bluuebunny/crossref_metadata_2025 huggingface dataset:
Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.
Authors: Etter E F , Minta A , Poenie M , Fay F S
Published: July 2002

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.
Disruption of the aldolase A tetramer into catalytically active monomers.
Authors: Beernink P T , Tolan D R
Published: July 2002

The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.
Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits.
Authors: Artemyev N O , Natochin M , Busman M , Schey K L , Hamm H E
Published: July 2002

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (Palpha and Pbeta) and two inhibitory gamma subunits (Pgamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pgamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pgamma were designed for cross-linking experiments to identify regions on Palpha and Pbeta subunits that bind to the Pgamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pgamma were replaced with a single cysteine. This mutant, Pgamma83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pgamma83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pgamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pgamma83CysMBP and PDE catalytic subunits. Palpha and the N-terminally truncated Pbeta (lacking 147 aa residues) cross-linked to Pgamma83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Palpha. The corresponding region of Pbeta, Pbeta-749-761, also may bind to the Pgamma C terminus. Our data suggest that Pgamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.
Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells.
Authors: Yeh S , Chang C
Published: July 2002

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Authors: Klingmüller U , Bergelson S , Hsiao J G , Lodish H F
Published: July 2002

Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y401 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless of which tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential.
Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.
Authors: Cheng S Y , Huang H J , Nagane M , Ji X D , Wang D , Shih C C , Arap W , Huang C M , Cavenee W K
Published: July 2002

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.
Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.
Authors: Naldini L , Blömer U , Gage F H , Trono D , Verma I M
Published: July 2002

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.
Human TOP3: a single-copy gene encoding DNA topoisomerase III.
Authors: Hanai R , Caron P R , Wang J C
Published: July 2002

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.
YY1 regulates vitamin D receptor/retinoid X receptor mediated transactivation of the vitamin D responsive osteocalcin gene
Authors: Guo Bo , Aslam Fauzia , van Wijnen André J. , Roberts Stefan G. E. , Frenkel Baruch , Green Michael R. , DeLuca Hector , Lian Jane B. , Stein Gary S. , Stein Janet L.
Published: July 2002

The responsiveness of genes to steroid hormones is principally
mediated by functional interactions between DNA-bound hormone receptors
and components of the transcriptional initiation machinery, including
TATA-binding protein, TFIIB, or other RNA polymerase II associated
factors. This interaction can be physiologically modulated by promoter
context-specific transcription factors to facilitate optimal
responsiveness of gene expression to hormone stimulation. One
postulated regulatory mechanism involves the functional antagonism
between hormone receptors and nonreceptor transcription factors
interacting at the same hormone response element. Here we demonstrate
that the multifunctional regulator YY1 represses 1,25-dihydroxyvitamin
D
3
(vitamin D)-induced transactivation of the bone
tissue-specific osteocalcin gene. We identify YY1 recognition sequences
within the vitamin D response element (VDRE) of the osteocalcin gene
that are critical for YY1-dependent repression of vitamin D-enhanced
promoter activity. We show that YY1 and vitamin D receptor
(VDR)/retinoid X receptor heterodimers compete for binding at the
osteocalcin VDRE. In addition, we find that YY1 interacts directly with
TFIIB, and that one of the two tandemly repeated polypeptide regions of
TFIIB spanning the basic domain is responsible for this interaction.
TFIIB and VDR can also interact directly, and these factors synergize
to mediate transactivation. Our results suggest that YY1 regulates
vitamin D enhancement of osteocalcin gene transcription
in
vivo
by interfering with the interactions of the VDR with both
the VDRE and TFIIB.
The N-terminal tail of histone H2A binds to two distinct sites within the nucleosome core
Authors: Lee Kyu-Min , Hayes Jeffrey J.
Published: July 2002

