bluuebunny/crossref_metadata_2025 huggingface dataset:
GaAs/AlGaAs optical waveguides on silicon substrates grown by molecular beam epitaxy
Authors: Kim Young-Soon , Ramaswamy Ramu V. , Sakai Shiro , Matyi R. J. , Shichijo H.
Published: July 2002

A single-heterostructure optical waveguide at 1.3 μm wavelength in GaAs/AlGaAs, grown on Si by molecular beam epitaxy, is demonstrated. 6-μm-wide ridge waveguides with 0.25 μm ridge height had single modes and the measured average propagation loss was 11.7 dB/cm. The principal loss was mainly due to the relatively large free-carrier concentration in the guiding layer. It is expected that lower free-carrier concentration (∼1015 cm−3) will lead to waveguides with propagation loss of less than 3 dB/cm.
Photoluminescence excitation spectroscopy of GaxIn1−xAsyP1−y/InP quantum wells
Authors: Montie E. A. , Thijs P. J. A. , ’t Hooft G. W.
Published: July 2002

Photoluminescence excitation measurements were performed on GaxIn1−xAsyP1−y/InP single quantum wells. The results were analyzed with a k⋅p approach. Using the effective masses reported in the literature, the ratio of the discontinuities in the conduction and valence band is found to be 35:65. This is in good agreement with a ratio of 37:63, obtained directly from an observed transition involving free holes.
Extrinsic p-type doping of HgCdTe grown by organometallic epitaxy
Authors: Ghandhi S. K. , Taskar N. R. , Parat K. K. , Terry D. , Bhat I. B.
Published: July 2002

In this letter, we report on the extrinsic p-type doping of mercury cadmium telluride (MCT), during growth by organometallic vapor phase epitaxy. Arsine gas in hydrogen was used as the dopant source. The layers were characterized by Hall effect and by resistivity measurements over the temperature range from 20 to 300 K. The acceptor ionization energy was obtained for different doping concentrations from the Hall coefficient data. Its value decreases with doping concentration, and is about 8.5 meV for a doping of 1.1×1016 cm−3. This is a factor of 2 lower than the ionization energy of mercury vacancies, for layers of this (27–30% cadmium) composition and doping level. Annealing at 205 °C for 15 h in a Hg-rich ambient did not produce significant changes in the measured doping concentration. This indicates that the acceptor level is extrinsic in nature, and that arsenic behaves as a stable acceptor dopant in MCT, grown by organometallic epitaxy.
Dynamin at actin tails
Authors: Lee Eunkyung , De Camilli Pietro
Published: July 2002

Dynamin, the product of the
shibire
gene of
Drosophila,
is a GTPase critically required for
endocytosis. Some studies have suggested a functional link between
dynamin and the actin cytoskeleton. This link is of special interest,
because there is evidence implicating actin dynamics in endocytosis.
Here we show that endogenous dynamin 2, as well as green
fluorescence protein fusion proteins of both dynamin 1 and 2, is
present in actin comets generated by
Listeria
or by type
I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is
further enriched at the interface between the tails and the moving
organelles. Dynamin mutants harboring mutations in the GTPase domain
inhibited nucleation of actin tails induced by PIPK and moderately
reduced their speed. Although dynamin localization to the tails
required its proline-rich domain, expression of a dynamin mutant
lacking this domain also diminished tail formation. In addition, this
mutant disrupted a membrane-associated actin scaffold (podosome
rosette) previously shown to include dynamin. These findings suggest
that dynamin is part of a protein network that controls nucleation of
actin from membranes. At endocytic sites, dynamin may couple the
fission reaction to the polymerization of an actin pool that functions
in the separation of the endocytic vesicles from the plasma membrane.
Structural diversity of heparan sulfate binding domains in chemokines
Authors: Lortat-Jacob Hugues , Grosdidier Aurélien , Imberty Anne
Published: July 2002

