bluuebunny/crossref_metadata_2025 huggingface dataset:
Aggregate sound velocities and acoustic Grüneisen parameter of iron up to 300 GPa and 1,200 K
Authors: Dubrovinsky L. S. , Dubrovinskaia N. A. , Le Bihan T.
Published: July 2002

Successful interpretation of available geophysical data requires experimental and theoretical information on the elasticity of solids under physical conditions of Earth's interior. Because iron is considered as major component in Earth's core, elastic properties of iron at high pressures and temperatures are very important for modeling its composition and dynamics. We usein situx-ray diffraction data on ɛ-iron at static pressures up to 300 GPa and temperatures to 1,200 K to determine the Debye–Waller temperature factors and calculate aggregate sound velocities and Grüneisen parameter of ɛ-iron by using an approach that is based on Rietveld refinement at high pressures and temperatures.
Expression and phylogeny of claudins in vertebrate primordia
Authors: Kollmar Richard , Nakamura Shashi Karia , Kappler James A. , Hudspeth A. J.
Published: July 2002

Claudins, the major transmembrane proteins of tight junctions, are members of the tetraspanin superfamily of proteins that mediate cellular adhesion and migration. Their functional importance is demonstrated by mutations in claudin genes that eliminate tight junctions in myelin and the testis, abolish Mg
2+
resorption in the kidney, and cause autosomal recessive deafness. Here we report that two paralogs among 15 claudin genes in the zebrafish,
Danio rerio
, are expressed in the otic and lateral-line placodes at their earliest stages of development. Related claudins in amphibians and mammals are expressed in a similar manner in vertebrate primordia such as sensory placodes, branchial arches, and limb buds. We also show that the claudin gene family may have expanded along the chordate stem lineage from urochordates to gnathostomes, in parallel with the elaboration of vertebrate characters. We propose that tight junctions not only form barriers in mature epithelia, but also participate in vertebrate morphogenesis.
Random matrices and quantum chaos
Authors: Kriecherbauer Thomas , Marklof Jens , Soshnikov Alexander
Published: July 2002

The theory of random matrices has far-reaching applications in many different areas of mathematics and physics. In this note, we briefly describe the state of the theory and two of the perhaps most surprising appearances of random matrices, namely in the theory of quantum chaos and in the theory of prime numbers.
Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer
Authors: Yamazaki Yukiko , Makino Hatsune , Hamaguchi-Hamada Kayoko , Hamada Shun , Sugino Hidehiko , Kawase Eihachiro , Miyata Takaki , Ogawa Masaharu , Yanagimachi Ryuzo , Yagi Takeshi
Published: July 2002

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells.
Inositol polyphosphate 4-phosphatase type I regulates cell growth downstream of transcription factor GATA-1
Authors: Vyas Paresh , Norris F. Anderson , Joseph Raji , Majerus Philip W. , Orkin Stuart H.
Published: July 2002

Megakaryocytes lacking transcription factor GATA-1 fail to complete
maturation
in vivo
and hyperproliferate. To define how
GATA-1 regulates megakaryocyte cell growth we searched for mRNA
transcripts expressed in primary wild-type, but not
GATA-1
−
, megakaryocytes. One differentially expressed
transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase
I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and
also has lesser activity against soluble analogues of this lipid,
inositol 3,4-bisphosphate and inositol 1,3,4-triphosphate.
Reintroduction of 4-Ptase I into both primary GATA-1
−
and
wild-type megakaryocytes significantly retards cell growth, suggesting
that absence of 4-Ptase I may contribute to the hyperproliferative
phenotype of GATA-1
−
megakaryocytes. Overexpression of
4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken
together, these data indicate that 4-Ptase I is a regulator of cell
proliferation.
On the origin of metacentric, attached-X (A-X) chromosomes in
Drosophila melanogaster
males
Authors: Green M. M. , Piergentili R.
Published: July 2002