Each of the core histone proteins within the nucleosome has a central “structured” domain that comprises the spool onto which the DNA superhelix is wrapped and an N-terminal “tail” domain in which the structure and molecular interactions have not been rigorously defined. Recent studies have shown that the N-terminal domains of core histones probably contact both DNA and proteins within the nucleus and that these interactions play key roles in the regulation of nuclear processes (such as transcription and replication) and are critical in the formation of the chromatin fiber. An understanding of these complex mechanisms awaits identification of the DNA or protein sites within chromatin contacted by the tail domains. To this end, we have developed a site-specific histone protein–DNA photocross-linking method to identify the DNA binding sites of the N-terminal domains within chromatin complexes. With this approach, we demonstrate that the N-terminal tail of H2A binds DNA at two defined locations within isolated nucleosome cores centered around a position ≈40 bp from the nucleosomal dyad and that this tail probably adopts a defined structure when bound to DNA.
Prp43: An RNA helicase-like factor involved in spliceosome disassembly
Authors: Arenas Jaime E. , Abelson John N.
Published: July 2002

The
Saccharomyces cerevisiae
genes
PRP2
,
PRP16
, and
PRP22
encode pre-mRNA splicing factors that belong to the highly conserved “DEAH” family of putative RNA helicases. We previously identified two additional members of this family,
JA1
and
JA2
. To investigate its biological function, we cloned the
JA1
gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family. A
ja1
allele carrying a mutation identical to that in the temperature-sensitive (ts)
prp22–1
gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement. Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats. Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes. The results presented indicate that
JA1
encodes a
p
re-m
R
NA
p
rocessing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA. We have therefore renamed this gene
PRP43
.
Dentition of
Proteopithecus sylviae
, an archaic anthropoid from the Fayum, Egypt
Authors: Miller Ellen R. , Simons Elwyn L.
Published: July 2002

Proteopithecus sylviae
is an archaic anthropoid from the late Eocene quarry L-41, Fayum Province, Egypt. The dentition of
Proteopithecus
is very primitive and does not closely resemble that of other, better known, primates from the Fayum (e.g., parapithecids and propliopithecids). The dental morphology, much of which is described herein, shows a platyrrhine-like level of organization, suggesting that
P. sylviae
may occupy a position near the base of the modern anthropoid radiation.
Comparative mapping of Andropogoneae:
Saccharum
L. (sugarcane) and its relation to sorghum and maize
Authors: Guimarães Claudia T. , Sills Gavin R. , Sobral Bruno W. S.
Published: July 2002

Comparative genetic maps of Papuan
Saccharum officinarum
L. (2
n
= 80) and
S. robustum
(2
n
= 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of
S. officinarum
and
S. robustum
were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and
Saccharum
genomes. There were no differences in marker order between
S. officinarum
and sorghum. Furthermore, there were no alterations in SDM order between
S. officinarum
and
S. robustum
. The
S. officinarum
and
S. robustum
maps also were compared with the map of the polysomic octoploid
S. spontaneum
‘SES 208’ (2
n
= 64,
x
= 8), thus permitting relations to homology groups (“chromosomes”) of
S. spontaneum
to be studied. Investigation of transmission genetics in
S. officinarum
and
S. robustum
confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce
S. officinarum
and
S. robustum
linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2
n
= 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.
A system for functional analysis of Ebola virus glycoprotein
Authors: Takada Ayato , Robison Clinton , Goto Hideo , Sanchez Anthony , Murti K. Gopal , Whitt Michael A. , Kawaoka Yoshihiro
Published: July 2002

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured
in vitro
.
Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: Implications for the treatment of multiple sclerosis
Authors: Hooper D. Craig , Bagasra Omar , Marini Joseph C. , Zborek Anna , Ohnishi S. Tsuyoshi , Kean Rhonda , Champion Jean M. , Sarker Ashit B. , Bobroski Lisa , Farber John L. , Akaike Takaaki , Maeda Hiroshi , Koprowski Hilary
Published: July 2002

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent
in situ
polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.
Decreased probability of neurotransmitter release underlies striatal long-term depression and postnatal development of corticostriatal synapses
Authors: Choi Sukwoo , Lovinger David M.
Published: July 2002

