bluuebunny/crossref_metadata_2025 huggingface dataset:
Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.
Authors: Lopez I , Anthony R G , Maciver S K , Jiang C J , Khan S , Weeds A G , Hussey P J
Published: July 2002

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.
Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.
Authors: Paschal B M , Delphin C , Gerace L
Published: July 2002

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.
Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness.
Authors: Lorenz U , Ravichandran K S , Burakoff S J , Neel B G
Published: July 2002

Protein tyrosine phosphorylation and dephosphorylation are key regulatory events in T-cell receptor (TCR) signaling. We investigated the role of the tyrosine phosphatase SHPTP1 in TCR signaling by analysis of TCR signal transduction in motheaten (me/me) mice, which lack SHPTP1 expression. As revealed by flow cytometric analysis, thymocyte development was normal in me/me mice. However, me/me thymocytes hyperproliferated (3-to 5-fold) in response to TCR stimulation, whereas their response to interleukin 2 stimulation was unchanged compared with normal thymocytes. TCR-induced hyperproliferation of me/me thymocytes was reproduced in purified single-positive thymocytes. Moreover, me/me thymocytes produced increased amounts of interleukin 2 production upon TCR stimulation. Biochemical analysis revealed that, in response to TCR or TCR/CD4 stimulation, thymocytes lacking SHPTP1 showed increased tyrosyl phosphorylation of several cellular substrates, which correlated with increased activation of the src-family kinases Lck and Fyn. Taken together, our data suggest that SHPTP1 is an important negative regulator of TCR signaling, acting at least in part to inactivate Lck and Fyn.
A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels.
Authors: Ismailov I I , Fuller C M , Berdiev B K , Shlyonsky V G , Benos D J , Barrett K E
Published: July 2002

Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)P4) has a direct biphasic (activation/inhibition) effect on an epithelial Ca(2+)-activated chloride channel. The effect of D-Ins(3,4,5,6)P4 is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)P4 on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an "orphan" messenger. They suggest that the molecular target for D-Ins(3,4,5,6)P4 is a plasma membrane Ca(2+)-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)P4 and Ca2+ may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis.
Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription.
Authors: Zhang H , Dornadula G , Alur P , Laughlin M A , Pomerantz R J
Published: July 2002

Reverse transcription of HIV-1, without detergent or amphipathic peptide-induced permeability of the viral envelope, has been demonstrated to occur in the intact HIV-1 virion. In this report, we demonstrate that the amphipathic domains in the C terminus of the transmembrane glycoprotein (gp41) account for the natural permeability of the HIV-1 envelope to deoxyribonucleoside triphosphates, the substrates for DNA polymerization. In addition, nonphysiological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorporate into, and irreversibly terminate reverse transcripts. As a result, viral infectivity is potently inhibited. Since the lentiviral envelope with these newly demonstrated characteristics can serve as a delivery pathway for anti-reverse transcription agents, we propose a unique strategy to prevent HIV-1 interand, possibly, intrahost transmission.
Evolutionary analyses of hedgehog and
Hoxd-10
genes in fish species closely related to the zebrafish
Authors: Zardoya Rafael , Abouheif Ehab , Meyer Axel
Published: July 2002

The study of development has relied primarily on the isolation of
mutations in genes with specific functions in development and on the
comparison of their expression patterns in normal and mutant
phenotypes. Comparative evolutionary analyses can complement these
approaches. Phylogenetic analyses of Sonic hedgehog
(
Shh
) and
Hoxd-10
genes from 18 cyprinid
fish species closely related to the zebrafish provide novel insights
into the functional constraints acting on
Shh
. Our
results confirm and extend those gained from expression and crystalline
structure analyses of this gene. Unexpectedly, exon 1 of
Shh
is found to be almost invariant even in third codon
positions among these morphologically divergent species suggesting that
this exon encodes for a functionally important domain of the hedgehog
protein. This is surprising because the main functional domain of
Shh
had been thought to be that encoded by exon 2.
Comparisons of
Shh
and
Hoxd-10
gene
sequences and of resulting gene trees document higher evolutionary
constraints on the former than on the latter. This might be indicative
of more general evolutionary patterns in networks of developmental
regulatory genes interacting in a hierarchical fashion. The presence of
four members of the hedgehog gene family in cyprinid fishes was
documented and their homologies to known hedgehog genes in other
vertebrates were established.
Molecular characterization of the human peroxisomal branchedchain acyl-CoA oxidase: cDNA cloning, chromosomal assignment, tissue distribution, and evidence for the absence of the protein in Zellweger syndrome
Authors: Baumgart Eveline , Vanhooren Johannes C. T. , Fransen Mark , Marynen Peter , Puype Magda , Vandekerckhove Joël , Leunissen Jack A. M. , Fahimi H. Dariush , Mannaerts Guy P. , Van Veldhoven Paul P.
Published: July 2002