We describe here the isolation and cytogenetic characterization of
a mutation
inseparabile
which generates in males a high
frequency of A-X females. The mutation, segregating in low frequency in
a laboratory stock, maps to cytological location 82F7–11 in the third
chromosome. The mutation acts premeiotically in the male germ line.
Disrupting the X chromosome centromeric heterochromatin suppresses the
formation of A-X chromosome, implying that the mutation is involved in
chromatid cohesion. The
inseparabile
mutation also
affects disjunction of the chromosome 4 in males. We suspect that the
mutation was responsible for the original A-X female found by L.
V. Morgan in 1921.
Histone tails modulate nucleosome mobility and regulate ATP-dependent nucleosome sliding by NURF
Authors: Hamiche Ali , Kang Ju-Gyeong , Dennis Cynthia , Xiao Hua , Wu Carl
Published: July 2002

Nu
cleosome
R
emodeling
F
actor (NURF) is an ATP-dependent nucleosome remodeling
complex that alters chromatin structure by catalyzing nucleosome
sliding, thereby exposing DNA sequences previously associated with
nucleosomes. We systematically studied how the unstructured N-terminal
residues of core histones (the N-terminal histone tails) influence
nucleosome sliding. We used bacterially expressed
Drosophila
histones to reconstitute hybrid nucleosomes
lacking one or more histone N-terminal tails. Unexpectedly, we found
that removal of the N-terminal tail of histone H2B promoted uncatalyzed
nucleosome sliding during native gel electrophoresis. Uncatalyzed
nucleosome mobility was enhanced by additional removal of other histone
tails but was not affected by hyperacetylation of core histones by
p300. In addition, we found that the N-terminal tail of the histone H4
is specifically required for ATP-dependent catalysis of nucleosome
sliding by NURF. Alanine scanning mutagenesis demonstrated that H4
residues 16-KRHR-19 are critical for the induction of nucleosome
mobility, revealing a histone tail motif that regulates NURF activity.
An exchange of histone tails between H4 and H3 impaired NURF-induced
sliding of the mutant nucleosome, indicating that the location of the
KRHR motif in relation to global nucleosome structure is functionally
important. Our results provide functions for the N-terminal
histone tails in regulating the mobility of nucleosomes.
Restricted chromosomal silencing in nucleolar dominance
Authors: Lewis Michelle S. , Pikaard Craig S.
Published: July 2002

Failure of one parent's chromosomes to organize nucleoli in an
interspecific hybrid is an epigenetic phenomenon known as nucleolar
dominance. Selective gene silencing on a scale of millions of bp
is known to be involved, but the full extent to which nucleolus
organizer region (NOR)-bearing chromosomes are inactivated beyond the
NORs is unknown. Aided by genome sequence data for
Arabidopsis
thaliana
, we have mapped the extent of nucleolar
dominance-induced silencing in
Arabidopsis suecica
, the
allotetraploid hybrid of
A. thaliana
and
Arabidopsis arenosa
. Using a sensitive reverse
transcription PCR assay, we show that the four
A.
thaliana
NORs, each ≈4 Mbp in size, are ≈99.5% silenced in
A. suecica
vegetative leaves, whereas the NORs inherited
from
A. arenosa
remain fully active. The two
A.
thaliana
NORs,
NOR2
and
NOR4,
abut the telomeres on chromosomes 2 and 4, thus there are no genes
distal to the NORs. The three protein-coding genes nearest
NOR4
on its centromere-proximal side, the closest of
which is only 3.1 kb from rRNA gene sequences, are shown to be
transcribed in the hybrid despite the silencing of the adjacent
≈4-Mbp NOR. These data argue against hypotheses in which NOR
inactivation is attributed to the spread of silencing from adjacent
chromosomal regions, but favor models in which NORs or rRNA genes are
the targets of regulation.
A new hemoglobin gene from soybean: a role for hemoglobin in all plants.
Authors: Anderson C R , Jensen E O , LLewellyn D J , Dennis E S , Peacock W J
Published: July 2002

We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.
Dynamic contrast-enhanced magnetic resonance imaging reveals stress-induced angiogenesis in MCF7 human breast tumors.
Authors: Furman-Haran E , Margalit R , Grobgeld D , Degani H
Published: July 2002

