bluuebunny/crossref_metadata_2025 huggingface dataset:
Three members of a novel small gene-family from
Arabidopsis thaliana
able to complement functionally an
Escherichia coli
mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity
Authors: Gutierrez-Marcos José F. , Roberts Michael A. , Campbell Edward I. , Wray John L.
Published: July 2002

Three different cDNAs, Prh-19, Prh-26, and Prh-43
[3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog],
have been isolated by complementation of an
Escherichia coli
cysH
mutant, defective in PAPS reductase activity, to
prototrophy with an
Arabidopsis thaliana
cDNA library in
the expression vector λYES. Sequence analysis of the cDNAs revealed
continuous open reading frames encoding polypeptides of 465, 458, and
453 amino acids, with calculated molecular masses of 51.3, 50.5, and
50.4 kDa, respectively, that have strong homology with fungal, yeast,
and bacterial PAPS reductases. However, unlike microbial PAPS
reductases, each PRH protein has an N-terminal extension,
characteristic of a plastid transit peptide, and a C-terminal extension
that has amino acid and deduced three-dimensional homology to
thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be
a much more efficient substrate than PAPS when the activity of the PRH
proteins was tested by their ability to convert
35
S-labeled
substrate to acid-volatile
35
S-sulfite. We speculate that
the thioredoxin-like domain is involved in catalytic function, and that
the PRH proteins may function as novel “APS reductase” enzymes.
Southern hybridization analysis showed the presence of a small
multigene family in the
Arabidopsis
genome. RNA blot
hybridization with gene-specific probes revealed for each gene the
presence of a transcript of ≈1.85 kb in leaves, stems, and roots that
increased on sulfate starvation. To our knowledge, this is the first
report of the cloning and characterization of plant genes that encode
proteins with APS reductase activity and supports the suggestion that
APS can be utilized directly, without activation to PAPS, as an
intermediary substrate in reductive sulfate assimilation.
Colored diffraction catastrophes.
Authors: Berry M V , Klein S
Published: July 2002

On fine scales, caustics produced with white light show vividly colored diffraction fringes. For caustics described by the elementary catastrophes of singularity theory, the colors are characteristic of the type of singularity. We study the diffraction colors of the fold and cusp catastrophes. The colors can be simulated computationally as the superposition of monochromatic patterns for different wavelengths. Far from the caustic, where the luminosity contrast is negligible, the fringe colors persist; an asymptotic theory explains why. Experiments with caustics produced by refraction through irregular bathroom-window glass show good agreement with theory. Colored fringes near the cusp reveal fine lines that are not present in any of the monochromatic components; these lines are explained in terms of partial decoherence between rays with widely differing path differences.
Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev.
Authors: Fridell R A , Fischer U , Lührmann R , Meyer B E , Meinkoth J L , Malim M H , Cullen B R
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.
Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins.
Authors: Krizek B A , Meyerowitz E M
Published: July 2002

The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG.
Foamy virus reverse transcriptase is expressed independently from the Gag protein.
Authors: Enssle J , Jordan I , Mauer B , Rethwilm A
Published: July 2002

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses.
Safety-modified episomal vectors for human gene therapy
Authors: Cooper Mark J. , Lippa Mara , Payne Jennifer M. , Hatzivassiliou Georgia , Reifenberg Erica , Fayazi Behnaz , Perales Jose C. , Morrison Laura J. , Templeton Dennis , Piekarz Richard L. , Tan June
Published: July 2002