Peroxisomes in human liver contain two distinct acyl-CoA
oxidases with different substrate specificities: (
i
)
palmitoyl-CoA oxidase, oxidizing very long straight-chain fatty acids
and eicosanoids, and (
ii
) a branched-chain acyl-CoA
oxidase (hBRCACox), involved in the degradation of long branched fatty
acids and bile acid intermediates. The accumulation of branched fatty
acids and bile acid intermediates leads to severe mental retardation
and death of the diseased children. In this study, we report the
molecular characterization of the hBRCACox, a prerequisite for studying
mutations in patients with a single enzyme deficiency. The composite
cDNA sequence of hBRCACox, derived from overlapping clones isolated via
immunoscreening and hybridization of human liver cDNA expression
libraries, consisted of 2225 bases and contained an open reading frame
of 2046 bases, encoding a protein of 681 amino acids with a calculated
molecular mass of 76,739 Da. The C-terminal tripeptide of the protein
is SKL, a known peroxisome targeting signal. Sequence comparison with
the other acyl-CoA oxidases and evolutionary analysis revealed that,
despite its broader substrate specificity, the hBRCACox is the human
homolog of rat trihydroxycoprostanoyl-CoA oxidase (rTHCCox) and that
separate gene duplication events led to the occurrence in mammals of
acyl-CoA oxidases with different substrate specificities. Northern blot
analysis demonstrated that—in contrast to the rTHCCox gene—the
hBRCACox gene is transcribed also in extrahepatic tissues such as
heart, kidney, skeletal muscle, and pancreas. The highest levels of the
2.6-kb mRNA were found in heart, followed by liver. The enzyme is
encoded by a single-copy gene, which was assigned to chromosome 3p14.3
by fluorescent
in situ
hybridization. It was absent from
livers of Zellweger patients as shown by immunoblot analysis and
immunocytochemistry.
Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in
Escherichia coli
Authors: Jayakumar Arumugam , Huang Wei-Yong , Raetz Beate , Chirala Subrahmanyam S. , Wakil Salih J.
Published: July 2002

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid
synthase (FAS; EC
2.3.1.85
) from the human brain FAS cDNA clones we
characterized previously. In the process of accomplishing this task, we
developed a novel PCR procedure, recombinant PCR, which is very useful
in joining two overlapping DNA fragments that do not have a common or
unique restriction site. The full-length cDNA was cloned in pMAL-c2 for
heterologous expression in
Escherichia coli
as a
maltose-binding protein fusion. The recombinant protein was purified by
using amylose-resin affinity and hydroxylapatite chromatography. As
expected from the coding capacity of the cDNA expressed, the chimeric
recombinant protein has a molecular weight of 310,000 and reacts with
antibodies against both human FAS and maltose-binding protein. The
maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate
synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of
the partial activities of FAS at levels comparable with those of the
native human enzyme purified from HepG2 cells. Like the native HepG2
FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and
minimal amounts of stearic and arachidic acids. Similarly, a human FAS
cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and
malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned
and expressed in
E. coli
using pMAL-c2. The expressed
fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity
(
M
r
190,000) and exhibited the activities of
the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase.
In addition, a human FAS cDNA encoding domains II and III (enoyl and
β-ketoacyl reductases, acyl carrier protein, and thioesterase) was
cloned in pET-32b(+) and expressed in
E. coli
as a
fusion protein with thioredoxin and six in-frame histidine residues.
The recombinant fusion protein, thioredoxin-human FAS domains II and
III, that was purified from
E. coli
had a molecular
weight of 159,000 and exhibited the activities of the enoyl and
β-ketoacyl reductases and the thioesterase. Both the MBP and the
thioredoxin-His-tags do not appear to interfere with the catalytic
activity of human FAS or its partial activities.
ZnT-3, a putative transporter of zinc into synaptic vesicles
Authors: Palmiter Richard D. , Cole Toby B. , Quaife Carol J. , Findley Seth D.
Published: July 2002