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T2-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.
Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.
Authors: Rickman D W , Rickman C B
Published: July 2002

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.
Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.
Authors: Parangi S , O'Reilly M , Christofori G , Holmgren L , Grosfeld J , Folkman J , Hanahan D
Published: July 2002

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.
Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.
Authors: Griscavage J M , Wilk S , Ignarro L J
Published: July 2002

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.
cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC6803.
Authors: Katoh A , Lee K S , Fukuzawa H , Ohyama K , Ogawa T
Published: July 2002

We have isolated mutants of Synechocystis PCC6803 that grew very slowly in a low-sodium medium, which is unfavorable for HCO3(-) transport, and examined two of these mutants (SC1 and SC2) for their ability to take up CO2 and HCO3(-) in the light. The CO2 transport activity of SC1 and SC2 was much lower than that of the wild type (WT), whereas there was no difference between the mutants and the WT in their activity of HCO3(-) transport. A clone containing a 3.9-kilobase-pair insert DNA that transforms both mutants to the WT phenotype was isolated from a genomic library of WT Synechocystis. Sequencing of the insert DNA in the region of mutations in SC1 and SC2 revealed an open reading frame (designated cotA), which showed significant amino-acid sequence homology to cemA encoding a protein found in the inner envelope membrane of chloroplasts. The cotA gene is present in a single copy and was not cotranscribed with any other gene(s). cotA encodes a protein of 247 amino acids containing four transmembrane domains. There was substitution of a single base in SC1 and two bases in SC2 in their cotA genes. A possible role of the cotA gene product in CO2 transport is discussed.
Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells.
Authors: James G , Goldstein J L , Brown M S
Published: July 2002

Benzodiazepine (BZA)-5B, a CAAX farnesyl-transferase inhibitor, was previously shown to block the farnesylation of H-Ras and to reverse the transformed morphology of Rat1 cells expressing oncogenic H-RasV12. Non-transformed Rat1 cells were not affected by BZA-5B, suggesting that they produce a form of Ras whose prenylation is not blocked by this compound. The likely candidate is K-RasB, which differs from H-Ras primarily in the terminal 24 amino acids. In the current study we examined the effect of BZA-5B on the prenylation of a chimeric oncogenic Ras protein designated H/K-RasBV12, consisting of the first 164 amino acids of H-RasV12 followed by the last 24 amino acids of K-RasB. BZA-5B failed to block the prenylation of this chimera and was thus unable to reverse the transformed morphology of Rat1 cells in which it was expressed. Another potent inhibitor of H-Ras farnesylation, L-739,749, also failed to block prenylation of H/K-RasBV12. Similar results were obtained in transfected cells expressing a widely used version of K-RasBV12 containing a 10-amino acid extension at its NH2 terminus. Neither BZA-5B nor L-739,749 reversed the transformed morphology of cells expressing H/K-RasBV12. The resistance of K-RasB to farnesyltransferase inhibition provides a likely explanation for the resistance of nontransformed cells to the growth inhibitory effects of BZA-5B and L-739,749.
Detection of residue contacts in a protein folding intermediate
Authors: Balbach Jochen , Forge Vincent , Lau Wai Shun , Jones Jonathan A. , van Nuland Nico A. J. , Dobson Christopher M.
Published: July 2002

Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with α-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.
Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver
Authors: Dabeva Mariana D. , Hwang Seong-Gyu , Vasa Srinivasa Rao G. , Hurston Ethel , Novikoff Phyllis M. , Hixson Douglas C. , Gupta Sanjeev , Shafritz David A.
Published: July 2002

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.
The
Xenopus
proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties
Authors: Irwin David M. , Satkunarajah Malathy , Wen Yi , Brubaker Patricia L. , Pederson Raymond A. , Wheeler Michael B.
Published: July 2002

