bluuebunny/crossref_metadata_2025 huggingface dataset:
Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution
in vitro
Authors: Yoda Kinya , Ando Satoshi , Morishita Setsuo , Houmura Kenichi , Hashimoto Keiji , Takeyasu Kunio , Okazaki Tuneko
Published: July 2002

Centromere protein A (CENP-A) is a variant of histone H3 with more than 60% sequence identity at the C-terminal histone fold domain. CENP-A specifically locates to active centromeres of animal chromosomes and therefore is believed to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. Here we report that CENP-A, highly purified from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitution system mediated by nucleosome assembly protein-1 (NAP-1). The structure of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H2B, and H4, and closed circular DNAs had the following properties. By atomic force microscopy, “beads on a string” images were obtained that were similar to those obtained with nucleosomes reconstituted with four standard histones. DNA ladders with repeats of approximately 10 bp were produced by DNase I digestion, indicating that the DNA was wrapped round the protein complex. Mononucleosomes isolated by glycerol gradient sedimentation had a relative molecular mass of ≈200 kDa and were composed of 120–150 bp of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B. Thus, we conclude that CENP-A forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA.
Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes
Authors: Hoth Markus , Button Donald C. , Lewis Richard S.
Published: July 2002

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca
2+
signals. In many cells, mitochondria act as Ca
2+
buffers by taking up and releasing Ca
2+
, but this simple buffering action by itself often cannot explain the organelle's effects on Ca
2+
signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca
2+
channels in T lymphocytes as a mechanism of mitochondrial Ca
2+
signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca
2+
stores causes sustained activation of the store-operated Ca
2+
current, I
CRAC
(CRAC, Ca
2+
release-activated Ca
2+
). Inhibition of mitochondrial Ca
2+
uptake by compounds that dissipate the intramitochondrial potential unmasks Ca
2+
-dependent inactivation of I
CRAC
. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca
2+
accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca
2+
uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca
2+
]
i
elevation, indicating a functional link between mitochondrial regulation of I
CRAC
and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca
2+
channel activity and signal transmission from the plasma membrane to the nucleus.
Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets
Authors: Naumov Yuri N. , Bahjat Keith S. , Gausling Rudolph , Abraham Roshini , Exley Mark A. , Koezuka Yasuhiko , Balk Steven B. , Strominger Jack L. , Clare-Salzer Michael , Wilson S. Brian
Published: July 2002

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of α-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.
The double
par
locus of virulence factor pB171: DNA segregation is correlated with oscillation of ParA
Authors: Ebersbach Gitte , Gerdes Kenn
Published: July 2002

Prokaryotic plasmids and chromosomes encode partitioning
(
par
) loci that segregate DNA to daughter cells before
cell division. Recent database analyses showed that almost all known
par
loci encode an ATPase and a DNA-binding protein, and
one or more cis-acting regions where the proteins act. All
par
-encoded ATPases belong to one of two protein
superfamilies, Walker-type and actin-like ATPases. This property
was recently used to divide
par
loci into Types I and II
loci. We show here that the
Escherichia coli
virulence factor pB171 encodes a double
par
locus that
consists of one Type I and one Type II locus. Separately, each locus
stabilized a test-plasmid efficiently. Together, the two loci mediated
even more efficient plasmid stabilization. The
par
loci
have a unique genetic organization in that they share a common central
region at which the two different DNA-binding proteins probably act.
Interestingly, a fusion protein consisting of the Walker-type ParA
ATPase and Gfp was functional and oscillated in nucleoid regions
on a time scale of minutes. ParA-green fluorescent protein (Gfp)
oscillation depended on both ParB and
parC
but was
independent of
minCDE
. Point mutations in the Walker A
box motif simultaneously abolished plasmid stabilization and ParA-Gfp
oscillation. These observations raise the possibility that ParA
oscillation is prerequisite for active plasmid segregation.
The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers.
Authors: Rothman N , Bhatnagar V K , Hayes R B , Zenser T V , Kashyap S K , Butler M A , Bell D A , Lakshmi V , Jaeger M , Kashyap R
Published: July 2002

