fos
-induced growth factor/vascular endothelial growth factor D induces angiogenesis
in vivo
and
in vitro
Authors: Marconcini Lucia , Marchiò Serena , Morbidelli Lucia , Cartocci Elena , Albini Adriana , Ziche Marina , Bussolino Federico , Oliviero Salvatore
Published: July 2002

c-
fos
-induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea
in vivo
in a dose-dependent manner.
In vitro
Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi’s sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that
Figf/Vegf-D
expression is under the control of the nuclear oncogene c-
fos
, our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion.
Serum choline activates mutant acetylcholine receptors that cause slow channel congenital myasthenic syndromes
Authors: Zhou Ming , Engel Andrew G. , Auerbach Anthony
Published: July 2002

We have found that mutant acetylcholine receptor channels (AChRs) that cause slow-channel congenital myasthenic syndromes are activated by serum and that the high frequency of openings in serum is reduced by treatment with choline oxidase. Thus, slow-channel congenital myasthenic syndrome AChRs at the neuromuscular junction are likely to be activated both by steady exposure to serum choline and by transient exposure to synaptically released transmitter. Single-channel kinetic analyses indicate that the increased response to choline is caused by a reduced intrinsic stability of the closed channel. The results suggest that a mutation that destabilizes the inactive conformation of the AChR, together with the sustained exposure of endplates to serum choline, results in continuous channel activity that contributes to the pathophysiology of the disease.
Evolutionary demographic models for mortality plateaus
Authors: Wachter Kenneth W.
Published: July 2002

Plateaus in the age pattern of hazard functions at extreme ages have been discovered in large populations of medflies,
Drosophila
, nematodes, and people. Mueller and Rose [(1996)
Proc. Natl. Acad. Sci. USA
93, 15249–15253] have proposed several age-structured demographic models to represent effects of mutation accumulation and antagonistic pleiotropy on randomly evolving schedules of demographic rates. They assert that “evolutionary theory [as embodied in their models] predicts late-life mortality plateaus.” This paper defines a class of Markovian models that includes those of Mueller and Rose and obtains a characterization of the possible limiting states. For the basic model, the result implies that schedules with late-life mortality plateaus above a minimal threshold are not limiting states. The models fail, but not for reasons previously conjectured. Transient states, visited early by the process, do display mortality plateaus. Other models from this class may have a role to play in reconciling observed plateaus with evolutionary theory.
Linkage disequilibrium test implies a large effective population number for HIV
in vivo
Authors: Rouzine I. M. , Coffin J. M.
Published: July 2002

The effective size of the HIV population
in vivo
, although critically important for the prediction of appearance of drug-resistant variants, is currently unknown. To address this issue, we have developed a simple virus population model, within which the relative importance of stochastic factors and purifying selection for genetic evolution differs over, at least, three broad intervals of the effective population size, with approximate boundaries given by the inverse selection coefficient and the inverse mutation rate per base per cycle. Random drift and selection dominate the smallest (stochastic) and largest (deterministic) population intervals, respectively. In the intermediate (selection–drift) interval, random drift controls weakly diverse populations, whereas strongly diverse populations are controlled by selection. To estimate the effective size of the HIV population
in vivo
, we tested 200
pro
sequences isolated from 11 HIV-infected patients for the presence of a linkage disequilibrium effect which must exist only in small populations. This analysis demonstrated a steady-state virus population of 10
5
infected cells or more, which is either in or at the border of the deterministic regime with respect to evolution of separate bases.
Thermodynamics of T cell receptor binding to peptide–MHC: Evidence for a general mechanism of molecular scanning
Authors: Boniface J. Jay , Reich Ziv , Lyons Daniel S. , Davis Mark M.
Published: July 2002