The effectiveness of ongoing gene therapy trials may be limited by the expression characteristics of viral and plasmid-based vectors. To enhance levels of heterologous gene expression, we have developed a safety-modified episomal expression vector that replicates extrachromosomally in human cells. This vector system employs a simian virus 40 (SV40) large T antigen mutant (107/402-T) that is deficient in binding to human tumor suppressor gene products, including p53, retinoblastoma, and p107, yet retains replication competence. These SV40-based episomes replicate to thousands of copies by 2–4 days after gene transfer in multiple types of human cell lines, with lower activity in hamster cells, and no detectable activity in dog, rat, and murine cell lines. Importantly, 107/402-T has enhanced replication activity compared with wild-type T antigen; this finding may be due, in part, to the inability of p53 and retinoblastoma to inactivate 107/402-T function. We demonstrate that the level and duration of 107/402-T expression regulates the observed episomal copy number per cell. Compared with standard plasmid constructs, episomes encoding 107/402-T yield approximately 10- to 100-fold enhanced levels of gene expression in unselected populations of transient transfectants. To determine if 107/402-T-based episomes replicate extrachromosomally
in vivo
, tumor explants in nude mice were directly injected with liposome/DNA complexes. Using a PCR-based assay, we demonstrate that SV40-based episomes replicate in human cells after direct
in vivo
gene transfer. These data suggest that safety-modified SV40-based episomes will be effective for cancer gene therapy because high level expression of therapeutic genes in transient transfectants should yield enhanced tumor elimination.
The human
XRCC9
gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells
Authors: Liu Nan , Lamerdin Jane E. , Tucker James D. , Zhou Zi-Q. , Walter Christi A. , Albala Joanna S. , Busch David B. , Thompson Larry H.
Published: July 2002

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated
XRCC9
, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in
XRCC9
cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The
XRCC9
genomic sequence was cloned and mapped to chromosome 9p13. The translated
XRCC9
sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb
XRCC9
mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues.
XRCC9
is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.
Late memory-related genes in the hippocampus revealed by RNA fingerprinting
Authors: Cavallaro Sebastiano , Meiri Noam , Yi Chu-Li , Musco Simone , Ma Wu , Goldberg Jesse , Alkon Daniel L.
Published: July 2002

Although long-term memory is thought to require a cellular program of gene expression and increased protein synthesis, the identity of proteins critical for associative memory is largely unknown. We used RNA fingerprinting to identify candidate memory-related genes (MRGs), which were up-regulated in the hippocampus of water maze-trained rats, a brain area that is critically involved in spatial learning. Two of the original 10 candidate genes implicated by RNA fingerprinting, the rat homolog of the ryanodine receptor type-2 and glutamate dehydrogenase (EC
1.4.1.3
), were further investigated by Northern blot analysis, reverse transcription–PCR, and
in situ
hybridization and confirmed as MRGs with distinct temporal and regional expression. Successive RNA screening as illustrated here may help to reveal a spectrum of MRGs as they appear in distinct domains of memory storage.
Supercoil-induced extrusion of a regulatory DNA hairpin
Authors: Dai Xing , Greizerstein Miriam B. , Nadas-Chinni Krisztina , Rothman-Denes Lucia B.
Published: July 2002

Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5- to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition. We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg
2+
-dependent manner. Specific sequences on the template strand are required for hairpin extrusion. These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes. In addition, a specific stable hairpin-inducing sequence can regulate transcription
in vivo
. Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.
Synthesis of functional eukaryotic ribosomal RNAs in trans: Development of a novel
in vivo
rDNA system for dissecting ribosome biogenesis
Authors: Liang Wen-Qing , Fournier Maurille J.
Published: July 2002

Active 18S and 25S ribosomal RNAs were produced in trans in yeast, from plasmids containing RNA polymerase II transcription signals and rDNA fragments with unique hybridization tags. Analyses were carried out in cells with temperature-sensitive RNA polymerase I. Functional rRNAs were derived from separate 18S and 5.8/25S rRNA coding units, however, active 25S rRNA could be produced only by cotranscription with 5.8S rRNA. The results demonstrate that the polycistronic organization of the large rDNA operon is not required for successful processing of rRNA or assembly of functional ribosomes. The split operon system should facilitate future efforts to dissect eukaryotic ribosome biogenesis.
The area code hypothesis revisited: Olfactory receptors and other related transmembrane receptors may function as the last digits in a cell surface code for assembling embryos
Authors: Dreyer William J.
Published: July 2002