Heparan sulfate (HS) molecules are ubiquitous in animal tissues where they function as ligands that are dramatically involved in the regulation of the proteins they bind. Of these, chemokines are a family of small proteins with many biological functions. Their well-conserved monomeric structure can associate in various oligomeric forms especially in the presence of HS. Application of protein surface analysis and energy calculations to all known chemokine structures leads to the proposal that four different binding modes are created by the folding and oligomerization of these proteins. So, based on the present state of our knowledge, four different clusters of amino acids should be involved in the recognition process. Our results help to rationalize how unique sequences of HS specifically bind any given chemokine. The conclusions open the route for a rational design of compounds of therapeutical interest that could influence chemokine activity.
The
Arf
tumor suppressor gene promotes hyaloid vascular regression during mouse eye development
Authors: McKeller Robyn N. , Fowler Jennifer L. , Cunningham Justine J. , Warner Nikita , Smeyne Richard J. , Zindy Frederique , Skapek Stephen X.
Published: July 2002

A key tumor suppressor mechanism that is disrupted frequently in human cancer involves the
ARF
and
p53
genes. In mouse fibroblasts, the
Arf
gene product responds to abnormal mitogenic signals to activate p53 and trigger either cell cycle arrest or apoptosis. Recent evidence indicates that
Arf
also has
p53
-independent functions that may contribute to its tumor suppressor activity. Using
Arf

−/−

and
p53

−/−

mice, we have discovered a
p53
-independent requirement for
Arf
in the developmental regression of the hyaloid vascular system (HVS) in the mouse eye.
Arf
is expressed in the vitreous of the eye and is induced before HVS regression in the first postnatal week. In the absence of
Arf
, failed HVS regression causes a pathological process that resembles persistent hyperplastic primary vitreous, a developmental human eye disease thought to have a genetic basis. These findings demonstrate an essential and unexpected role for
Arf
during mouse eye development, provide insights into the potential genetic basis for persistent hyperplastic primary vitreous, and indicate that
Arf
regulates vascular regression in a
p53
-independent manner. The latter finding raises the possibility that
Arf
may function as a tumor suppressor at least in part by regulating tumor angiogenesis.
Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA
Authors: Maeda Naoyoshi , Palmarini Massimo , Murgia Claudio , Fan Hung
Published: July 2002

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine
pulmonary carcinoma, a unique animal model for human bronchioalveolar
carcinoma. We previously isolated a JSRV proviral clone and showed that
it was both infectious and oncogenic. Thus JSRV is necessary and
sufficient for the development of ovine pulmonary carcinoma, but no
data are available on the mechanisms of transformation. Inspection of
the JSRV genome reveals standard retroviral genes, but no evidence for
a viral oncogene. However, an alternate ORF in
pol
(
orf-x
) might be a candidate for a transforming gene. We
tested whether the JSRV genome might encode a transforming gene by
transfecting an expression plasmid for JSRV [pCMVJS21, driven by the
cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3
cells. Foci of transformed cells appeared in the transfected cultures
2–3 weeks posttransfection; cloned transformants showed anchorage
independence for growth, and they expressed JSRV RNA. These results
indicate that the JRSV genome contains information with direct
transforming potential for NIH 3T3 cells. Transfection of a mutated
version of pCMVJS21 in which the
orf-x
protein was
terminated by two stop codons also gave transformed foci. Thus,
orf-x
was eliminated as the candidate transforming gene.
In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which
the
gag
,
pol
(and
orf-x
)
coding sequences were deleted also gave transformed foci. These results
indicate that the envelope gene carries the transforming potential.
This is an unusual example of a native retroviral structural protein
with transformation potential.
A decamer duplication in the 3′ region of the
BRI
gene originates an amyloid peptide that is associated with dementia in a Danish kindred
Authors: Vidal Ruben , Révész Tamas , Rostagno Agueda , Kim Eugene , Holton Janice L. , Bek Toke , Bojsen-Møller Marie , Braendgaard Hans , Plant Gordon , Ghiso Jorge , Frangione Blas
Published: July 2002