The murine
ZnT3
gene was cloned by virtue of its
homology to the
ZnT2
gene, which encodes a membrane
protein that facilitates sequestration of zinc in endosomal vesicles.
ZnT-3 protein is predicted to have six transmembrane domains and shares
52% amino acid identity with ZnT-2, with the homology extending
throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the
amino acid sequence is 86% identical to murine ZnT-3. The mouse
ZnT3
gene has 8 exons and maps to chromosome 5. Northern
blot and reverse transcriptase–PCR analyses demonstrate that murine
ZnT-3 expression is restricted to the brain and testis.
In
situ
hybridization reveals that within the brain, ZnT-3 mRNA is
most abundant in the hippocampus and cerebral cortex. Antibodies raised
against the C-terminal tail of mouse ZnT-3 react with the projections
from these neurons and produce a pattern similar to that obtained with
Timm’s reaction, which reveals histochemically reactive zinc within
synaptic vesicles. We propose that ZnT-3 facilitates the accumulation
of zinc in synaptic vesicles.
Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination
Authors: Russ Andreas P. , Friedel Christina , Ballas Karin , Kalina Uwe , Zahn Dirk , Strebhardt Klaus , von Melchner Harald
Published: July 2002

A strategy employing gene-trap mutagenesis and site-specific
recombination (Cre/
loxP
) has been developed to isolate
genes that are transcriptionally activated during programmed cell
death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells
(FDCP1) expressing a reporter plasmid that codes for herpes simplex
virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3
were transduced with a retroviral gene-trap vector carrying coding
sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre
expression from integrations into active genes resulted in a permanent
switching between the selectable marker genes that converted the FDCP1
cells to factor independence. Selection for autonomous growth yielded
recombinants in which Cre sequences in the U3 region were expressed
from upstream cellular promoters. Because the expression of the marker
genes is independent of the trapped cellular promoter, genes could be
identified that were transiently induced by IL-3 withdrawal.
The common
nodABC
genes of
Rhizobium meliloti
are host-range determinants
Authors: Roche Philippe , Maillet Fabienne , Plazanet Claire , Debellé Frédéric , Ferro Myriam , Truchet Georges , Promé Jean-Claude , Dénarié Jean
Published: July 2002

Symbiotic bacteria of the genus
Rhizobium
synthesize
lipo-chitooligosaccharides, called Nod factors (NFs), which act as
morphogenic signal molecules on legume hosts. The common
nodABC
genes, present in all
Rhizobium
species, are required for the synthesis of the core structure of NFs.
NodC is an
N
-acetylglucosaminyltransferase, and NodB is a
chitooligosaccharide deacetylase; NodA is involved in N-acylation of
the aminosugar backbone. Specific
nod
genes are involved
in diverse NF substitutions that confer plant specificity. We
transferred to
R. tropici
, a broad host-range tropical
symbiont, the ability to nodulate alfalfa, by introducing
nod
genes of
R. meliloti
. In addition to
the specific
nodL
and
nodFE
genes, the
common
nodABC
genes of
R. meliloti
were
required for infection and nodulation of alfalfa. Purified NFs of the
R. tropici
hybrid strain, which contained chitin
tetramers and were partly N-acylated with unsaturated C16 fatty acids,
were able to elicit nodule formation on alfalfa. Inactivation of the
R. meliloti nodABC
genes suppressed the ability of the
NFs to nodulate alfalfa. Studies of NFs from
nodA
,
nodB
,
nodC
, and
nodI
mutants indicate that (
i
) NodA of
R.
meliloti
, in contrast to NodA of
R. tropici
, is
able to transfer unsaturated C16 fatty acids onto the chitin backbone
and (
ii
) NodC of
R. meliloti
specifies
the synthesis of chitin tetramers. These results show that allelic
variation of the common
nodABC
genes is a genetic
mechanism that plays an important role in signaling variation and in
the control of host range.
Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.
Authors: Huang T , Campadelli-Fiume G
Published: July 2002

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.
Human cooperation in the simultaneous and the alternating Prisoner's Dilemma: Pavlov versus Generous Tit-for-Tat.
Authors: Wedekind C , Milinski M
Published: July 2002