Changes in synaptic efficacy are crucial for the development of appropriate neural circuits and brain information storage. We have investigated mechanisms underlying long-term depression (LTD) at glutamatergic synapses in the striatum, a brain region important in motor performance and cognition, and a target for Huntington and Parkinson diseases. Induction of striatal LTD is dependent on postsynaptic depolarization and calcium influx through L-type channels. Surprisingly, LTD maintenance appears to involve a decrease in the probability of neurotransmitter release from presynaptic terminals as evidenced by increases in paired-pulse facilitation and the coefficient of variation of synaptic responses that are tightly associated with LTD expression. Furthermore, both the apparent probability of neurotransmitter release and the magnitude of LTD decrease concomitantly during postnatal development, consistent with the idea that striatal LTD is involved in a developmental decrease in the probability of neurotransmitter release at corticostriatal synapses. The presynaptic changes that underlie striatal LTD may also be important for motor performance and certain forms of learning and memory.
Repair of plasmalemmal lesions by vesicles
Authors: Eddleman Christopher S. , Ballinger Martis L. , Smyers Mark E. , Godell Christopher M. , Fishman Harvey M. , Bittner George D.
Published: July 2002

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca
2+
-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca
2+
inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1–30 μm) plasmalemmal holes or a complete transection of the plasmalemma.
An 85-kb tandem triplication in the slow Wallerian degeneration (
Wld
s
) mouse
Authors: Coleman Michael P. , Conforti Laura , Buckmaster E. Anne , Tarlton Andrea , Ewing Robert M. , Brown Michael C. , Lyon Mary F. , Perry V. Hugh
Published: July 2002

Wallerian degeneration is the degeneration of the distal stump of an injured axon. It normally occurs over a time course of around 24 hr but it is delayed in the slow Wallerian degeneration mutant mouse (C57BL/
Wld
s
) for up to 3 weeks. The gene, which protects from rapid Wallerian degeneration,
Wld
, previously has been mapped to distal chromosome 4. This paper reports the fine genetic mapping of the
Wld
locus, the generation of a 1.4-Mb bacterial artificial chromosome and P1 artificial chromosome contig, and the identification of an 85-kb tandem triplication mapping within the candidate region. The mutation is unique to C57BL/
Wld
s
among 36 strains tested and therefore is a strong candidate for the mutation that leads to delayed Wallerian degeneration. There are very few reports of tandem triplications in a vertebrate and no evidence for a mutation mechanism so this unusual mutation was characterized in more detail. Sequence analysis of the boundaries of the repeat unit revealed a minisatellite array at the distal boundary and a matching 8-bp sequence at the proximal boundary. This finding suggests that recombination between short homologous sequences (“illegitimate” or “nonhomologous” recombination) was involved in the rearrangement. In addition, a duplication allele was identified in two
Wld
s
mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.
Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)
Authors: Achen Marc G. , Jeltsch Michael , Kukk Eola , Mäkinen Taija , Vitali Angela , Wilks Andrew F. , Alitalo Kari , Stacker Steven A.
Published: July 2002

We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However, VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.
A dominant-negative pleiotrophin mutant introduced by homologous recombination leads to germ-cell apoptosis in male mice
Authors: Zhang Nan , Yeh Hsiu-Jeng , Zhong Rong , Li Yue-Sheng , Deuel Thomas F.
Published: July 2002

Pleiotrophin (PTN) is an 18-kDa heparin-binding secretory growth/differentiation factor for different cell types. Its gene is differentially expressed in both mesenchyme and central nervous system during development and highly expressed in a number of different human tumors. Recently, a PTN mutant was found to act as a dominant-negative effector of PTN signaling. We have now used homologous recombination to introduce the dominant-negative PTN mutant into embryonic stem cells to generate chimeric mice. All highly chimeric male mice with germinal epithelium exclusively derived from embryonic stem cells with the heterologous PTN mutation were sterile. Their testes were uniformly atrophic, and the spermatocytes were strikingly apoptotic at all stages of development. The results support a central role of PTN signaling in normal spermatogenesis and suggest that interruption of PTN signaling may lead to sterility in males.
Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice
Authors: Hauser Wolfgang , Knobeloch Klaus-Peter , Eigenthaler Martin , Gambaryan Stepan , Krenn Veit , Geiger Jörg , Glazova Margarita , Rohde Elvira , Horak Ivan , Walter Ulrich , Zimmer Michael
Published: July 2002

Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell–cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and theListeria monocytogenessurface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin αIIbβ3inhibition in human platelets. To gain further insight into thein vivofunction of this protein, VASP-deficient mice were generated by homologous recombination. VASP−/− mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin αIIbβ3activation. Although the limited phenotypic differences between wild-type and VASP−/− mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.
Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties
Authors: Page Martin J. , Amess Bob , Townsend R. Reid , Parekh Raj , Herath Athula , Brusten Luc , Zvelebil Marketa J. , Stein Robert C. , Waterfield Michael D. , Davies Susan C. , O’Hare Michael J.
Published: July 2002

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.
Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome
c
oxidase
Authors: Fabian Marian , Wong William W. , Gennis Robert B. , Palmer Graham
Published: July 2002

The “peroxy” intermediate (P form) of bovine cytochrome
c
oxidase was prepared by reaction of the two-electron reduced mixed-valence CO complex with
18
O
2
after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for
18
O enrichment by mass spectrometry. It was found that approximately one oxygen atom (
18
O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen–oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water.
Detection of neuritic plaques in Alzheimer’s disease by magnetic resonance microscopy
Authors: Benveniste Helene , Einstein Gillian , Kim Katie R. , Hulette Christine , Johnson G. Allan
Published: July 2002

Magnetic resonance microscopy (MRM) theoretically provides the spatial resolution and signal-to-noise ratio needed to resolve neuritic plaques, the neuropathological hallmark of Alzheimer’s disease (AD). Two previously unexplored MR contrast parameters, T2* and diffusion, are tested for plaque-specific contrast to noise. Autopsy specimens from nondemented controls (
n
= 3) and patients with AD (
n
= 5) were used. Three-dimensional T2* and diffusion MR images with voxel sizes ranging from 3 × 10
−3
mm
3
to 5.9 × 10
−5
mm
3
were acquired. After imaging, specimens were cut and stained with a microwave king silver stain to demonstrate neuritic plaques. From controls, the alveus, fimbria, pyramidal cell layer, hippocampal sulcus, and granule cell layer were detected by either T2* or diffusion contrast. These structures were used as landmarks when correlating MRMs with histological sections. At a voxel resolution of 5.9 × 10
−5
mm
3
, neuritic plaques could be detected by T2*. The neuritic plaques emerged as black, spherical elements on T2* MRMs and could be distinguished from vessels only in cross-section when presented in three dimension. Here we provide MR images of neuritic plaques
in vitro
. The MRM results reported provide a new direction for applying this technology
in vivo
. Clearly, the ability to detect and follow the early progression of amyloid-positive brain lesions will greatly aid and simplify the many possibilities to intervene pharmacologically in AD.
Defects in transforming growth factor-β signaling cooperate with a
Ras
oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes
Authors: Glick Adam , Popescu Nicholas , Alexander Valarie , Ueno Hikaru , Bottinger Erwin , Yuspa Stuart H.
Published: July 2002

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an
in vitro
model of keratinocyte transformation that parallels
in vivo
malignant conversion to squamous cell carcinoma, we show that v-
ras
Ha
transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.
Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses
Authors: Song Ji-Ying , Ichtchenko Konstantin , Südhof Thomas C. , Brose Nils
Published: July 2002