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene
BRI
in familial British dementia. Molecular genetic analysis of the
BRI
gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795–796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This
de novo-
created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia.
Genetic consequences of sequential founder events by an island-colonizing bird
Authors: Clegg Sonya M. , Degnan Sandie M. , Kikkawa Jiro , Moritz Craig , Estoup Arnaud , Owens Ian P. F.
Published: July 2002

The importance of founder events in promoting evolutionary changes on islands has been a subject of long-running controversy. Resolution of this debate has been hindered by a lack of empirical evidence from naturally founded island populations. Here we undertake a genetic analysis of a series of historically documented, natural colonization events by the silvereye species-complex (
Zosterops lateralis
), a group used to illustrate the process of island colonization in the original founder effect model. Our results indicate that single founder events do not affect levels of heterozygosity or allelic diversity, nor do they result in immediate genetic differentiation between populations. Instead, four to five successive founder events are required before indices of diversity and divergence approach that seen in evolutionarily old forms. A Bayesian analysis based on computer simulation allows inferences to be made on the number of effective founders and indicates that founder effects are weak because island populations are established from relatively large flocks. Indeed, statistical support for a founder event model was not significantly higher than for a gradual-drift model for all recently colonized islands. Taken together, these results suggest that single colonization events in this species complex are rarely accompanied by severe founder effects, and multiple founder events and/or long-term genetic drift have been of greater consequence for neutral genetic diversity.
Stable isotope evidence for increasing dietary breadth in the European mid-Upper Paleolithic
Authors: Richards Michael P. , Pettitt Paul B. , Stiner Mary C. , Trinkaus Erik
Published: July 2002

New carbon and nitrogen stable isotope values for human remains
dating to the mid-Upper Paleolithic in Europe indicate significant
amounts of aquatic (fish, mollusks, and/or birds) foods in some of
their diets. Most of this evidence points to exploitation of inland
freshwater aquatic resources in particular. By contrast, European
Neandertal collagen carbon and nitrogen stable isotope values do not
indicate significant use of inland aquatic foods but instead show that
they obtained the majority of their protein from terrestrial
herbivores. In agreement with recent zooarcheological analyses, the
isotope results indicate shifts toward a more broad-spectrum
subsistence economy in inland Europe by the mid-Upper Paleolithic
period, probably associated with significant population increases.
Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation
Authors: Arora Shikha , Lapinski Philip Edward , Raghavan Malini
Published: July 2002

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurringin vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding siteper seare not essential for the TAP catalytic cycle.
Comprehensive survey of proteins targeted by chloroplast thioredoxin
Authors: Motohashi Ken , Kondoh Aiko , Stumpp Michael T. , Hisabori Toru
Published: July 2002

Possible target proteins of chloroplast thioredoxin (Trx) have been
investigated in the stroma lysate of spinach chloroplasts. For that
purpose, we immobilized a mutant of
m
-type Trx in which
an internal cysteine at the active site was substituted with serine, on
cyanogen bromide-activated resin. By using this resin, the target
proteins in chloroplast were efficiently acquired when they formed the
mixed-disulfide intermediates with the immobilized Trxs. We could
acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx),
which were recently identified as targets of chloroplast Trxs.
Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose
1,7-
bis
phosphatase, well-known thiol enzymes in the
Calvin cycle, also were recognized among the collected proteins,
suggesting the method is applicable for our purpose. Furthermore, four
proteins were identified from a homology search of the
NH
2
-terminal sequence of the acquired proteins: glutamine
synthetase, a protein homologous to chloroplast cyclophilin, a homolog
of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of
the recombinant cyclophilin and Prx-Q of
Arabidopsis
thaliana
were then examined. The method developed in the
present study is thus applicable to investigate the various redox
networks via Trxs and the related enzymes in the cell.
Ribozyme rescue of photoreceptor cells in P23H transgenic rats: Long-term survival and late-stage therapy
Authors: LaVail Matthew M. , Yasumura Douglas , Matthes Michael T. , Drenser Kimberly A. , Flannery John G. , Lewin Alfred S. , Hauswirth William W.
Published: July 2002