Several epidemiologic studies indicate that NAT2-related slow N-acetylation increases bladder cancer risk among workers exposed to aromatic amines, presumably because N-acetylation is important for the detoxification of these compounds. Previously, we showed that NAT2 polymorphisms did not influence bladder cancer risk among Chinese workers exposed exclusively to benzidine (BZ), suggesting that NAT2 N-acetylation is not a critical detoxifying pathway for this aromatic amine. To evaluate the biologic plausibility of this finding, we carried out a cross-sectional study of 33 workers exposed to BZ and 15 unexposed controls in Ahmedabad, India, to evaluate the presence of BZ-related DNA adducts in exfoliated urothelial cells, the excretion pattern of BZ metabolites, and the impact of NAT2 activity on these outcomes. Four DNA adducts were significantly elevated in exposed workers compared to controls; of these, the predominant adduct cochromatographed with a synthetic N-(3'- phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine standard and was the only adduct that was significantly associated with total BZ urinary metabolites (r = 0.68, P < 0.0001). To our knowledge this is the first report to show that BZ forms DNA adducts in exfoliated urothelial cells of exposed humans and that the predominant adduct formed is N-acetylated, supporting the concept that monofunctional acetylation is an activation, rather than a detoxification, step for BZ. However, because almost all BZ-related metabolites measured in the urine of exposed workers were acetylated among slow, as well as rapid, acetylators (mean +/- SD 95 +/- 1.9% vs. 97 +/- 1.6%, respectively) and NAT2 activity did not affect the levels of any DNA adduct measured, it is unlikely that interindividual variation in NAT2 function is relevant for BZ-associated bladder carcinogenesis.
Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.
Authors: Lu T T , Yan L G , Madri J A
Published: July 2002

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.
Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.
Authors: Hung C F , Holzmacher R , Connolly E , Berenbaum M R , Schuler M A
Published: July 2002

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.
Characterization of avian T-cell receptor γ genes
Authors: Six Adrien , Rast Jonathan P. , McCormack Wayne T. , Dunon Dominique , Courtois David , Li Yue , Chen Chen-lo H. , Cooper Max D.
Published: July 2002

In birds and mammals T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the γδ T-cell lineage, the present studies sought to define the chicken TCRγ locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR γ chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCRγ cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a γδ T-cell line, but not in B or αβ T-cell lines. Three multimember Vγ subfamilies, three Jγ gene segments, and a single constant region Cγ gene were identified in the avian TCRγ locus. Members of each of the three Vγ subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCRγ repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-Jγ recombination, nuclease activity, and P- and N-nucleotide additions to generate a diverse repertoire of avian TCRγ genes early in ontogeny.
Nitric oxide-related species inhibit evoked neurotransmission but enhance spontaneous miniature synaptic currents in central neuronal cultures
Authors: Pan Zhuo-Hua , Segal Michael M. , Lipton Stuart A.
Published: July 2002

Nitric oxide (NO
·
) does not react significantly
with thiol groups under physiological conditions, whereas a variety of
endogenous NO donor molecules facilitate rapid transfer to
thiol of nitrosonium ion (NO
+
, with one less electron than
NO
·
). Here, nitrosonium donors are shown to decrease
the efficacy of evoked neurotransmission while increasing the frequency
of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In
contrast, pure NO
·
donors have little effect (displaying
at most only a slight increase) on the amplitude of evoked EPSCs and
frequency of spontaneous mEPSCs in our preparations. These findings may
help explain heretofore paradoxical observations that the NO moiety can
either increase, decrease, or have no net effect on synaptic activity
in various preparations.
Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.
Authors: Sanchez Y , Lovell M , Marin M C , Wong P E , Wolf-Ledbetter M E , McDonnell T J , Killary A M
Published: July 2002

Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma.
Local densities orthogonal to beta-sheet amide planes: patterns of packing in globular proteins.
Authors: Beardsley D S , Kauzmann W J
Published: July 2002