The iterated Prisoner's Dilemma has become the paradigm for the evolution of cooperation among egoists. Since Axelrod's classic computer tournaments and Nowak and Sigmund's extensive simulations of evolution, we know that natural selection can favor cooperative strategies in the Prisoner's Dilemma. According to recent developments of theory the last champion strategy of "win--stay, lose--shift" ("Pavlov") is the winner only if the players act simultaneously. In the more natural situation of players alternating the roles of donor and recipient a strategy of "Generous Tit-for-Tat" wins computer simulations of short-term memory strategies. We show here by experiments with humans that cooperation dominated in both the simultaneous and the alternating Prisoner's Dilemma. Subjects were consistent in their strategies: 30% adopted a Generous Tit-for-Tat-like strategy, whereas 70% used a Pavlovian strategy in both the alternating and the simultaneous game. As predicted for unconditional strategies, Pavlovian players appeared to be more successful in the simultaneous game whereas Generous Tit-for-Tat-like players achieved higher payoffs in the alternating game. However, the Pavlovian players were smarter than predicted: they suffered less from defectors and exploited cooperators more readily. Humans appear to cooperate either with a Generous Tit-for-Tat-like strategy or with a strategy that appreciates Pavlov's advantages but minimizes its handicaps.
Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.
Authors: Hardenbol P , Van Dyke M W
Published: July 2002

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.
Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.
Authors: Liu B , Troxler R F
Published: July 2002

We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level.
Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.
Authors: Alexander-Miller M A , Leggatt G R , Berzofsky J A
Published: July 2002

The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.
Prediction of the stability of DNA triplexes.
Authors: Roberts R W , Crothers D M
Published: July 2002

We present rules that allow one to predict the stability of DNA pyrimidine.purine.pyrimidine (Y.R.Y) triple helices on the basis of the sequence. The rules were derived from van't Hoff analysis of 23 oligonucleotide triplexes tested at a variety of pH values. To predict the enthalpy of triplex formation (delta H degrees), a simple nearest-neighbor model was found to be sufficient. However, to accurately predict the free energy of the triplex (delta G degrees), a combination model consisting of five parameters was needed. These parameters were (i) the delta G degrees for helix initiation, (ii) the delta G degrees for adding a T-A.T triple, (iii) the delta G degrees for adding a C(+)-G.C triple, (iv) the penalty for adjacent C bases, and (v) the pH dependence of the C(+)-G.C triple's stability. The fitted parameters are highly consistent with thermodynamic data from the basis set, generally predicting both delta H degrees and delta G degrees to within the experimental error. Examination of the parameters points out several interesting features. The combination model predicts that C(+) -G.C. triples are much more stabilizing than T-A.T triples below pH 7.0 and that the stability of the former increases approximately equal to 1 kcal/mol per pH unit as the pH is decreased. Surprisingly though, the most stable sequence is predicted to be a CT repeat, as adjacent C bases partially cancel the stability of one another. The parameters successfully predict tm values from other laboratories, with some interesting exceptions.
Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity
Authors: Gosling Jennifa , Monteclaro Felipe S. , Atchison Robert E. , Arai Hidenori , Tsou Chia-Lin , Goldsmith Mark A. , Charo Israel F.
Published: July 2002

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.
Evolution of paired domains: Isolation and sequencing of jellyfish and hydra
Pax
genes related to
Pax-5
and
Pax-6
Authors: Sun Hongmin , Rodin Andrei , Zhou Yihong , Dickinson Douglas P. , Harper Donald E. , Hewett-Emmett David , Li Wen-Hsiung
Published: July 2002

Pax proteins are a family of transcription factors with a highly conserved paired domain; many members also contain a paired-type homeodomain and/or an octapeptide. Nine mammalian
Pax
genes are known and classified into four subgroups:
Pax-1/9
,
Pax-2/5/8
,
Pax-3/7
, and
Pax-4/6
. Most of these genes are involved in nervous system development. In particular,
Pax-6
is a key regulator that controls eye development in vertebrates and
Drosophila
. Although the
Pax-4/6
subgroup seems to be more closely related to
Pax-2/5/8
than to
Pax-3/7
or
Pax-1/9
, its evolutionary origin is unknown. We therefore searched for a
Pax-6
homolog and related genes in
Cnidaria
, which is the lowest phylum of animals that possess a nervous system and eyes. A sea nettle (a jellyfish) genomic library was constructed and two pax genes (
Pax-A
and
-B
) were isolated and partially sequenced. Surprisingly, unlike most known
Pax
genes, the paired box in these two genes contains no intron. In addition, the complete cDNA sequences of hydra
Pax-A
and
-B
were obtained. Hydra Pax-B contains both the homeodomain and the octapeptide, whereas hydra Pax-A contains neither. DNA binding assays showed that sea nettle Pax-A and -B and hydra Pax-A paired domains bound to a Pax-5/6 site and a Pax-5 site, although hydra Pax-B paired domain bound neither. An alignment of all available paired domain sequences revealed two highly conserved regions, which cover the DNA binding contact positions. Phylogenetic analysis showed that Pax-A and especially Pax-B were more closely related to Pax-2/5/8 and Pax-4/6 than to Pax-1/9 or Pax-3/7 and that the
Pax
genes can be classified into two supergroups:
Pax-A/Pax-B/Pax-2/5/8/4/6
and
Pax-1/9/3/7
. From this analysis and the gene structure, we propose that modern
Pax-4/6
and
Pax-2/5/8
genes evolved from an ancestral gene similar to cnidarian
Pax-B
, having both the homeodomain and the octapeptide.
Epigenetic changes at the insulin-like growth factor II/
H19
locus in developing kidney is an early event in Wilms tumorigenesis
Authors: Okamoto Keisei , Morison Ian M. , Taniguchi Takanobu , Reeve Anthony E.
Published: July 2002