Recent evidence emerging from several laboratories,
integrated with new data obtained by searching the genome databases,
suggests that the area code hypothesis provides a good heuristic model
for explaining the remarkable specificity of cell migration and tissue
assembly that occurs throughout embryogenesis. The area code hypothesis
proposes that cells assemble organisms, including their brains and
nervous systems, with the aid of a molecular-addressing code that
functions much like the country, area, regional, and local portions of
the telephone dialing system. The complexity of the information
required to code cells for the construction of entire organisms is so
enormous that we assume that the code must make combinatorial use of
members of large multigene families. Such a system would reuse the same
receptors as molecular digits in various regions of the embryo, thus
greatly reducing the total number of genes required. We present the
hypothesis that members of the very large families of olfactory
receptors and vomeronasal receptors fulfill the criteria proposed for
area code molecules and could serve as the last digits in such a code.
We discuss our evidence indicating that receptors of these families are
expressed in many parts of developing embryos and suggest that they
play a key functional role in cell recognition and targeting not only
in the olfactory system but also throughout the brain and numerous
other organs as they are assembled.
Mammalian cytidine 5′-monophosphate
N
-acetylneuraminic acid synthetase: A nuclear protein with evolutionarily conserved structural motifs
Authors: Münster Anja-K. , Eckhardt Matthias , Potvin Barry , Mühlenhoff Martina , Stanley Pamela , Gerardy-Schahn Rita
Published: July 2002

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5′-monophosphate
N
-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary
lec32
mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the
lec32
mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative
Escherichia coli
mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.
A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins
Authors: Djellas Yasmine , Antonakis Kostas , Le Breton Guy C.
Published: July 2002

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A
2
receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A
2
receptors. This effect (
i
) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (
ii
) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (
iii
) was G protein-mediated because it was blocked by an G
αq
C terminus antibody; and (
iv
) was associated with a net increase in G
αq
coupling to thromboxane A
2
receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common G
α
subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases G
αq
association with thromboxane A
2
receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.
Inhibition of myogenesis by transforming growth factor β is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm
Authors: De Angelis Luciana , Borghi Serena , Melchionna Roberta , Berghella Libera , Baccarani-Contri Miranda , Parise Flavia , Ferrari Stefano , Cossu Giulio
Published: July 2002

Transforming growth factor β (TGF-β) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-β. Within 30 min of treatment, TGF-β induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-β. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-β, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-β. We propose a mechanism in which the inhibition of myogenesis by TGF-β is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.
Origins and antiquity of X-linked triallelic color vision systems in New World monkeys
Authors: Boissinot Stephane , Tan Ying , Shyue Song-Kun , Schneider Horacio , Sampaio Iracilda , Neiswanger Katherine , Hewett-Emmett David , Li Wen-Hsiung
Published: July 2002

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.
A homeobox gene with potential developmental control function in the meristem of the conifer
Picea abies
Authors: Sundås-Larsson A. , Svenson M. , Liao H. , Engström P.
Published: July 2002

Many homeobox genes control essential developmental processes in animals and plants. In this report, we describe the first cDNA corresponding to a homeobox gene isolated from a gymnosperm, the
HBK1
gene from the conifer
Picea abies
(L.) Karst (Norway spruce). The sequence shows distinct similarities specifically to the KNOX (
kno
tted-like homeobo
x
) class of homeobox genes known from different angiosperm plants. The deduced amino acid sequence of
HBK1
is strikingly similar within the homeodomain (84% identical) to the maize gene
Knotted1 (Kn1)
, which acts to regulate cell differentiation in the shoot meristem. This similarity suggested that the phylogenetic association of
HBK1
with the KNOX genes might be coupled to a conservation of gene function. In support of this suggestion, we have found
HBK1
to be expressed in the apical meristem in the central population of nondifferentiated stem cells, but not in organ primordia developing at the flanks of the meristem. This pattern of expression is similar to that of
Kn1
in the maize meristem. We show further that
HBK1
, when expressed ectopically in transgenic
Arabidopsis
plants, causes aberrations in leaf development that are similar to the effects of ectopic expression of angiosperm KNOX genes on
Arabidopsis
development. Taken together, these data suggest that
HBK1
has a role, similar to the KNOX genes in angiosperms, in the control of cellular differentiation in the apical meristem of spruce. The data also indicate that KNOX-gene regulation of vegetative development is an ancient feature of seed plants that was present in the last common ancestor of conifers and angiosperms.
Farnesyl transferase inhibitors cause enhanced mitotic sensitivity to taxol and epothilones
Authors: Moasser Mark M. , Sepp-Lorenzino Laura , Kohl Nancy E. , Oliff Allen , Balog Aaron , Su Dai-Shi , Danishefsky Samuel J. , Rosen Neal
Published: July 2002