Ribozyme-directed cleavage of mutant mRNAs appears to be a
potentially effective therapeutic measure for dominantly inherited
diseases. We previously demonstrated that two ribozymes targeted to the
P23H mutation in rhodopsin slow photoreceptor degeneration in
transgenic rats for up to 3 months of age when injected before
significant degeneration at postnatal day (P) 15. We now have explored
whether ribozyme rescue persists at older ages, and whether ribozymes
are effective when injected later in the degeneration after significant
photoreceptor cell loss. Recombinant adeno-associated virus (rAAV)
vectors incorporating a proximal bovine rod opsin promoter were used to
transfer either hairpin or hammerhead ribozyme genes to photoreceptors.
For the study of long-term survival, rAAV was administered by
subretinal injection at P15, and the rats were allowed to live up to 8
months of age. For the study of late-stage gene transfer, rAAV was
administered at P30 or P45, when 40–45% of the photoreceptors already
had degenerated. Eyes were examined functionally by the
electroretinogram and structurally by morphometric analysis. When
injected at P15, expression of either ribozyme markedly slowed the rate
of photoreceptor degeneration for at least 8 months and resulted in
significantly greater electroretinogram amplitudes at least up to P180.
When injected at P30 or P45, virtually the same number of
photoreceptors survived at P130 as when injected at P15. Ribozyme
rescue appears to be a potentially effective, long-term therapy for
autosomal dominant retinal degeneration and is highly effective even
when the gene transfer is done after significant photoreceptor cell
loss.
The anticancer drug–DNA complex: Femtosecond primary dynamics for anthracycline antibiotics function
Authors: Qu Xiaogang , Wan Chaozhi , Becker Hans-Christian , Zhong Dongping , Zewail Ahmed H.
Published: July 2002

The anthracycline–DNA complex, which is a potent agent for cancer
chemotherapy, has a unique intercalating molecular structure with
preference to the GC bases of DNA, as shown by Rich's group in studies
of single-crystal x-ray diffraction. Understanding cytotoxicity
and its photoenhancement requires the unraveling of the dynamics under
the solution-phase, physiological condition. Here we report our first
study of the primary processes of drug function. In a series of
experiments involving the drug (daunomycin and adriamycin) in water,
the drug–DNA complexes, the complexes with the four nucleotides (dGTP,
dATP, dCTP, and dTTP), and the drug-apo riboflavin-binding protein, we
show the direct involvement of molecular oxygen and DNA base-drug
charge-separation—the rates for the reduction of the drug and
dioxygen indicate the crucial role of drug/base/O
2
in the
efficient and catalytic redox cycling. These dynamical steps, and the
subsequent reactions of the superoxide product(s), can account for the
photoenhanced function of the drug in cells, and potentially for
the cell death.
Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.
Authors: Choi T , Rulong S , Resau J , Fukasawa K , Matten W , Kuriyama R , Mansour S , Ahn N , Vande Woude G F
Published: July 2002

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.
Adenine phosphoribosyltransferase-deficient mice develop 2,8-dihydroxyadenine nephrolithiasis.
Authors: Engle S J , Stockelman M G , Chen J , Boivin G , Yum M N , Davies P M , Ying M Y , Sahota A , Simmonds H A , Stambrook P J , Tischfield J A
Published: July 2002