We have investigated the efficiency of packing by calculating intramolecular packing density above and below peptide planes of internal beta-pleated sheet residues in five globular proteins. The orientation of interest was chosen to allow study of regions that are approximately perpendicular to the faces of beta-pleated sheets. In these locations, nonbonded van der Waals packing interactions predominate over hydrogen bonding and solvent interactions. We observed considerable variability in packing densities within these regions, confirming that the interior packing of a protein does not result in uniform occupation of the available space. Patterns of fluctuation in packing density suggest that the regular backbone-to-backbone network of hydrogen bonds is not likely to be interrupted to maximize van der Waals interactions. However, high-density packing tends to occur toward the ends of beta-structure strands where hydrogen bonds are more likely to involve nonpolar side-chain groups or solvent molecules. These features result in internal protein folding with a central low-density core surrounded by a higher-density subsurface shell, consistent with our previous calculations regarding overall protein packing density.
Ligand-induced overexpression of a constitutively active β
2
-adrenergic receptor: Pharmacological creation of a phenotype in transgenic mice
Authors: Samama Philippe , Bond Richard A. , Rockman Howard A. , Milano Carmelo A. , Lefkowitz Robert J.
Published: July 2002

Transgenic overexpression (40- to 100-fold) of the wild-type human β
2
-adrenergic receptor in the hearts of mice leads to a marked increase in cardiac contractility, which is apparently due to the low level of spontaneous (i.e., agonist-independent) activity inherent in the receptor. Here we report that transgenic mice expressing a mutated constitutively active form of the receptor (CAM) show no such phenotype, owing to its modest expression (3-fold above endogenous cardiac β-adrenergic receptor levels). Surprisingly, treatment of the animals with a variety of β-adrenergic receptor ligands leads to a 50-fold increase in CAM β
2
-adrenergic receptor expression, by stabilizing the CAM β
2
-adrenergic receptor protein. Receptor up-regulation leads in turn to marked increases in adenylate cyclase activity, atrial tension determined
in vitro
, and indices of cardiac contractility determined
in vivo
. These results illustrate a novel mechanism for regulating physiological responses, i.e., ligand-induced stabilization of a constitutively active but inherently unstable protein.
The antisense
bcl-2
–
IgH
transcript is an optimal target for synthetic oligonucleotides
Authors: Morelli Susanna , Delia Domenico , Capaccioli Sergio , Quattrone Alessandro , Schiavone Nicola , Bevilacqua Anna , Tomasini Silvia , Nicolin Angelo
Published: July 2002

In most human follicular B cell lymphomas the
bcl-2
gene is up-regulated as a result of the t(14;18) chromosomal translocation generating a hybrid
bcl-2–IgH
mRNA. Recently, we have identified in t(14;18)-positive cells a
bcl-2–IgH
mRNA in the antisense orientation, putatively responsible for the overexpression of
bcl-2
. Herein we show that this chimeric antisense transcript is an optimal target for synthetic oligodeoxynucleotides (ODNs). A variety of sense-oriented oligonucleotides have been designed that target the antisense transcript in regions endowed with a sequence specificity presumably restricted to an individual cell line (the
bcl-2–IgH
fusion regions) or extended to all t(14;18) cells (the ectopic
bcl-2
segment upstream from the major breakpoint region and the
IgH
segment). All sense-oriented ODNs complementary to the antisense transcript induced an early strong inhibition of cell growth and a late fulminant cell death. As expected, the activity of ODNs targeting the fusion region was restricted to each individual cell line, whereas the activity of all ODNs targeting the common
bcl-2
and
IgH
segments was extended to all t(14;18) cell lines tested. These sense ODNs were not effective in untranslocated cell lines. Antisense-oriented ODNs, complementary to the
bcl-2–IgH
mRNA, and control ODNs (scrambled, inverted, or mismatched) were biologically ineffective. The selectivity and efficacy of all sense ODNs tested provide support for the development of therapeutic ODNs targeting the
bcl-2–IgH
antisense transcript expressed in human follicular lymphomas.
Responses in the brain of estrogen receptor α-disrupted mice
Authors: Shughrue Paul J. , Lubahn Dennis B. , Negro-Vilar Andres , Korach Kenneth S. , Merchenthaler Istvan
Published: July 2002