An important class of cellular proteins, which includes members of the p21ras family, undergoes posttranslational farnesylation, a modification required for their partition to membranes. Specific farnesyl transferase inhibitors (FTIs) have been developed that selectively inhibit the processing of these proteins. FTIs have been shown to be potent inhibitors of tumor cell growth in cell culture and in murine models and at doses that cause little toxicity to the animal. These data suggest that these drugs might be useful therapeutic agents. We now report that, when FTI is combined with some cytotoxic antineoplastic drugs, the effects on tumor cells are additive. No interference is noted. Furthermore, FTI and agents that prevent microtubule depolymerization, such as taxol or epothilones, act synergistically to inhibit cell growth. FTI causes increased sensitivity to induction of metaphase block by these agents, suggesting that a farnesylated protein may regulate the mitotic check point. The findings imply that FTI may be a useful agent for the treatment of tumors with wild-type
ras
that are sensitive to taxanes.
A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target
in vivo
with near-perfect efficiency
Authors: Samarsky Dmitry A. , Ferbeyre Gerardo , Bertrand Edouard , Singer Robert H. , Cedergren Robert , Fournier Maurille J.
Published: July 2002

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a “snorbozyme,” is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency
in vivo
. This is the most efficient
in vivo
cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage
in vivo
. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.
Passive entry of a DNA molecule into a small pore
Authors: de Gennes Pierre-Gilles
Published: July 2002

I consider a vesicle with an open pore of small radius
r
p
, exposed to a DNA solution. The crucial moment is the entry, when a chain end faces the pore and enters it. I discuss qualitatively the following three characteristic times: (
i
) the duration of the entry of one chain end (defining the minimum lifetime of the pore) τ
e
∼ 10
−4
sec, (
ii
) the transfection time τ
t
(the time required to be sure that one chain has gone in) τ
t
∼ hours, and (
iii
) the sliding time τ

S

(the time between entry of one end and entry of the other end) ∼ 1 sec. A fortunate feature is that sliding may proceed even if the pore tends to close itself after entry.
Steroid receptor coactivator-1 (SRC-1) enhances ligand-dependent and receptor-dependent cell-free transcription of chromatin
Authors: Liu Zheng , Wong Jiemin , Tsai Sophia Y. , Tsai Ming-Jer , O’Malley Bert W.
Published: July 2002

Progesterone receptor (PR) functions as a transcription factor that modulates the transcription of target genes in response to progesterone and other signals. The transcriptional activity of PR requires the involvement of coactivators such as steroid receptor coactivator-1 (SRC-1). To dissect the role of SRC-1 in PR transactivation, we established anin vitrotranscription system with chromatin templates, in which PR induced transcription in a ligand-dependent and PRE-dependent manner. In the presence of ligand, purified PR bound to chromatin templates, resulting in chromatin remodeling. With this system, the ability of purified SRC-1 to act as a coactivator of PR was examined. SRC-1 potentiated transcription by ligand-activated PR, whereas it had no effect on transcription in the absence of ligands. As SRC-1 possesses intrinsic histone acetyltransferase activity, we tested the role of acetylation in PR-mediated transcription by using a histone deacetylase inhibitor, trichostatin A (TSA). We found that addition of TSA strongly enhanced PR-dependent transcription on chromatin but not on naked DNA template, and the effects of SRC-1 and TSA on PR transactivation were partially redundant. In addition, SRC-1 was able to potentiate PR transactivation with nonchromatin templates. Thus, our results substantiate a two-step mechanism whereby recruitment of coactivator SRC-1 by the ligand-activated PRin vivoleads to (i) chromatin remodeling through histone acetylation and (ii) recruitment/stabilization of the preinitiation complex.
The
HER-2/neu
receptor tyrosine kinase gene encodes a secreted autoinhibitor
Authors: Doherty Joni K. , Bond Chris , Jardim Armando , Adelman John P. , Clinton Gail M.
Published: July 2002

HER-2/neu
(
erbB-2
) encodes an 185-kDa orphan receptor tyrosine kinase that is constitutively active as a dimer and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of ≈68 kDa, designated herstatin, as the product of an alternative
HER-2
transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-2 followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to
HER
-2-transfected cells with a
K
D
of ≈14 nM and was chemically crosslinked to p185HER-2, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-2 mRNA in carcinoma cells that contain an amplified
HER-2
gene. Herstatin appears to be an inhibitor of p185HER-2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that overexpress
HER-2
.
Rapid treadmilling of brain microtubules free of microtubule-associated proteins
in vitro
and its suppression by tau
Authors: Panda Dulal , Miller Herbert P. , Wilson Leslie
Published: July 2002

