bluuebunny/crossref_metadata_2025 huggingface dataset:
High-speed planar GaAs photoconductors with surface implant layers
Authors: Anderson Gordon Wood , Papanicolaou Nicolas A. , Thompson Phillip E. , Boos John B. , Carruthers Thomas F. , Ma David I , Mack Ingham A. G. , Modolo John A. , Kub Francis J.
Published: July 2002

Selective implantation of silicon into GaAs is demonstrated as a simple method for modifying the response characteristics of low-doped planar GaAs photoconductors for optoelectronic circuits with varying requirements. Response times and sensitivities of the photoconductors were strongly dependent on the implantation dose and energy. Rise times and full width at half-maximum (FWHM) values of devices receiving low-dose implants were of the order of 40–150 ps. Rise times and FWHM values of devices which received higher dose implants were in the ranges 50–140 ps and 1–5 ns, respectively. The sensitivity of devices which received higher dose implants was about a factor of 100 (20 dB in optical power) greater than that of devices which received lower dose implants.
Advanced lift-off planarization process for Josephson integrated circuits
Authors: Ishida Ichiro , Tahara Syuichi , Wada Yoshifusa
Published: July 2002

An advanced lift-off planarization process utilizing an undercut technique of a photoresist etching mask has been developed to achieve planarization of thin-sputtered and fine-patterned films that are necessary for high-performance Josephson integrated circuits (IC’s). A stack of the same kind of photoresist layers, including the modified layer between them, has been utilized as an etching mask providing fine-patterned film profiles with minimized resist degradation by the top photoresist protection layer. This advanced planarization process brings about smooth surfaces having no residues and no grooves along pattern edges. 30 nm deviation from planarity has been demonstrated on a 200-nm-thick planarized Nb superconducting layer. A four-level interconnection of Josephson IC’s was successfully fabricated by this process.
Influence of metal interlayers on Schottky barrier formation for Au/ZnSe (100) and Al/ZnSe (100)
Authors: Vos M. , Xu F. , Weaver J. H. , Cheng H.
Published: July 2002

Schottky barrier formation for Al/ZnSe (100) and Au/ZnSe (100) was studied using photoelectron spectroscopy. The initial Fermi level position for sputter-annealed ZnSe (100) surfaces was 2.05 eV above the valence-band maximum (VBM). The final Fermi level position, established after the deposition of several monolayers of metal adatoms, was very different for Al (2.17 eV) and Au (1.25 eV, relative to the VBM). The deposition of Au interlayers for Al/Au/ZnSe and Al interlayers for Au/Al/ZnSe showed that it is possible to ‘‘tune’’ the Schottky barrier height between these extremes by choosing interlayers of definite thickness.
Suppression of sidelobes in the far-field radiation patterns of optical waveguide arrays
Authors: Abeles J. H. , Deri R. J.
Published: July 2002

Arrays of coherent optical emitters are attractive for high-speed, wide-angle optical beamsteering or deflection, but uniform arrays radiate undesirable sidelobes. It is shown that dramatic suppression of sidelobes can be accomplished by configuring emitters in a nonuniform pattern. A nearly optimum radiation pattern for scanning results from gradually doubling the interelement spacing across a one-dimensional array. As an example, a 100-element waveguide array of minimum interelement spacing 5 μm radiates a 0.08° main lobe (full width at half-maximum) with a 30× suppression of sidelobe intensity.
Photochromism and anomalous crystallite orientation of ZnO films prepared by a sputtering-type electron cyclotron resonance microwave plasma
Authors: Matsuoka Morito , Ono Ken’ichi
Published: July 2002

This letter describes the crystal structure and the photochromism of ZnO films, which are prepared by a sputtering-type electron cyclotron resonance microwave plasma. c-axis preferential orientation and (101) preferential orientation are realized in the Zn oxide films, which are deposited on a glass substrate at a low temperature below 200 °C. Both films have an excellent crystallite orientation that is the best reported to date. The films with anomalous crystallite orientation exhibit typical photochromic characteristics, which are induced by x-ray irradiation. The photochromism is caused by a color center, that is, the O2− vacancy in ZnO films. The absorption center exists in an energy range of 1.5–4 eV.
Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor
Authors: Sawada Hitoshi , Sakai Naoyuki , Abe Yukichi , Tanaka Etsuko , Takahashi Youko , Fujino Junko , Kodama Eri , Takizawa Satoshi , Yokosawa Hideyoshi
Published: July 2002