The proglucagon gene encodes several hormones that have key roles in the regulation of metabolism. In particular, glucagon-like peptide (GLP-1), a potent stimulus of insulin secretion, is being developed as a therapy for the treatment of non-insulin-dependent diabetes mellitus. To define structural moieties of the molecule that convey its insulinotropic activity, we have cloned and characterized the proglucagon gene from the amphibian,
Xenopus laevis
. Unexpectedly, these cDNAs were found to encode three unique glucagon-like-1 peptides, termed xenGLP-1A, xenGLP-1B, and xenGLP-1C in addition to the typical proglucagon-derived hormones glucagon and GLP-2. xenGLP-1A, -1B, and -1C were synthesized and tested for their ability to bind and activate the human GLP-1 receptor (hGLP-1R), and to stimulate insulin release from rat pancreas. All three
Xenopus
GLP-1-like peptides bind effectively to the hGLP-1R and stimulate cAMP production. Surprisingly, xenGLP-1B(1–30) demonstrated higher affinity for the hGLP-1R than hGLP-1 (IC
50
of 1.1 ± 0.4 nM vs. 4.4 ± 1.0 nM, respectively,
P
< 0.02) and was equipotent to hGLP-1 in stimulating cAMP production (EC
50
of 0.17 ± 0.02 nM vs. 0.6 ± 0.2 nM, respectively,
P
> 0.05). Further studies demonstrated that hGLP-1, xenGLP-1A, -1B, and -1C stimulate comparable insulin release from the pancreas. These results demonstrate that despite an average of nine amino acid differences between the predicted
Xenopus
GLPs and hGLP-1, all act as hGLP-1R agonists.
Opioid receptors from a lower vertebrate (
Catostomus commersoni
): Sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution
Authors: Darlison Mark G. , Greten Florian R. , Harvey Robert J. , Kreienkamp Hans-Jürgen , Stühmer Thorsten , Zwiers Henk , Lederis Karl , Richter Dietmar
Published: July 2002

The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish
Catostomus commersoni
. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian μ-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the μ-selective agonist [
d
-Ala
2
, MePhe
4
-Gly-ol
5
]enkephalin (DAMGO) (
K
d
= 0.63 ± 0.15 nM) and for the nonselective antagonist naloxone (
K
d
= 3.1 ± 1.3 nM). The receptor had negligible affinity for U50488 and [
d
-Pen
2
,
d
-Pen
5
]enkephalin (DPDPE), which are κ- and δ-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 μM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in
Xenopus
oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the μ-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of ≈400 million years implying that it fulfills an important physiological role.
Oxytocin stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamic explants by releasing nitric oxide
Authors: Rettori V. , Canteros G. , Renoso R. , Gimeno M. , McCann S. M.
Published: July 2002

Oxytocin induces mating behavior in rats of both sexes. Previous experiments revealed that progesterone-induced sex behavior in ovariectomized, estrogen-primed rats was caused by release of NO from NOergic neurons that stimulated the release of luteinizing hormone-releasing hormone (LHRH). The LHRH activated brain-stem neurons that initiated the lordosis reflex. We hypothesized that oxytocin might similarly release NO in the medial basal hypothalamic region that would stimulate release of LHRH into the hypophyseal portal vessels to release luteinizing hormone. To investigate this hypothesis, medial basal hypothalamic explants were preincubated in Krebs–Ringer bicarbonate buffer for 30 min, followed by a 30-min incubation in fresh Krebs–Ringer bicarbonate buffer containing the compounds to be tested. Oxytocin stimulated LHRH release 3- to 4-fold at the lowest concentration tested (10
−10
M). Values remained at a plateau as the concentration was increased to 10
−7
M and then declined in a concentration-dependent manner, so that there was no stimulation with a concentration of 10
−5
M. Oxytocin (10
−7
M) stimulated release of prostaglandin E
2
into the medium, a finding consistent with a role of NO in the response. That NO indeed mediated the action of oxytocin was supported by blockade of the action of oxytocin by the competitive inhibitor of NO synthase (NOS),
N
G
-monomethyl-
l
-arginine (300 μM). Furthermore, oxytocin (10
−9
to 10
−7
M) activated NOS as measured at the end of the experiments. Oxytocin appeared to act to stimulate norepinephrine terminals in the medial basal hypothalamus, which activated NOS by α
1
-adrenergic receptors, because prazocine, an α
1
receptor blocker, inhibited the LHRH-releasing action of oxytocin. Finally, incubation of neural lobe explants with sodium nitroprusside, a NO releasor, revealed that nitroprusside (300–600 μM, but not 900 μM) inhibited oxytocin release. Therefore, the NO released by oxytocin also diffuses into the oxytocin neuronal endings and inhibits oxytocin release, forming a negative feedback loop. The results indicate that oxytocin is important not only in induction of mating, but also in stimulating LHRH release with subsequent luteinizing hormone discharge that plays a crucial role in reproduction.
Resistance to apoptosis in CTLL-2 cells constitutively expressing c-Myb is associated with induction of BCL-2 expression and Myb-dependent regulation ofbcl-2 promoter activity
Authors: Salomoni Paolo , Perrotti Danilo , Martinez Robert , Franceschi Claudio , Calabretta Bruno
Published: July 2002