These studies sought to determine if neurons in the estrogen receptor-α knockout (ERαKO) mouse brain concentrated 16α-[
125
I]iodo-11β-methoxy-17β-estradiol (
125
I-estrogen), and if so, whether estrogen binding augmented the expression of progesterone receptor (PR) mRNA. Mice were injected with
125
I-estrogen and cryostat sections thaw mounted onto emulsion-coated slides. After 30–90 days of exposure, cells with a nuclear uptake and retention of
125
I-estrogen were observed in a number of ERαKO mouse brain regions including the preoptic nucleus and arcuate nucleus of the hypothalamus, bed nucleus of the stria terminalis, and amygdala, although the number of labeled cells and intensity of nuclear concentration was markedly attenuated when compared with wild-type littermates. Competition studies with excess 17β-estradiol, diethylstilbestrol, or moxestrol, but not with R5020 or dihydrotestosterone, prevented the nuclear concentration of
125
I-estrogen. To determine if the low level of estrogen binding was capable of regulating gene expression,
in situ
hybridization was used to evaluate PR mRNA in the brain. ERαKO and wild-type mice were ovariectomized and treated with vehicle or 17β-estradiol, and brains were sectioned and hybridized with a PR cRNA probe. Analysis of hybridization signal revealed a similar, low level of PR mRNA in ovariectomized wild-type and homozygous mice, and a marked increase in expression after treatment of ovariectomized animals with 17β-estradiol, with the level of hybridization signal being significantly higher in wild-type animals when compared with ERαKO mice. The results demonstrate that estrogen binds in the ERαKO brain and is capable of modulating PR gene expression, thus supporting the presence and functionality of a nonclassical estrogen receptor.
Australian lungfish neurohypophysial hormone genes encode vasotocin and [Phe
2
]mesotocin precursors homologous to tetrapod-type precursors
Authors: Hyodo Susumu , Ishii Susumu , Joss Jean M. P.
Published: July 2002

In view of the well-established role of neurohypophysial hormones in osmoregulation of terrestrial vertebrates, lungfishes are a key group for study of the molecular and functional evolution of the hypothalamo-neurohypophysial system. Here we report on the primary structure of the precursors encoding vasotocin (VT) and [Phe
2
]mesotocin ([Phe
2
]MT) of the Australian lungfish,
Neoceratodus forsteri
. Genomic sequence analysis and Northern blot analysis confirmed that [Phe
2
]MT is a native oxytocin family peptide in the Australian lungfish, although it has been reported that the lungfish neurohypophysis contains MT. The VT precursor consists of a signal peptide, VT, that is connected to a neurophysin by a Gly-Lys-Arg sequence, and a copeptin moiety that includes a Leu-rich core segment and a glycosylation site. In contrast, the [Phe
2
]MT precursor does not contain a copeptin moiety. These structural features of the lungfish precursors are consistent with those in tetrapods, but different from those in teleosts where both VT and isotocin precursors contain a copeptin-like moiety without a glycosylation site at the carboxyl terminals of their neurophysins. Comparison of the exon/intron organization also supports homology of the lungfish [Phe
2
]MT gene with tetrapod oxytocin/MT genes, rather than with teleost isotocin genes. Moreover, molecular phylogenetic analysis shows that neurohypophysial hormone genes of the lungfish are closely related to those of the toad. The present results along with previous morphological findings indicate that the hypothalamo-neurohypophysial system of the lungfish has evolved along the tetrapod lineage, whereas the teleosts form a separate lineage, both within the class
Osteichthyes
.
In vitro
reconstruction of the
Aspergillus
(=
Emericella
)
nidulans
genome
Authors: Prade Rolf A. , Griffith James , Kochut Krys , Arnold Jonathan , Timberlake William E.
Published: July 2002

A physical map of the 31-megabase
Aspergillus nidulans
genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the
A. nidulans
genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.
Behavioral effects of estrogen receptor gene disruption in male mice
Authors: Ogawa Sonoko , Lubahn Dennis B. , Korach Kenneth S. , Pfaff Donald W.
Published: July 2002

Gonadal steroid hormones regulate sexually dimorphic development of brain functions and behaviors. Their nuclear receptors offer the opportunity to relate molecular events in neurons to simple instinctive mammalian behaviors. We have determined the role of estrogen receptor (ER) activation by endogenous estrogen in the development of male-typical behaviors by the use of transgenic estrogen-receptor-deficient (ERKO) mice. Surprisingly, in spite of the fact that they are infertile, ERKO mice showed normal motivation to mount females but they achieved less intromissions and virtually no ejaculations. Aggressive behaviors were dramatically reduced and male-typical offensive attacks were rarely displayed by ERKO males. Moreover, ER gene disruption demasculinized open-field behaviors. In the brain, despite the evident loss of functional ER protein, the androgen-dependent system appears to be normally present in ERKO mice. Together, these findings indicate that ER gene expression during development plays a major role in the organization of male-typical aggressive and emotional behaviors in addition to simple sexual behaviors.
Detection of changes in pairwise interactions during allosteric transitions: Coupling between local and global conformational changes in GroEL
Authors: Aharoni Amir , Horovitz Amnon
Published: July 2002