The ubiquitin–proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata)Halocynthia roretzion sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin–proteasome system during fertilization ofH. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated bothin vitroandin vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin–proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin–proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.
Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts
Authors: Saffran Douglas C. , Raitano Arthur B. , Hubert Rene S. , Witte Owen N. , Reiter Robert E. , Jakobovits Aya
Published: July 2002

Prostate stem-cell antigen (PSCA) is a cell-surface antigen
expressed in normal prostate and overexpressed in prostate cancer
tissues. PSCA expression is detected in over 80% of patients with
local disease, and elevated levels of PSCA are correlated with
increased tumor stage, grade, and androgen independence, including high
expression in bone metastases. We evaluated the therapeutic efficacy of
anti-PSCA mAbs in human prostate cancer xenograft mouse models by using
the androgen-dependent LAPC-9 xenograft and the androgen-independent
recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8
(IgG1κ) and 3C5 (IgG2aκ), inhibited formation of s.c. and
orthotopic xenograft tumors in a dose-dependent manner. Furthermore,
administration of anti-PSCA mAbs led to retardation of established
orthotopic tumor growth and inhibition of metastasis to distant sites,
resulting in a significant prolongation in the survival of
tumor-bearing mice. These studies suggest PSCA as an attractive target
for immunotherapy and demonstrate the therapeutic potential of
anti-PSCA mAbs for the treatment of local and metastatic prostate
cancer.
Plasticity in the organization and sequences of human KIR/ILT gene families
Authors: Wilson Michael J. , Torkar Michaela , Haude Anja , Milne Sarah , Jones Tania , Sheer Denise , Beck Stephan , Trowsdale John
Published: July 2002

The ≈1-Mb leukocyte receptor complex at 19q13.4 is a key polymorphic immunoregion containing all of the natural killer-receptorKIRand relatedILTgenes. When the organization of the leukocyte receptor complex was compared from two haplotypes, the gene content in theKIRregion varied dramatically, with framework loci flanking regions of widely variable gene content. TheILTgenes were more stable in number except forILT6, which was present only in one haplotype. Analysis ofAlurepeats and comparison ofKIRgene sequences, which are over 90% identical, are consistent with a recent origin.KIRgenesis was followed by extensive duplication/deletion as well as intergenic sequence exchange, reminiscent of MHC class I genes, which provide KIR ligands.
Conserved and divergent aspects of terminal patterning in the beetle
Tribolium castaneum
Authors: Schröder Reinhard , Eckert Christoph , Wolff Christian , Tautz Diethard
Published: July 2002

To infer similarities and differences in terminal pattern formation in insects, we analyzed several of the key genes of this process in the beetle
Tribolium castaneum
. We cloned two genes of the terminal pattern cascade, namely
tailless
(
tll
) and
forkhead
(
fkh
), from
Tribolium
and studied their expression patterns. In addition, we analyzed the pattern of MAP kinase activation at blastoderm stage as a possible signature for
torso-
dependent signaling. Further, we analyzed the late expression of the previously cloned
Tribolium caudal
(
Tc-cad
) gene. Finally, we used the upstream region of
Tc-tll
to drive a reporter gene construct in
Drosophila
. We find that this construct is activated at the terminal regions in
Drosophila
, suggesting that the
torso-
dependent pathway is conserved between the species. We show that most of the expression patterns of the genes studied here are similar in
Drosophila
and
Tribolium
, suggesting conserved functions. There is, however, one exception, namely the early function of
Tc-tll
at the posterior pole. In
Drosophila
, the posterior
tll
expression is involved in the direct regulation of the target genes of the terminal pathway. In
Tribolium
, posterior
Tc-tll
expression occurs only for a short time and ceases before the target genes known from
Drosophila
are activated. Thus, we infer that
Tc-tll
does not function as a direct regulator of segmentation genes at the posterior end. It is more likely to be involved in the early specification of a group of “terminal” cells, which begin to differentiate only at a later stage of embryogenesis, when much of the abdominal segmentation process is complete. Thus, there appears to have been a major shift in
tll
function during the evolutionary transition from short germ to long germ embryogenesis.
The endocytic catalysts, Rab5a and Rab7, are tandem regulators of thyroid hormone production
Authors: Croizet-Berger Karine , Daumerie Chantal , Couvreur Marianne , Courtoy Pierre J. , van den Hove Marie-France
Published: July 2002