Adenine phosphoribosyltransferase (APRT) deficiency in humans is an autosomal recessive syndrome characterized by the urinary excretion of adenine and the highly insoluble compound 2,8-dihydroxyadenine (DHA) that can produce kidney stones or renal failure. Targeted homologous recombination in embryonic stem cells was used to produce mice that lack APRT. Mice homozygous for a null Aprt allele excrete adenine and DHA crystals in the urine. Renal histopathology showed extensive tubular dilation, inflammation, necrosis, and fibrosis that varied in severity between different mouse backgrounds. Thus, biochemical and histological changes in these mice mimic the human disease and provide a suitable model of human hereditary nephrolithiasis.
A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis.
Authors: Sato Y , Hong S K , Tagiri A , Kitano H , Yamamoto N , Nagato Y , Matsuoka M
Published: July 2002

Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis.
The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato.
Authors: Whitham S , McCormick S , Baker B
Published: July 2002

It has been proposed that cloned plant disease resistance genes could be transferred from resistant to susceptible plant species to control important crop plant diseases. The recently cloned N gene of tobacco confers resistance to the viral pathogen, tobacco mosaic virus. We generated transgenic tomato plants bearing the N gene and demonstrate that N confers a hypersensitive response and effectively localizes tobacco mosaic virus to sites of inoculation in transgenic tomato, as it does in tobacco. The ability to reconstruct the N-mediated resistance response to tobacco mosaic virus in tomato demonstrates the utility of using isolated resistance genes to protect crop plants from diseases, and it demonstrates that all the components necessary for N-mediated resistance are conserved in tomato.
Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.
Authors: Arts E J , Stetor S R , Li X , Rausch J W , Howard K J , Ehresmann B , North T W , Wöhrl B M , Goody R S , Wainberg M A , Grice S F
Published: July 2002

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.
A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.
Authors: Mizoguchi T , Irie K , Hirayama T , Hayashida N , Yamaguchi-Shinozaki K , Matsumoto K , Shinozaki K
Published: July 2002

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.
Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector.
Authors: Tripathy S K , Svensson E C , Black H B , Goldwasser E , Margalith M , Hobart P M , Leiden J M
Published: July 2002

Erythropoietin (Epo)-responsive anemia is a common and debilitating complication of chronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 micrograms of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 +/- 0.4% to levels of 64 +/- 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 micrograms of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent elevations in hematocrits. Mice injected once with 300 micrograms of pVRmEpo displayed 5-fold increased serum Epo levels and elevated hematocrits of 79 +/- 3.3% at 45 days after injection. The i.m. injected plasmid DNA remained localized to the site of injection as assayed by the PCR. We conclude that i.m. injection of plasmid DNA represents a viable nonviral gene transfer method for the treatment of acquired and inherited serum protein deficiencies.
Superoxide accelerates DNA damage by elevating free-iron levels
Authors: Keyer Kay , Imlay James A.
Published: July 2002

Superoxide promotes hydroxyl-radical formation and consequent DNA
damage in cells of all types. The long-standing hypothesis that it
primarily does so by delivering electrons to adventitious iron on DNA
was refuted by recent studies in
Escherichia coli
.
Alternative proposals have suggested that superoxide may accelerate
oxidative DNA damage by leaching iron from storage proteins or enzymic
[4Fe-4S] clusters. The released iron might then deposit on the
surface of the DNA, where it could catalyze the formation of DNA
oxidants using other electron donors. The latter model is affirmed by
the experiments described here. Whole-cell electron paramagnetic
resonance demonstrated that the level of loose iron in
superoxide-stressed cells greatly exceeds that of unstressed cells.
Bacterial iron storage proteins were not the major source for free
iron, since superoxide also increased iron levels in mutants lacking
these iron storage proteins. However, overproduction of an enzyme
containing a labile [4Fe-4S] cluster dramatically increased the free
iron content of cells when they were growing in air. The rates of
spontaneous mutagenesis and DNA damage from exogenous
H
2
O
2
increased commensurately. It is striking
that both growth defects and DNA damage caused by superoxide ensue from
its ability to damage a subset of iron–sulfur clusters.
The mutation rate and cancer
Authors: Tomlinson I. P. M. , Novelli M. R. , Bodmer W. F.
Published: July 2002