Antigen-dependent activation of T lymphocytes requires T cell receptor (TCR)-mediated recognition of specific peptides, together with the MHC molecules to which they are bound. To achieve this recognition in a reasonable time frame, the TCR must scan and discriminate rapidly between thousands of MHC molecules differing from each other only in their bound peptides. Kinetic analysis of the interaction between a TCR and its cognate peptide–MHC complex indicates that both association and dissociation depend heavily on the temperature, indicating the presence of large energy barriers in both phases. Thermodynamic analysis reveals changes in heat capacity and entropy that are characteristic of protein–ligand associations in which local folding is coupled to binding. Such an “induced-fit” mechanism is characteristic of sequence-specific DNA-binding proteins that must also recognize specific ligands in the presence of a high background of competing elements. Here, we propose that induced fit may endow the TCR with its requisite discriminatory capacity and suggest a model whereby the loosely structured antigen-binding loops of the TCR rapidly explore peptide–MHC complexes on the cell surface until some critical structural complementarity is achieved through localized folding transitions. We further suggest that conformational changes, implicit in this model, may also propagate beyond the TCR antigen-binding site and directly affect self-association of ligated TCRs or TCR–CD3 interactions required for signaling.
All males are not created equal: Fertility differences depend on gamete recognition polymorphisms in sea urchins
Authors: Palumbi Stephen R.
Published: July 2002

Behaviors, morphologies, and genetic loci directly involved in reproduction have been increasingly shown to be polymorphic within populations. Explaining how such variants are maintained by selection is crucial to understanding the genetic basis of fertility differences, but direct tests of how alleles at reproductive loci affect fertility are rare. In the sea urchin genus
Echinometra
, the protein bindin mediates sperm attachment to eggs, evolves quickly, and is polymorphic within species. Eggs exposed to experimental sperm mixtures show strong discrimination on the basis of the males’ bindin genotype. Different females produce eggs that nonrandomly select sperm from different males, showing that variable egg–sperm interactions determine fertility. Eggs select sperm with a bindin genotype similar to their own, suggesting strong linkage between female choice and male trait loci. These experiments demonstrate that alleles at a single locus can have a strong effect on fertilization and that reproductive loci may retain functional polymorphisms through epistatic interactions between male and female traits. They also suggest that positive selection at gamete recognition loci like bindin involves strong selection within species on mate choice interactions.
Vocal imitation in zebra finches is inversely related to model abundance
Authors: Tchernichovski Ofer , Lints Thierry , Mitra Partha P. , Nottebohm Fernando
Published: July 2002

A juvenile male zebra finch,Taeniopygia guttata, kept singly with its father develops a fairly complete imitation of the father’s song. The imitation is less complete when other male siblings are present, possibly because as imitation commences, model abundance increases. Here we examine the consequences of allowing more or less access to a song model. Young males heard a brief song playback when they pecked at a key, but different males were allowed to hear different numbers of playbacks per day. Using an automated procedure that scored the similarity between model and pupil songs, we discovered that 40 playbacks of the song motif per day, lasting a total of 30 sec, resulted in a fairly complete imitation. More exposure led to less complete imitation. Vocal imitation often may reflect the interaction of diverse influences. Among these, we should now include the possible inhibitory effect of model overabundance, which may foster individual identity and explain the vocal diversity found in zebra finches and other songbirds.
Compromised disease resistance in saponin-deficient plants
Authors: Papadopoulou K. , Melton R. E. , Leggett M. , Daniels M. J. , Osbourn A. E.
Published: July 2002

Saponins are glycosylated plant secondary metabolites found in many major food crops [Price, K. R., Johnson, I. T. & Fenwick, G. R. (1987)
CRC Crit. Rev. Food Sci. Nutr.
26, 27–133]. Because many saponins have potent antifungal properties and are present in healthy plants in high concentrations, these molecules may act as preformed chemical barriers to fungal attack. The isolation of plant mutants defective in saponin biosynthesis represents a powerful strategy for evaluating the importance of these compounds in plant defense. The oat root saponin avenacin A-1 fluoresces under ultraviolet illumination [Crombie, L., Crombie, W. M. L. & Whiting, D. A. (1986)
J. Chem. Soc. Perkins 1
, 1917–1922], a property that is extremely rare among saponins. Here we have exploited this fluorescence to isolate saponin-deficient (
sad
) mutants of a diploid oat species,
Avena strigosa
. These
sad
mutants are compromised in their resistance to a variety of fungal pathogens, and a number of lines of evidence suggest that this compromised disease resistance is a direct consequence of saponin deficiency. Because saponins are widespread throughout the plant kingdom, this group of secondary metabolites may have general significance as antimicrobial phytoprotectants.
Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction
Authors: Wang Ting-Fang , Kleckner Nancy , Hunter Neil
Published: July 2002