Relaxation of imprinting at the insulin-like growth factor II (
IFG-II
)/
H19
locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of
IFG-II
imprinting, we determined the frequency of epigenetic change at the
IFG-II/H19
locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of
IFG-II
imprinting and biallelic
H19
methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the
IFG-II/H19
locus.
Resource competition as a mechanism for B cell homeostasis
Authors: McLean A. R. , Rosado M. M. , Agenes F. , Vasconcellos R. , Freitas A. A.
Published: July 2002

Cellular competition for survival signals offers a cogent and appealing mechanism for the maintenance of cellular homeostasis [Raff, M. C. (1992)Nature (London)356, 397–400]. We present a theoretical and experimental investigation of the role of competition for resources in the regulation of peripheral B cell numbers. We use formal ecological competition theory, mathematical models of interspecific competition, and competitive repopulation experiments to show that B cells must compete to persist in the periphery and that antigen forms a part of the resources over which B cells compete.
Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain
Authors: Liang Zicai , Sottrup-Jensen Lars , Aspán Anna , Hall Martin , Söderhäll Kenneth
Published: July 2002

A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish,
Pacifastacus leniusculus
, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper,
Locusta migratoria
. The nine light chain domains and the
Locusta
inhibitors share a characteristic cysteine array (Cys-Xaa
9–12
-Cys-Xaa
2
-Cys-Xaa-Cys-Xaa
6–8
-Cys-Xaa
4
-Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor.
Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease
Authors: Hunot Stephane , Brugg Bernard , Ricard Damien , Michel Patrick P. , Muriel Marie-Paule , Ruberg Merle , Faucheux Baptiste A. , Agid Yves , Hirsch Etienne C.
Published: July 2002

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal
in vitro
models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown
in vitro
with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C
2
-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.
In vitro
reconstitution of the photosystem I light-harvesting complex LHCI-730: Heterodimerization is required for antenna pigment organization
Authors: Schmid Volkmar H. R. , Cammarata Kirk V. , Bruns Brigitte U. , Schmidt Gregory W.
Published: July 2002

Here we describe the
in vitro
reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato
Lhca
genes in
Escherichia coli
. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.
The observed global warming record: What does it tell us?
Authors: Wigley T. M. L. , Jones P. D. , Raper S. C. B.
Published: July 2002

Global, near-surface temperature data sets and their derivations
are discussed, and differences between the Jones and Intergovernmental
Panel on Climate Change data sets are explained. Global-mean
temperature changes are then interpreted in terms of anthropogenic
forcing influences and natural variability. The inclusion of aerosol
forcing improves the fit between modeled and observed changes but does
not improve the agreement between the implied climate sensitivity value
and the standard model-based range of 1.5–4.5°C equilibrium warming
for a CO
2
doubling. The implied sensitivity goes from below
the model-based range of estimates to substantially above this range.
The addition of a solar forcing effect further improves the fit and
brings the best-fit sensitivity into the middle of the model-based
range. Consistency is further improved when internally generated
changes are considered. This consistency, however, hides many
uncertainties that surround observed data/model comparisons. These
uncertainties make it impossible currently to use observed global-scale
temperature changes to narrow the uncertainty range in the climate
sensitivity below that estimated directly from climate models.
Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF
Authors: McEwan Iain J. , Gustafsson Jan-Åke
Published: July 2002

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity
in vitro
revealed that AR
142–485
fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR
142–485
. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.
Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases
Authors: Luo Yuan , Bond Joshua D. , Ingram Vernon M.
Published: July 2002