c-Myb, the cellular homologue of the transforming gene of the avian myeloblastosis virus, is preferentially expressed in all hematopoietic lineages, including T and B lymphocyte lineages. In T lymphocytes, c-Myb expression appears to be required for cell cycle progression and proliferation. To further investigate the role of c-Myb in T cell proliferation and survival, interleukin (IL) 2-dependent CTLL-2 cells were transfected with a constitutively activec-mybor with ac-mybantisense construct able to down-regulate endogenous Myb levels, and the transfectants were assessed for proliferation and survival in low concentrations of IL-2 and for susceptibility to dexamethasone-induced apoptosis. Compared with control cells, CTLL-2 cells constitutively expressing c-Myb proliferate in low concentrations of IL-2 and are less susceptible to apoptosis induced by IL-2 deprivation or treatment with dexamethasone. In contrast, cells transfected with an antisensec-mybconstruct do not proliferate in low concentrations of IL-2 and undergo apoptosis upon IL-2 deprivation or dexamethasone treatment more rapidly than parental cells. Overexpression of c-Myb was accompanied by up-regulation of BCL-2 expression. In transient transfection assays, the murinebcl-2promoter was efficiently transactivated by c-Myb, but such effect was observed also in cells transfected with a DNA binding-deficientc-mybconstruct. Moreover, in gel retardation assays, a 38-bp oligomer in the shortestbcl-2promoter segment regulated by c-Myb formed a specific complex with nuclear extracts from c-Myb-transfected CTLL-2 cells. Thus, these results strongly suggest that c-Myb, in addition to regulating T cell proliferation, protects T lymphocytes from apoptosis by induction of BCL-2 expression, which involves a c-Myb-dependent mechanism of promoter regulation.
A structural model for GroEL–polypeptide recognition
Authors: Buckle Ashley M. , Zahn Ralph , Fersht Alan R.
Published: July 2002

A monomeric peptide fragment of GroEL, consisting of residues 191–376, is a mini-chaperone with a functional chaperoning activity. We have solved the crystal structure at 1.7 Å resolution of GroEL(191–376) with a 17-residue N-terminal tag. The N-terminal tag of one molecule binds in the active site of a neighboring molecule in the crystal. This appears to mimic the binding of a peptide substrate molecule. Seven substrate residues are bound in a relatively extended conformation. Interactions between the substrate and the active site are predominantly hydrophobic, but there are also four hydrogen bonds between the main chain of the substrate and side chains of the active site. Although the preferred conformation of a bound substrate is essentially extended, the flexibility of the active site may allow it to accommodate the binding of exposed hydrophobic surfaces in general, such as molten globule-type structures. GroEL can therefore help unfold proteins by binding to a hydrophobic region and exert a binding pressure toward the fully unfolded state, thus acting as an “unfoldase.” The structure of the mini-chaperone is very similar to that of residues 191–376 in intact GroEL, so we can build it into GroEL and reconstruct how a peptide can bind to the tetradecamer. A ring of connected binding sites is noted that can explain many aspects of substrate binding and activity.
“Autoimmune rejection” of neonatal heart transplants in experimental Chagas disease is a parasite-specific response to infected host tissue
Authors: Tarleton R. L. , Zhang L. , Downs M. O.
Published: July 2002