At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic membranes into synaptic junctions are unknown. Neuroligins and β-neurexins are neuronal cell-surface proteins that bind to each other and form asymmetric intercellular junctions. To test whether the neuroligin/β-neurexin junction is related to synapses, we generated and characterized monoclonal antibodies to neuroligin 1. With these antibodies, we show that neuroligin 1 is synaptic. The neuronal localization, subcellular distribution, and developmental expression of neuroligin 1 are similar to those of the postsynaptic marker proteins PSD-95 and NMDA-R1 receptor. Quantitative immunogold electron microscopy demonstrated that neuroligin 1 is clustered in synaptic clefts and postsynaptic densities. Double immunofluorescence labeling revealed that neuroligin 1 colocalizes with glutamatergic but not γ-aminobutyric acid (GABA)ergic synapses. Thus neuroligin 1 is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. In addition, the neuroligin/β-neurexin junction may be involved in the specification of excitatory synapses.
The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis
Authors: Bishop Gerard J. , Nomura Takahito , Yokota Takao , Harrison Kate , Noguchi Takahiro , Fujioka Shozo , Takatsuto Suguru , Jones Jonathan D. G. , Kamiya Yuji
Published: July 2002

Brassinosteroids (BRs) are steroidal plant hormones essential for
normal plant growth and development. Mutants in the biosynthesis or
perception of BRs are usually dwarf. The tomato
Dwarf
gene
(
D
), which was predicted to encode a cytochrome P450 enzyme
(P450) with homology to other P450s involved in BR biosynthesis, was
cloned previously. Here, we show that DWARF catalyses the C-6 oxidation
of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate
precursor of brassinolide. To do this, we first confirmed that the
D
cDNA complemented the mutant light- and dark-grown
phenotypes of the
extreme dwarf
(

d
x

) allele of tomato. To identify a substrate
for the DWARF enzyme, exogenous application of BR intermediates to

d
x

plants was carried out. C-6 oxoBR
intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR,
6-deoxoCS, had little effect. Quantitative analysis of
endogenous BR levels in tomato showed mainly the presence
of 6-deoxoBRs. Furthermore,

d
x

plants were
found to lack CS and had a high level of 6-deoxoCS in comparison to
D
plants that had CS and a lower level of 6-deoxoCS.
Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS
was obtained by functional expression of DWARF in yeast. In these
experiments, the intermediate 6α-hydroxycastasterone was identified,
indicating that DWARF catalyzes two steps in BR biosynthesis. These
data show that DWARF is involved in the C-6 oxidation in BR
biosynthesis.
Long-range transport of mineral dust in the global atmosphere: Impact of African dust on the environment of the southeastern United States
Authors: Prospero Joseph M.
Published: July 2002

Soil dust is a major constituent of airborne particles in the
global atmosphere. Dust plumes frequently cover huge areas of the
earth; they are one of the most prominent and commonly visible features
in satellite imagery. Dust is believed to play a role in many
biogeochemical processes, but the importance of dust in these processes
is not well understood because of the dearth of information about the
global distribution of dust and its physical, chemical, and
mineralogical properties. This paper describes some features of the
large-scale distribution of dust and identifies some of the geological
characteristics of important source areas. The transport of dust from
North Africa is presented as an example of possible long-range dust
effects, and the impact of African dust on environmental processes in
the western North Atlantic and the southeastern United States is
assessed. Dust transported over long distances usually has a mass
median diameter <10 μm. Small wind-borne soil particles show signs
of extensive weathering; consequently, the physical and chemical
properties of the particles will greatly depend on the weathering
history in the source region and on the subsequent modifications that
occur during transit in the atmosphere (typically a period of a week or
more). To fully understand the role of dust in the environment and in
human health, mineralogists will have to work closely with scientists
in other disciplines to characterize the properties of mineral
particles as an ensemble and as individual particles especially with
regard to surface characteristics.
Loss of fibroblast growth factor receptor 2 ligand-binding specificity in Apert syndrome
Authors: Yu Kai , Herr Andrew B. , Waksman Gabriel , Ornitz David M.
Published: July 2002