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide
p
-(trifluoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. Comparison of the time courses of the rapid rise in cytosolic calcium with the slower increase in ERK1 and ERK2 activation suggests one or more intermediate stages in this pathway. Chelation of cytosolic calcium with dimethyl bis-(
o
-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid abolished the FCCP-stimulated rise in internal calcium, as well as the tyrosine phosphorylation and the activation of the ERKs. Surprisingly, caffeine, which releases calcium from different internal stores, did not increase the tyrosine phosphorylation and did not activate the ERKs. The FCCP effect on calcium storage may be related to mitochondrial dysfunction in Alzheimer disease, which might result in ineffective buffering of cytosolic calcium that leads to mitogen-activated protein kinase activation and subsequent protein phosphorylations.
Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines
Authors: Allen Patrick B. , Ouimet Charles C. , Greengard Paul
Published: July 2002

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity
in vitro
. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.
Altered 3′-terminal RNA structure in phage Qβ adapted to host factor-less
Escherichia coli
Authors: Schuppli Daniel , Miranda Giovanni , Tsui Ho-Ching Tiffany , Winkler Malcolm E. , Sogo José M. , Weber Hans
Published: July 2002

The RNA phage Qβ requires for the replication of its genome an RNA binding protein called Qβ host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3′-end of the Qβ plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qβ was adapted to an
Escherichia coli
Q13 host strain with an inactivated host factor (
hfq
) gene. This strain initially produced phage at a titer ≈10,000-fold lower than the wild-type strain and with minute plaque morphology, but after 12 growth cycles, phage titer and plaque size had evolved to levels near those of the wild-type host. RNAs isolated from adapted Qβ mutants were efficient templates for replicase without host factor
in vitro
. Electron microscopy showed that mutant RNAs, in contrast to wild-type RNA, efficiently interacted with replicase at the 3′-end in the absence of host factor. The same set of four mutations in the 3′-terminal third of the genome was found in several independently evolved phage clones. One mutation disrupts the base pairing of the 3′-terminal CCC
oh
sequence, suggesting that the host factor stimulates activity of the wild-type RNA template by melting out its 3′-end.
Dinor-oxo-phytodienoic acid: A new hexadecanoid signal in the jasmonate family
Authors: Weber Hans , Vick Brady A. , Farmer Edward E.
Published: July 2002

Jasmonic acid and its precursors are potent regulatory molecules in plants. We devised a method for the simultaneous extraction of these compounds from plant leaves to quantitate changes in the levels of jasmonate family members during health and on wounding. During our study, we identified a novel 16-carbon cyclopentenoic acid in leaf extracts from
Arabidopsis
and potato. The new compound, a member of the jasmonate family of signals, was named dinor-oxo-phytodienoic acid. Dinor-oxo-phytodienoic acid was not detected in the
Arabidopsis
mutant
fad5
, which is incapable of synthesizing 7Z,10Z,13Z-hexadecatrienoic acid (16:3), suggesting that the metabolite is derived directly from plastid 16:3 rather than by β-oxidation of the 18-carbon 12-oxo-phytodienoic acid. Simultaneous quantitation of jasmonate family members in healthy leaves of
Arabidopsis
and potato suggest that different plant species have different relative levels of jasmonic acid, oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. We term these profiles “oxylipin signatures.” Dinor-oxo-phytodienoic acid levels increased dramatically in
Arabidopsis
and potato leaves on wounding, suggesting roles in wound signaling. Treatment of
Arabidopsis
with micromolar levels of dinor-oxo-phytodienoic acid increased the ability of leaf extracts to transform linoleic acid into the α-ketol 13-hydroxy-12-oxo-9(
Z
) octadecenoic acid indicating that the compound can regulate part of its own biosynthetic pathway. Tightly regulated changes in the relative levels of biologically active jasmonates may permit sensitive control over metabolic, developmental, and defensive processes in plants.
Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation
Authors: Li Jianke , Meyer April N. , Donoghue Daniel J.
Published: July 2002

M-phase promoting factor or maturation promoting factor, a key regulator of the G
2
→ M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. We have previously shown that
Xenopus
cyclin B1 has five sites of Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism is still unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala to abolish phosphorylation, the mutant cyclin B1
Ala
is inactivated; activity can be enhanced by mutation of these residues to Glu to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Here we show that biological activity can be restored to cyclin B1
Ala
by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity was detected in association with cyclin B1
Ala
targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Furthermore, given the similar
in vitro
substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase–cyclin complexes.