The yeast genome encodes four proteins (Pms1 and Mlh1–3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexesin vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in apms1mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in themlh3mutant is reduced to ≈70% of wild-type levels but correction of heteroduplex is normal. In amlh2mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, themlh1mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.
Impaired motor coordination and Purkinje cell excitability in mice lacking calretinin
Authors: Schiffmann Serge N. , Cheron Guy , Lohof Ann , d’Alcantara Pablo , Meyer Michael , Parmentier Marc , Schurmans Stéphane
Published: July 2002

In the cerebellum, the parallel fiber-Purkinje cell synapse can undergo long-term synaptic plasticity suggested to underlie motor learning and resulting from variations in intracellular calcium concentration ([Ca
2+
]
i
). Ca
2+
binding proteins are enriched in the cerebellum, but their role in information processing is not clear. Here, we show that mice deficient in calretinin (Cr
−/−
) are impaired in tests of motor coordination. An impairment in Ca
2+
homeostasis in Cr
−/−
Purkinje cells was supported by the high Ca
2+
-saturation of calbindin-D28k in these cells. The firing behavior of Purkinje cells is severely affected in Cr
−/−
alert mice, with alterations of simple spike firing rate, complex spike duration, and simple spike pause. In contrast, in slices, transmission at parallel fiber- or climbing fiber-Purkinje cell synapses is unaltered, indicating that marked modifications of the firing behavior
in vivo
can be undetectable in slice. Thus, these results show that calretinin plays a major role at the network level in cerebellar physiology.
DER signaling restricts the boundaries of the wing field during
Drosophila
development
Authors: Baonza Antonio , Roch Fernando , Martín-Blanco Enrique
Published: July 2002

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation.
Drosophila
epidermal growth factor receptor (DER) signaling has many functions in fly development. Here we show that antagonizing DER signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. DER signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, DER signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells.
The multitype zinc-finger protein U-shaped functions in heart cell specification in the
Drosophila
embryo
Authors: Fossett Nancy , Zhang Qian , Gajewski Kathleen , Choi Cheol Yong , Kim Yongsok , Schulz Robert A.
Published: July 2002

Multitype zinc-finger proteins of the Friend of GATA/U-shaped (Ush) class function as transcriptional regulators of gene expression through their modulation of GATA factor activity. To better understand intrinsic properties of these proteins, we investigated the expression and function of the
ush
gene during
Drosophila
embryogenesis.
ush
is dynamically expressed in the embryo, including several cell types present within the mesoderm. The gene is active in the cardiogenic mesoderm, and a loss of function results in an overproduction of both cardial and pericardial cells, indicating a requirement for the gene in the formation of these distinct cardiac cell types. Conversely, ectopic expression of
ush
results in a decrease in the number of cardioblasts in the heart and the inhibition of a cardial cell enhancer normally regulated by the synergistic activity of the Pannier and Tinman cardiogenic factors. These findings suggest that, similar to its known function in thoracic bristle patterning, Ush functions in the control of heart cell specification through its modulation of Pannier transcriptional activity.
ush
is also required for mesodermal cell migration early in embryogenesis, where it shows a genetic interaction with the Heartless fibroblast growth factor receptor gene. Taken together, these results demonstrate a critical role for the Ush transcriptional regulator in several diverse processes of mesoderm differentiation and heart formation.