Rab proteins are small GTPases that control distinct vesicular transport steps. Along the endocytic pathway, Rab5a is a rate-limiting catalyst of internalization, and Rab7 controls trafficking through late endosomes to lysosomes. The dependence of thyroid hormone production by thyrocytes on thyroglobulin endocytosis and intracellular processing in late endosomes/lysosomes suggests that its rate can be regulated by the expression or function of these endocytic catalysts. We compared the expression level and membrane recruitment of Rab5a and Rab7 in autonomous thyroid adenomas (where the cAMP cascade is constitutively activated) and surrounding quiescent tissues. The concentrations of Rab5a and Rab7, but not of Rab8, were coordinately increased up to 6-fold in adenomas, and correlated with a proportionate decrease in soluble thyroglobulin content (reflecting colloid depletion by accelerated endocytic uptake in hyperactive tissue). In adenomas, a higher proportion of Rab5a and Rab7 was membrane associated, and the equilibrium density of particulate Rab7 and iodine shifted toward lysosomal fractions, indicating that progression along the degradation pathway also was promoted. In cultures of polarized human thyrocytes from normal patients, thyroid-stimulating hormone or forskolin increased, to a similar extent, Rab5a and Rab7 but not Rab8 expression, apical endocytosis of thyroglobulin and lucifer yellow, and basolateral secretion of T
3
and T
4
. Taken together, these
in vivo
and
in vitro
observations demonstrate that thyroid-stimulating hormone, via cAMP, coordinately enhances the expression of Rab5a and Rab7, which promote Tg endocytosis and transfer to lysosomes, respectively, resulting in accelerated thyroid hormone production.
A pentacyclic reaction intermediate of riboflavin synthase
Authors: Illarionov Boris , Eisenreich Wolfgang , Bacher Adelbert
Published: July 2002

The S41A mutant of riboflavin synthase from
Escherichia coli
catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various
13
C- and
15
N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.
A two-hybrid system for transactivator bait proteins
Authors: Hirst Martin , Ho Cynthia , Sabourin Luc , Rudnicki Michael , Penn Linda , Sadowski Ivan
Published: July 2002

We describe a two-hybrid strategy for detection of interactions with transactivator proteins. This repressed transactivator (RTA) system employs the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80
in vitro
. Furthermore, repression of
GAL
transcription by TUP1-GAL80 requires
SRB10
, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix–loop–helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins
in vivo
.
Snapshots of usher-mediated protein secretion and ordered pilus assembly
Authors: Saulino Evan T. , Bullitt Esther , Hultgren Scott J.
Published: July 2002

Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane. The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis. We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili. We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum. Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation. Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly. Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.
Autocatalytic proteolysis of the transcription factor-coactivator C1 (HCF): A potential role for proteolytic regulation of coactivator function
Authors: Vogel Jodi L. , Kristie Thomas M.
Published: July 2002

Site-specific proteolysis is an important biological mechanism for the regulation of cellular processes such as gene expression, cell signaling, development, and apoptosis. In transcriptional regulation, specific proteolysis regulates the localization and activity of many regulatory factors. The C1 factor (HCF), a cellular transcription factor and coactivator, undergoes site-specific proteolytic processing at a series of unusual amino acid reiterations to generate a family of amino- and carboxyl-terminal polypeptides that remain tightly associated. Expression and purification of bacterially expressed domains of the C1 factor identifies an autocatalytic activity that is responsible for the specific cleavage of the reiterations. In addition, coexpression of the autocatalytic domain with a heterologous protein containing a target cleavage site demonstrates that the C1 protease may also function in trans.
Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts
Authors: Khodarev Nikolai N. , Park James O. , Yu Jianqing , Gupta Nalin , Nodzenski Edwardine , Roizman Bernard , Weichselbaum Ralph R.
Published: July 2002

U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown
in vitro
were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation. The xenografts were harvested at 1, 5, or 24 h after irradiation and subjected to the same analyses. We observed and report on (
i
) cell-type common and cell-type specific responses, (
ii
) genes induced at low levels of irradiation but not at higher doses, (
iii
) temporal patterns of gene response in U87 xenografts that varied depending on radiation dose and temporal patterns of response that were similar at all doses tested, (
iv
) significantly higher up-regulation of cells in xenografts than in
in vitro
cultures, and (
v
) genes highly up-regulated by radiation. The responding genes could be grouped into nine functional clusters. The representation of the nine clusters was to some extent dependent on dose and time after irradiation. The results suggest that clinical outcome of ionizing radiation treatment may benefit significantly by taking into account both cell-type and radiation-dose specificity of cellular responses.
The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: Implications for the evolution of mammalian sex determination
Authors: Pask Andrew , Renfree Marilyn B. , Marshall Graves Jennifer A.
Published: July 2002