A protein engineering approach for detecting and measuring local conformational changes that accompany allosteric transitions in proteins is described. Using this approach, we can identify interactions that are made or broken during allosteric transitions. The method is applied to probe for changes in pairwise interactions in the chaperonin GroEL during its ATP-induced allosteric transitions. Two pairwise interactions are investigated: one between subunits (Asp-41 with Thr-522) and the other within subunits (Glu-409 with Arg-501). We find that the intraring intersubunit interaction between Asp-41 and Thr-522 changes little during the allosteric transitions of GroEL, indicating that the hydrogen bond between these residues is maintained. In contrast, the intrasubunit salt bridge between Glu-409 and Arg-501 becomes significantly weaker during the ATP-induced allosteric transitions of GroEL. Our results are consistent with the electron microscopy observations of an ATP-induced hinge movement of the apical domains relative to the equatorial domains.
Minus-strand origin of filamentous phage versus transcriptional promoters in recognition of RNA polymerase
Authors: Higashitani Atsushi , Higashitani Nahoko , Horiuchi Kensuke
Published: July 2002

Replication of complementary-strand DNA in filamentous phages is initiated by a primer RNA that is synthesized at the minus-strand origin on the viral single-stranded DNA by
Escherichia coli
RNA polymerase holoenzyme containing the σ
70
subunit. We have demonstrated that the affinity of RNA polymerase
in vitro
to the origin is about 16-fold higher than that to the
lac
UV5 promoter. We have also shown that the temperature dependence of the primer RNA synthesis is much lower than that of
lac
UV5 transcription. The high affinity of RNA polymerase to the origin depends on the single strandedness of the “−10 region.” A nucleotide sequence of the nontemplate strand in the −10 region was found to be important for the function, but that of the template strand was not. These observations suggest that σ
70
subunit directly interacts with the single-stranded nontemplate strand containing adenine residue(s) at the −10 region of promoter.
Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate “hit-and-run” oncogenic transformation in cooperation with the adenovirus E1A proteins
Authors: Shen Yuqiao , Zhu Hua , Shenk Thomas
Published: July 2002

Some epidemiological studies have suggested a possible link between human cytomegalovirus (HCMV) infection and various malignancies, and HCMV has been shown to transform cultured cells. However, viral DNA is not detected in most transformants, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. Here we show that the HCMV immediate early
1
and
2
genes can cooperate with the adenovirus
E1A
gene to generate transformed foci of primary baby rat kidney cells. HCMV gene expression is transient and viral DNA is not present in clonal cell lines derived from the transformed foci. We find that the HCMV immediate early proteins are mutagenic, and we propose that HCMV has the potential to contribute to oncogenesis through a “hit-and-run” mechanism, by inducing mutations in cellular genes.
Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin-dependent diabetes mellitus
Authors: Rondinone Cristina M. , Wang Ling-Mei , Lonnroth Peter , Wesslau Christian , Pierce Jacalyn H. , Smith Ulf
Published: July 2002

The large docking protein IRS-1 is a major substrate for the insulin receptor and other tyrosine kinases. It plays a key role in eliciting many of insulin’s actions, including binding and activation of phosphatidylinositol (PI) 3-kinase and the subsequent increase in glucose transport. Gene disruption of IRS-1 in mice is associated with an impaired insulin-stimulated glucose disposal
in vivo
and glucose transport
in vitro
, but the survival of the animals and residual insulin sensitivity is dependent on the presence of the alternative docking protein IRS-2. We examined the expression and function of IRS-1 and IRS-2 in adipocytes from healthy and diabetic individuals. Cells from subjects with non-insulin-dependent diabetes mellitus (NIDDM), but not with insulin-dependent diabetes mellitus, had an impaired insulin effect and a marked reduction (70 ± 6%) in the expression of IRS-1 protein, whereas IRS-2 was unchanged. In normal cells, IRS-1 was the main docking protein for the binding and activation of insulin-stimulated PI 3-kinase; IRS-2 was also functional but required a higher insulin concentration for a similar binding and activation of PI 3-kinase. In contrast in NIDDM cells with a low IRS-1 content, IRS-2 became the main docking protein. These findings may provide important reasons for the insulin resistance in NIDDM.
Control of retinoblastoma protein-independent hematopoietic cell cycle by the pRB-related p130
Authors: Hoshikawa Yutaka , Mori Akio , Amimoto Keiko , Iwabe Koji , Hatakeyama Masanori
Published: July 2002