The selection of advantageous mutations underlies tumorigenesis.
The growth of a tumor is therefore a form of evolution at the somatic
level, in which the population is comprised of individual cells within
the tumor. Models of tumorigenesis have considered the relative
importance of mutation and selection. We show that selection is more
important than an increased mutation rate in the growth of a tumor.
Some cancers may acquire a “mutator phenotype,” probably leading
to faster growth, but mutator phenotypes are not necessary for
carcinogenesis.
Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display
Authors: Lorimer Ian A. J. , Keppler-Hafkemeyer Andrea , Beers Richard A. , Pegram Charles N. , Bigner Darell D. , Pastan Ira
Published: July 2002

EGFRvIII is a mutant epidermal growth factor receptor found in
glioblastoma, and in carcinoma of the breast, ovary, and lung. The
mutant receptor has a deletion in its extracellular domain that results
in the formation of a new, tumor-specific extracellular sequence. Mice
were immunized with a synthetic peptide corresponding to this sequence
and purified EGFRvIII. A single chain antibody variable domain (scFv)
phage display library of 8 × 10
6
members was made
from the spleen of one immunized mouse. A scFv specific for EGFRvIII
was isolated from this library by panning with successively decreasing
amounts of synthetic peptide. This was used to make an immunotoxin by
fusing the scFv DNA sequence to sequences coding for domains II and III
of Pseudomonas exotoxin A. Purified immunotoxin had a
K
d
of 22 nM for peptide and a
K
d
of 11 nM for cell-surface EGFRvIII. The
immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an
IC
50
of 1 ng/ml (16 pM) on mouse fibroblasts transfected
with EGFRvIII and an IC
50
of 7–10 ng/ml (110–160 pM) on
transfected glioblastoma cells. There was no cytotoxic activity at 1000
ng/ml on the untransfected parent glioblastoma cell line. The
immunotoxin was completely stable upon incubation at 37°C for 24
h in human serum. The combination of good affinity, cytotoxicity and
stability make this immunotoxin a candidate for further preclinical
evaluation.
Muramic lactam in peptidoglycan of
Bacillus subtilis
spores is required for spore outgrowth but not for spore dehydration or heat resistance
Authors: Popham David L. , Helin Jari , Costello Catherine E. , Setlow Peter
Published: July 2002

Bacterial endospores derive much of their longevity and resistance
properties from the relative dehydration of their protoplasts. The
spore cortex, a peptidoglycan structure surrounding the protoplasm,
maintains, and is postulated to have a role in attaining, protoplast
dehydration. A structural modification unique to the spore cortex is
the removal of all or part of the peptide side chains from the majority
of the muramic acid residues and the conversion of 50% of the muramic
acid to muramic lactam. A mutation in the
cwlD
gene of
Bacillus subtilis
, predicted to encode a
muramoyl-
l
-alanine amidase, results in the production of
spores containing no muramic lactam. These spores have normally
dehydrated protoplasts but are unable to complete the
germination/outgrowth process to produce viable cells. Addition of
germinants resulted in the triggering of germination with loss of spore
refractility and the release of dipicolinic acid but no degradation of
cortex peptidoglycan. Germination in the presence of lysozyme allowed
the
cwlD
spores to produce viable cells and showed that
they have normal heat resistance properties. These results
(
i
) suggest that a mechanical activity of the cortex
peptidoglycan is not required for the generation of protoplast
dehydration but rather that it simply serves as a static structure to
maintain dehydration, (
ii
) demonstrate that degradation
of cortex peptidoglycan is not required for spore solute release or
partial spore core rehydration during germination, (
iii
)
indicate that muramic lactam is a major specificity determinant of
germination lytic enzymes, and (
iv
) suggest the
mechanism by which the spore cortex is degraded during germination
while the germ cell wall is left intact.
Geochemical challenge to earthquake prediction.
Authors: Wakita H
Published: July 2002