We have determined the treadmilling rate of brain microtubules (MTs) free of MT-associated proteins (MAPs) at polymer mass steady state
in vitro
by using [
3
H]GTP-exchange. We developed buffer conditions that suppressed dynamic instability behavior by ≈10-fold to minimize the contribution of dynamic instability to total tubulin-GTP exchange. The MTs treadmilled rapidly under the suppressed dynamic instability conditions, at a minimum rate of 0.2 μm/min. Thus, rapid treadmilling is an intrinsic property of MAP-free MTs. Further, we show that tau, an axonal stabilizing MAP involved in Alzheimer’s disease, strongly suppresses the treadmilling rate. These results indicate that tau’s function in axons might involve suppression of axonal MT treadmilling. We describe mathematically how treadmilling and dynamic instability are mechanistically distinct MT behaviors. Finally, we present a model that explains how small changes in the critical tubulin subunit concentration at MT minus ends, caused by intrinsic differences in rate constants or regulatory proteins, could produce large changes in the treadmilling rate.
Odorants induce the phosphorylation of the cAMP response element binding protein in olfactory receptor neurons
Authors: Moon Cheil , Sung Young-Kwan , Reddy Radhika , Ronnett Gabriele V.
Published: July 2002

Although odorants are known to activate olfactory receptor neurons through cAMP, the long-term effects of odorant detection are not known. Our recent findings indicate that there is also a delayed and sustained cAMP response, with kinetics sufficient to mediate long-term cellular responses. This cAMP response is mediated by cGMP through activation of adenylyl cyclase by protein kinase G (PKG). Therefore, we investigated the ability of odorants to regulate gene expression in rat olfactory epithelium. The cAMP-responsive binding protein (CREB) is a well-characterized transcription factor regulated by cAMP. We examined CREB activity in rat olfactory epithelium and olfactory receptor neurons (ORNs) after stimulation with odorants. Odorants increased levels of phosphorylated CREB in olfactory epithelium
in vivo
, and this increase was localized to ORNs
in vitro
. Incubation with 8-bromo-cGMP or sodium nitroprusside, a guanylyl cyclase activator, also increased phosphorylated CREB.
In vitro
, cAMP-dependent protein kinase phosphorylated CREB. In contrast, PKG failed to phosphorylate CREB directly
in vitro
. Our results demonstrate that the delayed odorant-induced cAMP signal activates CREB, which in turn may modulate gene expression in ORNs. In addition, cGMP indirectly affects CREB activation. This effect of cGMP on CREB activity through cAMP provides another mechanism for the modulation of CREB.
Three proteins involved in
Caenorhabditis elegans
vulval invagination are similar to components of a glycosylation pathway
Authors: Herman Tory , Horvitz H. Robert
Published: July 2002

We have molecularly analyzed three genes,
sqv-3
,
sqv-7
, and
sqv-8
, that are required for wild-type vulval invagination in
Caenorhabditis elegans
. The predicted SQV-8 protein is similar in sequence to two mammalian β(1,3)-glucuronyltransferases, one of which adds glucuronic acid to protein-linked galactose-β(1,4)-
N
-acetylglucosamine. SQV-3 is similar to a family of glycosyltransferases that includes vertebrate β(1,4)-galactosyltransferases, which create galactose-β(1,4)-
N
-acetylglucosamine linkages. One model is therefore that SQV-8 uses a SQV-3 product as a substrate. SQV-7 is similar to members of a family of nucleotide-sugar transporters. The
sqv
genes therefore are likely to encode components of a conserved glycosylation pathway that assembles a
C. elegans
carbohydrate moiety, the absence of which perturbs vulval invagination.
Long-range signaling within growing neurites mediated by neurotrophin-3
Authors: Chang Sunghoe , Popov Sergey V.
Published: July 2002

In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing
Xenopus
embryo neurons in culture. Within minutes after a focal exposure of a soma or a small (≈30 μm in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located ≈300–400 μm away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.
Triple-helix formation of DNA oligomers with methylthiourea-linked nucleosides (DNmt): A kinetic and thermodynamic analysis
Authors: Arya Dev P. , Bruice Thomas C.
Published: July 2002