The retinoblastoma tumor suppressor protein (pRB) is a potent inhibitor of mammalian cell growth and the functional inactivation of pRB is widely presumed to be essential for progression of the cell cycle from G
1
phase. In this work, the generality of pRB-based cell cycle control in mammalian cells was addressed by conditionally expressing pRB in cytokine-dependent hematopoietic cells. We show herein that these cells are able to progress through the cell cycle in response to cytokine despite the continued presence of supraphysiological amounts of wild-type pRB or phosphorylation-resistant pRB mutants. However, their growth was strongly blocked by ectopic expression of the pRB-related pocket protein, p130. This growth inhibition required the E2F-binding pocket domain but not the cyclin-binding domain of p130. Furthermore, increased amounts of the p130-controlled E2F, termed E2F-4, potentiated the mitogenic response of the cells to cytokine and the constitutive overexpression of E2F-4 rendered the cells cytokine-independent. Our results indicate the existence of a non-pRB-based cell cycle whose operation depends primarily on the interplay between p130 and E2F-4 in certain hematopoietic cells.
Inhibition of intrathymic T cell development by expression of a transgenic antagonist peptide
Authors: Levelt Christiaan N. , Mizoguchi Emiko , Huang Xin , Zacks Rebecca , Bhan Atul K. , Tonegawa Susumu
Published: July 2002

The mature T cell receptor (TCR) repertoire is shaped by positive- and negative-selection events taking place during T cell development. These events are regulated by interactions between the TCR and major histocompatibility complex molecules presenting self-peptides. It has been shown that many antagonist peptides are efficient at mediating positive selection. In this study we analyzed the effects of a transgene encoding an antagonist peptide (influenza NP34) that is presented by H-2Dbin a Tap-1-independent fashion in mice expressing the influenza NP68-specific TCR F5. We find that the transgenic peptide does not mediate positive or negative selection in F5+Tap-1−/−mice, but inhibits maturation of CD8+single positive thymocytes in F5+Tap-1+mice without inducing signs of negative selection. We conclude that antagonism of antigen recognition occurs not only at the level of mature T cells but also in T cell development.
Activation and repression of transcription by auxin-response factors
Authors: Ulmasov Tim , Hagen Gretchen , Guilfoyle Tom J.
Published: July 2002

Auxin-response factors (ARFs) bind with specificity to TGTCTC auxin-response elements (AuxREs), which are found in promoters of primary/early auxin-response genes. Nine different ARFs have been analyzed for their capacity to activate or repress transcription in transient expression assays employing auxin-responsive
GUS
reporter genes. One ARF appears to act as a repressor. Four ARFs function as activators and contain glutamine-rich activation domains. To achieve transcriptional activation on TGTCTC AuxREs in transient expression assays, ARFs require a conserved dimerization domain found in both ARF and Aux/IAA proteins, but they do not absolutely require their DNA-binding domains. Our results suggest that ARFs can activate or repress transcription by binding to AuxREs directly and that selected ARFs, when overexpressed, may potentiate activation further by associating with an endogenous transcription factor(s) (e.g., an ARF) that is bound to AuxREs. Transfection experiments suggest that TGTCTC AuxREs are occupied regardless of the auxin status in cells and that these occupied AuxREs are activated when exogenous auxin is applied to cells or when ARF activators are overexpressed. The results provide new insight into mechanisms involved with auxin regulation of primary/early-response genes.
Detoxification of environmental mutagens and carcinogens: Structure, mechanism, and evolution of liver epoxide hydrolase
Authors: Argiriadi Maria A. , Morisseau Christophe , Hammock Bruce D. , Christianson David W.
Published: July 2002