Infection with the protozoan parasiteTrypanosoma cruzioften results in chronic heart- and gut-associated disease known as Chagas disease. In this study we show that contrary to previous reports, neonatal hearts transplanted into mice chronically infected withT. cruzido not exhibit signs of autoimmune-type rejection or any significant inflammatory response. In addition to an absence of inflammation, these syngeneic heart transplants survive for more than 1 year and are absolutely free of parasites as determined byin situPCR analysis. However, if well-established transplanted hearts in chronically infected mice are directly injected with live parasites, a rapid and dramatic inflammatory response ensues that results in cessation of heart function. Likewise, transplanted hearts established in mice prior to systemic infection withT. cruzior hearts transplanted into mice during the acute stage ofT. cruziinfection become parasitized and develop inflammatory foci. In these cases where the transplanted hearts become parasitized, the ensuing inflammatory response is nearly identical to that observed in the native hearts ofT. cruzi-infected mice in terms of cell types present and adhesion molecules and cytokines expressed. Importantly, this response is strikingly different from that observed in the allogeneic heart rejection. These results clearly document that parasitization of heart tissue is both necessary and sufficient for the induction of tissue damage in Chagas disease and strongly argue against a principal autoimmune etiology for this disease.
Neuronal coincidence detection by voltage-sensitive electrical synapses
Authors: Edwards Donald H. , Yeh Shih-Rung , Krasne Franklin B.
Published: July 2002

Coincidence detection is important for functions as diverse as Hebbian learning, binaural localization, and visual attention. We show here that extremely precise coincidence detection is a natural consequence of the normal function of rectifying electrical synapses. Such synapses open to bidirectional current flow when presynaptic cells depolarize relative to their postsynaptic targets and remain open until well after completion of presynaptic spikes. When multiple input neurons fire simultaneously, the synaptic currents sum effectively and produce a large excitatory postsynaptic potential. However, when some inputs are delayed relative to the rest, their contributions are reduced because the early excitatory postsynaptic potential retards the opening of additional voltage-sensitive synapses, and the late synaptic currents are shunted by already opened junctions. These mechanisms account for the ability of the lateral giant neurons of crayfish to sum synchronous inputs, but not inputs separated by only 100 μsec. This coincidence detection enables crayfish to produce reflex escape responses only to very abrupt mechanical stimuli. In light of recent evidence that electrical synapses are common in the mammalian central nervous system, the mechanisms of coincidence detection described here may be widely used in many systems.
Evidence for a role of NF-κB in the survival of hematopoietic cells mediated by interleukin 3 and the oncogenic TEL/platelet-derived growth factor receptor β fusion protein
Authors: Besançon Françoise , Atfi Azeddine , Gespach Christian , Cayre Yvon E. , Bourgeade Marie-Françoise
Published: July 2002

Interleukin 3 (IL-3) and other hematopoietic cytokines transduce signals that stimulate DNA synthesis and cell survival. In certain chronic myelomonocytic leukemias, a TEL/platelet-derived growth factor receptor β (PDGFRβ) fusion protein is produced as a consequence of the t(5;12) translocation. It contains the amino terminus of the transcription factor TEL fused to the transmembranous and cytoplasmic domains of the PDGFRβ. It is oncogenic as it substitutes for IL-3, thus promoting cell growth and preventing apoptotic cell death. The mechanism by which TEL/PDGFRβ generates survival signals remains undefined. Here, we report that both IL-3 and TEL/PDGFRβ initiate a signaling cascade that leads to the activation of the transcriptional factor NF-κB. In fact, either cytokine deprivation of IL-3-dependent Ba/F3 cells or exposure of TEL/PDGFRβ-expressing cells to the specific inhibitor of the PDGFR tyrosine kinase, CGP53716, caused a strong decrease in NF-κB activity followed by extensive cell death. Further, treatment with the proteasome inhibitor Z-IE(
O-t-
Bu)A-leucinal suppressed IL-3 and TEL/PDGFRβ-dependent survival. The same result was seen upon overexpression of an unphosphorylable form of IκBα. Because both conditions inactivate NF-κB by preventing its translocation into the nucleus, that process seems to be essential for cell survival in response to IL-3 and TEL/PDGFRβ. Moreover, overexpression of a dominant-negative mutant of the protooncogene c-Myc, a downstream target of NF-κB, had a similar effect. We conclude that NF-κB plays an important role in maintaining cell survival in response to IL-3 and TEL/PDGFRβ and that c-Myc may be a downstream effector mediating this effect.
Targeted disruption of the Ca
2+
channel β
3
subunit reduces N- and L-type Ca
2+
channel activity and alters the voltagedependent activation of P/Q-type Ca
2+
channels in neurons
Authors: Namkung Yoon , Smith Stephen M. , Lee Seong Beom , Skrypnyk Nataliya V. , Kim Hyung-Lae , Chin Hemin , Scheller Richard H. , Tsien Richard W. , Shin Hee-Sup
Published: July 2002