Complementary short-strand DNA homooligomers and methylthiourea-linked homonucleosides associate and form triplexes in solution. The melting temperatures,
T
m
, the association and dissociation kinetic and thermodynamic parameters, and activation energies were determined by UV thermal analysis for the triplexes of short-strand DNA homooligomers [d(pA)
10
–d(pA)
23
] and poly(dA) with the methylthiourea-linked nucleoside [5′-NH
3
+
-d(Tmt)
4
-T-OH {DNmt
5
}]. Circular dichroism studies show evidence of triple-helical association dependent on the length of the target homooligomer. The melting and cooling curves exhibit hysteresis behavior in the temperature range of 10–95°C at 0.13 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (
k
on
,
E
on
) and dissociation (
k
off
,
E
off
) were obtained.
T
m
decreases with the ionic strength and increases with increase in length of the monomers. The rate constants
k
on
and
k
off
at a given temperature (288 K–310 K) are dependent on the DNA strand length and also decrease and increase respectively with the ionic strength. The energies of activation for the association and dissociation processes are in the range of −18 to −38 kcal/mol and 3 to 18 kcal/mol, respectively. The equilibrium constant for the formation of the triplexes [5′-NH
3
+
-d(Tmt)
4
-T-OH)
2
⋅d(pA)

x

,
x
= 10–23] is several orders of magnitude greater when compared with the triplexes of DNA. The number of base triplets in the nucleus of the DNmt
2
⋅DNA triple-helix (nucleation–zipping model) increases with decreased DNA oligomer length and with increased ionic strength. The values of Δ
H
° calculated from the activation parameters are between −30 and −50 kcal/(mol base) and the values of Δ
G
° are between −6 and −11 kcal/(mol base) for short-strand DNA.
Activator of G protein signaling 3 is a guanine dissociation inhibitor for Gα
i
subunits
Authors: De Vries Luc , Fischer Thierry , Tronchère Hélène , Brothers Greg M. , Strockbine Bentley , Siderovski David P. , Farquhar Marilyn Gist
Published: July 2002

Activator of G protein signaling 3 (AGS3) is a newly identified
protein shown to act at the level of the G protein itself. AGS3 belongs
to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that
is a Gα
i/o
binding motif. AGS3 interacts only with
members of the Gα
i/o
subfamily. By surface plasmon
resonance, we found that AGS3 binds exclusively to the GDP-bound form
of Gα
i3
. In GTPγS binding assays, AGS3 behaves as a
guanine dissociation inhibitor (GDI), inhibiting the rate of exchange
of GDP for GTP by Gα
i3
. AGS3 interacts with both
Gα
i3
and Gα
o
subunits, but has GDI activity
only on Gα
i3
, not on Gα
o
. The fourth GoLoco
motif of AGS3 is a major contributor to this activity. AGS3 stabilizes
Gα
i3
in its GDP-bound form, as it inhibits the increase
in tryptophan fluorescence of the Gα
i3
-GDP subunit
stimulated by AlF
4
−
. AGS3 is widely expressed as it is
detected by immunoblotting in brain, testis, liver, kidney, heart,
pancreas, and in PC-12 cells. Several different sizes of the protein
are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs
in heart and brain, respectively, suggesting tissue-specific
alternative splicing. Taken together, our results demonstrate that AGS3
is a GDI. To the best of our knowledge, no other GDI has been
described for heterotrimeric G proteins. Inhibition of the Gα subunit
and stimulation of heterotrimeric G protein signaling, presumably by
stimulating Gβγ, extend the possibilities for modulating signal
transduction through heterotrimeric G proteins.
A topological invariant to predict the three-dimensional writhe of ideal configurations of knots and links
Authors: Cerf Corinne , Stasiak Andrzej
Published: July 2002

We present herein a topological invariant of oriented alternating knots and links that predicts the three-dimensional (3D) writhe of the ideal geometrical configuration of the considered knot/link. The fact that we can correlate a geometrical property of a given configuration with a topological invariant supports the notion that the ideal configuration contains important information about knots and links. The importance of the concept of ideal configuration was already suggested by the good correlation between the 3D writhe of ideal knot configurations and the ensemble average of the 3D writhe of random configurations of the considered knots. The values of the new invariant are quantized: multiples of 4/7 for links with an odd number of components (including knots) and 2/7 plus multiples of 4/7 for links with an even number of components.
Effect of nicotinamide administration on ethanol consumption and on liver and brain acetaldehyde oxidation rate, by UChB rats
Authors: TAMPIER LUTSKE , QUINTANILLA MARÍA ELENA , MARDONES JORGE
Published: July 2002