Craniosynostosis syndromes are autosomal dominant human skeletal
diseases that result from various mutations in fibroblast growth factor
receptor genes (
Fgfrs
). Apert syndrome (AS) is one of
the most severe craniosynostosis syndromes and is associated with
severe syndactyly of the hands and feet and with central nervous system
malformations. AS is caused by specific missense mutations in one of
two adjacent amino acid residues (S252W or P253R) in the highly
conserved region linking Ig-like domains II and III of FGFR2. Here we
demonstrate that these mutations break one of the cardinal rules
governing ligand specificity of FGFR2. We show that the S252W mutation
allows the mesenchymal splice form of FGFR2 (FGFR2c) to bind and to be
activated by the mesenchymally expressed ligands FGF7 or FGF10 and the
epithelial splice form of FGFR2 (FGFR2b) to be activated by FGF2, FGF6,
and FGF9. These data demonstrate loss of ligand specificity of FGFR2
with retained ligand dependence for receptor activation. These data
suggest that the severe phenotypes of AS likely result from ectopic
ligand-dependent activation of FGFR2.
The Interruption of Income Convergence and Income Growth in Large Cities in the 1980s
Authors: Drennan Matthew P. , Tobier Emanuel , Lewis Jonathan
Published: July 2002

Recent studies have documented the divergence of per capita personal income among regions of the US in the 1980s following many decades of income convergence. This paper establishes the same pattern of divergence for median family income in the 1980s and argues that regional specialisation in producer services contributed to upward divergence of regions with above average income. Because producer services are concentrated in cities, the growth of median household income in the 51 largest US cities over the 1980s is analysed, for all households and for black households. Regression results indicate that cities more specialised in producer services at the beginning of the decade had much better growth than cities more specialised in manufacturing. The results for black households were very similar to the results for all households.
Territorial Competition and Globalisation: Scylla and Charybdis of European Cities
Authors: Budd Leslie
Published: July 2002

Policy-makers in Europe have been concerned with developing and sustaining the competitiveness of their cities. This concern comes from the view that the determing parameter of urban regions is globalisation. By engaging in a process of territorial competition, the economic and social welfare of cities' constituent territories can be maintained. What this paper argues is that there is a danger that territorial competition is as much an abstraction as globalisation and its application to policy-making will bring about distortions in economic development. By examining the specification of globalisation and territorial competition and the relevant literature, a debate may be initiated in which the consequences for the economic environment of European cities are addressed. The discussion is given some context by drawing on the cursory examples, one global and one local, of London and Lille.
Olfactory Mucosal Necrosis in Male CD Rats Following Acute Inhalation Exposure to Hydrogen Sulfide: Reversibility and the Possible Role of Regional Metabolism
Authors: Brenneman Karrie A. , Meleason David F. , Sar Madhabananda , Marshall Marianne W. , James R. Arden , Gross Elizabeth A. , Martin Joseph T. , Dorman David C.
Published: July 2002

Hydrogen sulfide (H 2S) is a potent inhibitor of cytochrome oxidase (CO) and is associated with dysosmia and anosmia in humans and nasal lesions in exposed rodents. An improved understanding of the pathogenesi s of these lesions is needed to determine their toxicological relevance. We exposed 10-week-old male CD rats to 0, 30, 80, 200, or 400 ppm H2S for 3 hours/day for 1 or 5 days consecutively. The nose was histologically examined 24 hours after H2S exposure, and lesion recovery was assessed at 2 and 6 weeks following the 5-day exposure. A single 3-hour exposure to ≥80 ppm H 2S resulted in regeneration of the respiratory mucosa and full thickness necrosis of the olfactory mucosa localized to the ventral and dorsal meatus, respectively. Repeated exposure to the same concentrations caused necrosis of the olfactory mucosa with early mucosal regeneration that extended from the dorsal medial meatus to the caudal regions of the ethmoid recess. Acute exposure to 400 ppm H2S induced severe mitochondrial swelling in sustentacular cells and olfactory neurons, which progressed to olfactory epithelial necrosis and sloughing. CO immunoreactive cells were more frequently observed in regions of the olfactory mucosa commonly affected by H2S than in regions that were not. These findings demonstrate that acute exposure to ≥80 ppm H2S resulted in reversible lesions in the respiratory and olfactory mucosae of the CD rat and that CO immunoreactivity may be a susceptibility factor for H2S-induced olfactory toxicity in the rat.