The current status of geochemical and groundwater observations for earthquake prediction in Japan is described. The development of the observations is discussed in relation to the progress of the earthquake prediction program in Japan. Three major findings obtained from our recent studies are outlined. (i) Long-term radon observation data over 18 years at the SKE (Suikoen) well indicate that the anomalous radon change before the 1978 Izu-Oshima-kinkai earthquake can with high probability be attributed to precursory changes. (ii) It is proposed that certain sensitive wells exist which have the potential to detect precursory changes. (iii) The appearance and nonappearance of coseismic radon drops at the KSM (Kashima) well reflect changes in the regional stress state of an observation area. In addition, some preliminary results of chemical changes of groundwater prior to the 1995 Kobe (Hyogo-ken nanbu) earthquake are presented.
Mutation of gene-proximal regulatory elements disrupts human ɛ-, γ-, and β-globin expression in yeast artificial chromosome transgenic mice
Authors: Liu Qinghui , Bungert Jörg , Engel James Douglas
Published: July 2002

Previous studies have defined transcriptional control elements, in
addition to the promoters, that both lie near individual human
β-globin locus genes and have been implicated in their differential
stage-specific regulation during development (i.e., are believed to
directly participate in hemoglobin switching). We have reinvestigated
the activities during erythropoiesis that might be conferred by two of
the more intensively analyzed of these elements, the ɛ-globin gene 5′
silencer and the β-globin gene 3′ enhancer, by deleting them from a
yeast artificial chromosome that spans the human β-globin locus, and
then analyzing transgenic mice for expression of all of the human
genes. These studies show that sequences within the ɛ-globin
“silencer” are not only required for silencing but are also
required for activation of ɛ-globin transcription; furthermore,
deletion of the silencer simultaneously reduced γ-globin
transcription during the yolk sac stage of erythroid development.
Analysis of the adult β-globin gene 3′ enhancer deletion showed that
its deletion affects only that gene.
A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis
Authors: Nocentini Giuseppe , Giunchi Linda , Ronchetti Simona , Krausz Ludovic Tibor , Bartoli Andrea , Moraca Rosalba , Migliorati Graziella , Riccardi Carlo
Published: July 2002

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.
Aggregation and disaggregation of senile plaques in Alzheimer disease
Authors: Cruz L. , Urbanc B. , Buldyrev S. V. , Christie R. , Gómez-Isla T. , Havlin S. , McNamara M. , Stanley H. E. , Hyman B. T.
Published: July 2002

We quantitatively analyzed, using laser scanning confocal microscopy, the three-dimensional structure of individual senile plaques in Alzheimer disease. We carried out the quantitative analysis using statistical methods to gain insights about the processes that govern
A
β peptide deposition. Our results show that plaques are complex porous structures with characteristic pore sizes. We interpret plaque morphology in the context of a new dynamical model based on competing aggregation and disaggregation processes in kinetic steady-state equilibrium with an additional diffusion process allowing
A
β deposits to diffuse over the surface of plaques.
An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains
Authors: Iovine Nicole M. , Elsbach Peter , Weiss Jerrold
Published: July 2002

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain ofEscherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.
Antileukemia effect of c-
myc
N3′→P5′ phosphoramidate antisense oligonucleotides
in vivo
Authors: Skorski Tomasz , Perrotti Danilo , Nieborowska-Skorska Malgorzata , Gryaznov Sergei , Calabretta Bruno
Published: July 2002