The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC
3.3.2.3
) has been determined at 2.8-Å resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the α/β hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.
Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies
Authors: Xu Rong , Ayers Brenda , Cowburn David , Muir Tom W.
Published: July 2002

A convenient
in vitro
chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with
15
N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation
in vitro
, will increase the size limits for protein structural determination in solution with NMR methods.
In vitro
chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of
Rhodobacter sphaeroides
Authors: van Brederode Marion E. , van Mourik Frank , van Stokkum Ivo H. M. , Jones Michael R. , van Grondelle Rienk
Published: July 2002

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium
Rhodobacter sphaeroides
. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (B
A
*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P
+
H
A
−
. In experiments in which P or B
A
was selectively excited at 880 nm or 796 nm, respectively, the formation of P
+
H
A
−
was associated with similar time constants of 1.5 ps and 1.7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P
+
B
A
−
and P* is formed in parallel from B
A
* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (B
B
) and the high exciton component of P (P
+
) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.
Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells
Authors: Heinrich Ralf , Cromarty Stuart I. , Hörner Michael , Edwards Donald H. , Kravitz Edward A.
Published: July 2002

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.
Intercellular adhesion molecule-1 inhibits interleukin 4 production by naive T cells
Authors: Luksch Claudia R. , Winqvist Ola , Ozaki Minette E. , Karlsson Lars , Jackson Michael R. , Peterson Per A. , Webb Susan R.
Published: July 2002

The type of cytokines produced during T cell responses determines susceptibility or resistance to many pathogens and influences the development of autoimmunity and allergy. To define the role of individual accessory molecules in cytokine production during primary immune responses,Drosophilacell lines expressing murine major histocompatibility complex class II molecules with defined combinations of accessory molecules were used to present peptide antigen to naive T cell receptor transgenic T cells. Significantly, expression of B7.1 or B7.2 without additional accessory molecules led to very high production of interleukin (IL)-4, which contrasted with minimal IL-4 production elicited by conventional antigen presenting cells (APC). However, coexpression of ICAM-1 and B7 onDrosophilaAPC induced little IL-4, suggesting an inhibitory role for intercellular adhesion molecule-1 (ICAM-1). In support of this idea, stimulation of T cell receptor transgenic T cells with peptide presented by splenic APC devoid of ICAM-1 (from ICAM-1-deficient mice) led to high IL-4 production. Thus, the level of IL-4 production by naive CD4+T cells during typical primary responses appears to be controlled, at least in part, by T–APC interactions involving ICAM-1.
Generation of
in vivo
activating factors in the ischemic intestine by pancreatic enzymes
Authors: Mitsuoka Hiroshi , Kistler Erik B. , Schmid-Schönbein Geert W.
Published: July 2002

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl
p
-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.
Seminar In Practical Management: A practical guide for those involved in office‐based methadone and other prescribing for addiction and pain management
Authors: BYRNE A. J.
Published: July 2002

This article aims to demonstrate that general practitioners can be involved in dependency treatment including methadone prescription in the normal course of their practice. As long as specialist and hospital support is available for different cases, GPs can look after dependency and chronic pain problems just as they look after schizophrenia, diabetes and depression.
Allocating livers to substance and alcohol misusers
Authors: NEUBERGER JAMES
Published: July 2002

The success of liver transplantation for the treatment of patients with end‐stage liver disease has resulted in a widening gap between the number of potential recipients and the numbers of grafts available. Allocation of these scarce resources to people who have developed liver failure as a consequence of their own and often illegal behaviour has attracted much controversy. For patients with alcoholic liver disease, there is relatively little evidence that many patients return to a damaging pattern of alcohol consumption and, at least in the short term, the outcome is no different compared with patients grafted for other causes of cirrhosis. There are well‐validated markers which predict abstinence. For abusers of other substances, there is relatively little experience. Of concern is the variation between the priority‐setting by the medical profession and the general public. The latter, who, in the UK, can be considered as providers of the donated organs and pay for the costs of the procedure, tend to rate patients with alcoholic liver disease and who have drug or substance abuse at a lower priority than those who develop liver failure from other causes. These differences need further debate and resolution.