In comparison to the well characterized role of the principal subunit of voltage-gated Ca
2+
channels, the pore-forming, antagonist-binding α
1
subunit, considerably less is understood about how β subunits contribute to neuronal Ca
2+
channel function. We studied the role of the Ca
2+
channel β
3
subunit, the major Ca
2+
channel β subunit in neurons, by using a gene-targeting strategy. The β
3
deficient (β
3
−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β
3
−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca
2+
channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β
3
subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β
3
−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca
2+
channels. The reduction in the expression of N-type Ca
2+
channels in the β
3
−/− mice may be expected to impair Ca
2+
entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.
In situ
synthesis of β-glucan microfibrils on tobacco plasma membrane sheets
Authors: Hirai Noriko , Sonobe Seiji , Hayashi Takahisa
Published: July 2002

A major concern in plant morphogenesis is whether cortical microtubules are responsible for the arrangement and action of β-glucan synthases in the plasma membrane. We prepared isolated plasma membrane sheets with cortical microtubules attached and tested whether β-glucan synthases penetrated through the membrane to form microfibrils and whether these synthases moved in the fluid membrane along the cortical microtubules. This technique enabled us to examine synthesis of β-glucan as a fiber with a two-dimensional structure. The synthesis of β-glucan microfibrils was directed in arrays by cortical microtubules at many loci on the membrane sheets. The microfibrils were mainly arranged along the microtubules, but the distribution of microfibrils was not always parallel to that of the microtubules. The rate of β-glucan elongation as determined directly on the exoplasmic surface was 620 nm per min. When the assembly of microtubules was disrupted by treatment with propyzamide, the β-glucans were not deposited in arrays but in masses. This finding shows that the arrayed cortical microtubules are not required for β-glucan synthesis but are required for the formation of arranged microfibrils on the membrane sheet.
A distinct cyclin-dependent kinase-activating kinase of
Arabidopsis thaliana
Authors: Umeda Masaaki , Bhalerao Rishikesh P. , Schell Jeff , Uchimiya Hirofumi , Koncz Csaba
Published: July 2002

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an
Arabidopsis
cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA,
cak1At
, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant.
cak1At
encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An
Arabidopsis
CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13
suc1
, but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At.
cak1At
is a single-copy gene in
Arabidopsis
and is highly expressed in proliferating cells of suspension cultures.
Branching morphogenesis independent of mesenchymal–epithelial contact in the developing kidney
Authors: Qiao Jizeng , Sakurai Hiroyuki , Nigam Sanjay K.
Published: July 2002