In vitro
, uniformly modified oligonucleotide N3′→P5′ phosphoramidates are apparently more potent antisense agents than phosphorothioate derivatives. To determine whether such compounds are also effective
in vivo
, severe combined immunodeficiency mice injected with HL-60 myeloid leukemia cells were treated systemically with equal doses of either phosphoramidate or phosphorothioate c-
myc
antisense or mismatched oligonucleotides. Compared with mice treated with mismatched oligodeoxynucleotides, the peripheral blood leukemic load of mice treated with the antisense sequences was markedly reduced, and such effects were associated with significantly prolonged survival of the antisense-treated mice. Moreover, with each of three different treatment schedules (100, 300, or 900 μg/day for 6 consecutive days), survival of the phosphoramidate-treated mice was significantly longer than that of the phosphorothioate-treated mice. Both phosphoramidate and phosphorothioate oligonucleotides were efficiently taken up by leukemic cells
in vivo
and were capable of specifically down-regulating c-Myc expression. Moreover, tissue distribution of the phosphoramidate derivatives was undistinguishable from that of the phosphorothioate derivatives. Collectively, these studies suggest that phosphoramidate oligonucleotides can serve as potent and specific antisense agents in the treatment of human leukemia and probably of other malignancies.
Differential utilization of CCR5 by macrophage and T cell tropic simian immunodeficiency virus strains
Authors: Edinger Aimee L. , Amedee Angela , Miller Karen , Doranz Benjamin J. , Endres Michael , Sharron Matthew , Samson Michel , Lu Zhao-hai , Clements Janice E. , Murphey-Corb Michael , Peiper Stephen C. , Parmentier Marc , Broder Christopher C. , Doms Robert W.
Published: July 2002

Certain chemokine receptors serve as cofactors for HIV type 1 envelope (env)-mediated cell–cell fusion and virus infection of CD4-positive cells. Macrophage tropic (M-tropic) HIV-1 isolates use CCR5, and T cell tropic (T-tropic) strains use CXCR4. To investigate the cofactors used by simian immunodeficiency viruses (SIV), we tested four T-tropic and two M-tropic SIV env proteins for their ability to mediate cell–cell fusion with cells expressing CD4 and either human or nonhuman primate chemokine receptors. Unlike HIV-1, both M- and T-tropic SIV envs used CCR5 but not CXCR4 or the other chemokine receptors tested. However, by testing a panel of CCR5/CCR2b chimeras, we found that the structural requirements for CCR5 utilization by M-tropic and T-tropic SIV strains were different. T-tropic SIV strains required the second extracellular loop of CCR5 whereas a closely related M-tropic SIV strain could, like M-tropic HIV-1 strains, use the amino-terminal domain of CCR5. As few as two amino acid changes in the SIV env V3 domain affected the regions of CCR5 that were critical for fusogenic activity. Receptor signaling was not required for either fusion or infection. Our results suggest that viral tropism may be influenced not only by the coreceptors used by a given virus strain but also by how a given coreceptor is used.
Expression of a borage desaturase cDNA containing an N-terminal cytochrome
b
5
domain results in the accumulation of high levels of Δ
6
-desaturated fatty acids in transgenic tobacco
Authors: Sayanova Olga , Smith Mark A. , Lapinskas Peter , Stobart A. Keith , Dobson Gary , Christie William W. , Shewry Peter R. , Napier Johnathan A.
Published: July 2002

γ-Linolenic acid (GLA; C18:3 Δ
6,9,12
) is a component of the seed oils of evening primrose (
Oenothera
spp.), borage (
Borago officinalis
L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a Δ
6
-fatty acid desaturase using linoleic acid (C18:2 Δ
9,12
) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ
6
-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 Δ
6,9,12,15
) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ
6
-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome
b
5
.
Insulin secretion is regulated by the glucose-dependent production of islet β cell macrophage migration inhibitory factor
Authors: Waeber Gérard , Calandra Thierry , Roduit Raphael , Haefliger Jacques-Antoine , Bonny Christophe , Thompson Nancy , Thorens Bernard , Temler Evelyne , Meinhardt Andreas , Bacher Michael , Metz Christine N. , Nicod Pascal , Bucala Richard
Published: July 2002

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting β cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet β cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.