The activities of liver and brain aldehyde dehydrogenase, an NAD+ dependent enzyme, which controls acetaldehyde oxidation have been reported to play a role in voluntary ethanol consumption. It has been reported that nicotinamide administration to rats increases NAD+ levels, that may increase acetaldehyde oxidation rates if basal NAD+ levels are not saturating for the enzyme. In the present paper the effect of nicotinamide administration on voluntary ethanol consumption by genetically high ethanol consumer UChB rats and brain and liver mitochondrial acetaldehyde oxidation were studied. Administration of nicotinamide 250 or 500 mg/kg i.p. to UChB rats, produced a significant reduction in their voluntary ethanol consumption and increased brain acetaldehyde oxidation in brain but not liver homogenates.These results suggest that basal NAD+ levels are not saturating for brain aldehyde dehydrogenase and that the reduction of ethanol consumption by UChB rats may be the consequence of a change in the brain redox state, rather than the local level of acetaldehyde.
Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4
Authors: Bijlmakers Marie-José J.E. , Isobe-Nakamura Misako , Ruddock Lindsay J. , Marsh Mark
Published: July 2002

In T lymphocytes, the Src-family protein tyrosine kinase p56lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles.

A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.
Loss of Matrix Adhesion Triggers Rapid Transformation-Selective Apoptosis in Fibroblasts
Authors: McGill Gaël , Shimamura Akiko , Bates Richard C. , Savage Robert E. , Fisher David E.
Published: July 2002

Cell–matrix and cell–cell adhesion are recognized physiological determinants of cell growth and survival. In epithelial and endothelial cell systems, oncogenic transformation has in several cases been shown to confer resistance to apoptosis upon depriving cells of substrate adhesion. We examined the effects of oncogenic transformation in adherent versus adhesion- deprived primary embryonic fibroblasts. Whereas untransformed early passage fibroblasts undergo cell cycle arrest, their Myc/Ras- or E1A/Ras-transformed counterparts rapidly enter apoptosis when placed into suspension. This phenomenon also occurs upon incubation with a soluble, RGD-containing integrin ligand and is blocked by a peptide antagonist to ICE family proteases or by aggregation of cells plated at high density. Loss of wild-type p53 modulates the kinetics but does not abrogate this death pathway. Transformation with activated Src rather than Ras rendered fibroblasts selectively resistant to adhesion-dependent apoptosis, an effect likely related to Src's role in integrin signaling, while simultaneously sensitizing the cells to radiation-induced apoptosis. Thus cell adhesion events regulate transformation-selective apoptosis in fibroblasts and provide potentially important targets for understanding and interfering with tumor cell viability.
Human Cytomegalovirus Inhibits Major Histocompatibility Complex Class II Expression By Disruption of the Jak/Stat Pathway
Authors: Miller Daniel M. , Rahill Brian M. , Boss Jeremy M. , Lairmore Michael D. , Durbin Joan E. , Waldman James W. , Sedmak Daniel D.
Published: July 2002

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon γ (IFN-γ)–stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-γ signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-γ–stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-γ signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.
Impaired Interleukin 4 Signaling in T Helper Type 1 Cells
Authors: Huang Hua , Paul William E.
Published: July 2002

Cluster of differentation (CD)4+ T helper cells (Th)1s fail to produce interleukin (IL)-4. Even if restimulated in the presence of IL-4, a condition that induces IL-4–producing capacity in naive CD4+ T cells, Th1s fail to become IL-4 producers. We report that Th1 cells have a major impairment in IL-4 signaling. When compared to both Th2s and naive T cells, they display a striking diminution in phosphorylation of Stat6. They also show reduced phosphorylation of Janus kinase (JAK)-3 and insulin receptor substrate (IRS)-2 when compared to Th2s. Stat6 and JAK-3 are present in equivalent amounts in Th1s and Th2s, but IRS-2 protein levels are much lower in Th1s than in Th2s. Altered sensitivity to IL-4, the major inducer of the Th2 phenotype, may explain the stability of the Th1 state.