Whether mesenchymal–epithelial interactions leading to branching morphogenesis in developing epithelial tissues such as the kidney require direct cell–cell contact or are due to soluble mediators elaborated by the inducing tissue has been the subject of much debate. Here we demonstrate that ureteric bud (UB) epithelium, from which the kidney collecting system and upper urinary tract are derived, can undergo impressive three-dimensional branching morphogenesis when cultured in the appropriate extracellular matrix context in the absence of direct contact with mesenchymal tissue, indicating that the program for branching morphogenesis is inherent to the UB. Both a soluble factor in BSN cell-conditioned medium (BSN-CM) derived from an immortalized cell line thought to originate in the early metanephric mesenchyme and glial cell line-derived neurotrophic factor (GDNF) were required for early and later events in branching morphogenesis. In the absence of BSN-CM, the isolated UB did not survive; a similar result was obtained in the presence of neutralizing antibodies against glial cell line-derived neurotrophic factor. Preliminary analysis of key activity present in BSN-CM indicates that it is a heat-sensitive, heparin-binding factor with a probable molecular mass greater than 100 kDa. When the
in vitro
cultured UB was recombined with freshly isolated metanephric mesenchyme, nephric units were induced in the mesenchyme, and the UB branches underwent elongation. Our data suggest that, although UB branching morphogenesis
per se
does not require direct mesenchymal contact, such contact may play a key role in regulating branch elongation and establishing the pattern of branching. The results also suggest an approach to
in vitro
engineering of nephron.
Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals
Authors: Qu Cheng-Kui , Yu Wen-Mei , Azzarelli Biagio , Feng Gen-Sheng
Published: July 2002

By using both genetic and biochemical approaches, we have investigated the physiological role of Shp-2, a cytoplasmic tyrosine phosphatase with two Src homology 2 domains, in signaling pathways downstream of epidermal growth factor receptor (EGF-R). In previous studies, a targeted deletion mutation in the SH2-N domain of Shp-2 was introduced into the murine
Shp-2
locus, which resulted in embryonic lethality of homozygous mutant (
Shp-2
−/−
) mice at midgestation. By aggregating
Shp-2
−/−
embryonic stem cells with wild-type embryos, we created
Shp-2
−/−
/wild-type chimeric animals. Most chimeras had open eyelids at birth and abnormal skin development, a phenotype characteristic of mice with mutations in EGF-R signaling components. In genetic crosses, a heterozygous
Shp-2
mutation dominantly enhanced the phenotype of a weak mutant allele of EGF-R (
wa-2
), resulting in distinctive growth retardation, developmental defects in the skin, lung, and intestine, and perinatal mortality that are reminiscent of EGF-R knockout mice. Biochemical analysis revealed that signal propagation proximal to the EGF-R upon EGF stimulation was significantly attenuated in
wa-2
fibroblast cells, which was exacerbated by the additional
Shp-2
mutation. Thus, we provide biological evidence here that protein-tyrosine phosphatase Shp-2 acts to enhance information flow from the EGF-R in mouse growth and development.
Striking activation of oxidative enzymes suspended in nonaqueous media
Authors: Dai Lizhong , Klibanov Alexander M.
Published: July 2002

The catalytic activity of four lyophilized oxidative
enzymes—horseradish peroxidase, soybean peroxidase,
Caldariomyces fumago
chloroperoxidase, and mushroom
polyphenol oxidase—is much lower when directly suspended in organic
solvents containing little water than when they are introduced into the
same largely nonaqueous media by first dissolving them in water and
then diluting with anhydrous solvents. The lower the water content of
the medium, the greater this discrepancy becomes. The mechanism of this
phenomenon was found to arise from reversible denaturation of the
oxidases on lyophilization: because of its conformational rigidity, the
denatured enzyme exhibits very limited activity when directly suspended
in largely nonaqueous media but renatures and thus yields much higher
activity if first redissolved in water. Two independent means were
discovered for dramatically minimizing the lyophilization-induced
inactivation, both involving the addition of certain types of
excipients to the aqueous enzyme solution before lyophilization. The
first group of excipients consists of phenolic and aniline substrates
as well as other hydrophobic compounds; these presumably bind to the
hydrophobic pocket of the enzyme active site, thereby preventing its
collapse during dehydration. The second group consists of general
lyoprotectants such as polyols and polyethylen glycol that apparently
preserve the overall enzyme structure during dehydration. The
activation effects of such excipients can reach into the tens and
hundreds of fold. Moreover, the activations afforded by the two
excipient groups are additive, resulting in up to a complete protection
against lyophilization-induced inactivation when representatives of the
two are present together.