Mutations in the
ATRX
gene on the human X chromosome
cause X-linked α-thalassemia and mental retardation. XY patients with
deletions or mutations in this gene display varying degrees of sex
reversal, implicating
ATRX
in the development of the
human testis. To explore further the role of
ATRX
in
mammalian sex differentiation, the homologous gene was cloned and
characterized in a marsupial. Surprisingly, active homologues of
ATRX
were detected on the marsupial Y as well as the X
chromosome. The Y-borne copy (
ATRY
) displays
testis-specific expression. This, as well as the sex reversal of ATRX
patients, suggests that
ATRY
is involved in testis
development in marsupials and may represent an ancestral
testis-determining mechanism that predated the evolution of
SRY
as the primary mammalian male sex-determining gene.
There is no evidence for a Y-borne
ATRX
homologue in
mouse or human, implying that this gene has been lost in eutherians and
its role supplanted by the evolution of
SRY
from
SOX3
as the dominant determiner of male differentiation.
A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.
Authors: David C , McPherson P S , Mundigl O , de Camilli P
Published: July 2002

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.
The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.
Authors: Carlsson L M , Marklund S L , Edlund T
Published: July 2002

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.
High mobility group I(Y)-like DNA-binding domains on a bacterial transcription factor.
Authors: Nicolas F J , Cayuela M L , Martínez-Argudo I M , Ruiz-Vazquez R M , Murillo F J
Published: July 2002

The bacterium Myxococcus xanthus responds to blue light by producing carotenoids. It also responds to starvation conditions by developing fruiting bodies, where the cells differentiate into myxospores. Each response entails the transcriptional activation of a separate set of genes. However, a single gene, carD, is required for the activation of both light- and starvation-inducible genes. Gene carD has now been sequenced. Its predicted amino acid sequence includes four repeats of a DNA-binding domain present in mammalian high mobility group I(Y) proteins and other nuclear proteins from animals and plants. Other peptide stretches on CarD also resemble functional domains typical of eukaryotic transcription factors, including a very acidic region and a leucine zipper. High mobility group yI(Y) proteins are known to bind the minor groove of A+T-rich DNA. CarD binds in vitro an A+T-rich element that is required for the proper operation of a carD-dependent promoter in vivo.
Sematech: Purpose and Performance
Authors: Irwin Douglas A. , Klenow Peter J.
Published: July 2002

In previous research, we have found a steep learning curve in the
production of semiconductors. We estimated that most production
knowledge remains internal to the firm, but that a significant fraction
“spills over” to other firms. The existence of such spillovers
may justify government actions to stimulate research on semiconductor
manufacturing technology. The fact that not all production knowledge
spills over, meanwhile, creates opportunities for firms to form joint
ventures and slide down their learning curves more efficiently. With
these considerations in mind, in 1987 14 leading U.S. semiconductor
producers, with the assistance of the U.S. government in the form of
$100 million in annual subsidies, formed a research and development
(R&D) consortium called Sematech. In previous research, we estimated
that Sematech has induced its member firms to lower their R&D spending.
This may reflect more sharing and less duplication of research, i.e.,
more research being done with each R&D dollar. If this is the case,
then Sematech members may wish to replace any funding withdrawn by the
U.S. government. This in turn would imply that the U.S. government’s
contributions to Sematech do not induce more semiconductor research
than would otherwise occur.
Arabidopsis thaliana
defense-related protein ELI3 is an aromatic alcohol:NADP
+
oxidoreductase
Authors: Somssich Imre E. , Wernert Petra , Kiedrowski Siegrid , Hahlbrock Klaus
Published: July 2002

We expressed a cDNA encoding the
Arabidopsis
thaliana
defense-related protein ELI3-2 in
Escherichia
coli
to determine its biochemical function. Based on a protein
database search, this protein was recently predicted to be a mannitol
dehydrogenase [Williamson, J. D., Stoop, J. M. H., Massel, M. O.,
Conkling, M. A. & Pharr, D. M. (1995)
Proc. Natl. Acad. Sci.
USA
92, 7148–7152]. Studies on the substrate specificity now
revealed that ELI3-2 is an aromatic alcohol: NADP
+
oxidoreductase (benzyl alcohol dehydrogenase). The enzyme showed a
strong preference for various aromatic aldehydes as opposed to the
corresponding alcohols. Highest substrate affinities were observed for
2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, and
benzaldehyde, in this order, whereas mannitol dehydrogenase activity
could not be detected. These and previous results support the notion
that ELI3-2 has an important role in resistance-related aromatic
acid-derived metabolism.
A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.
Authors: Rodermel S , Haley J , Jiang C Z , Tsai C H , Bogorad L
Published: July 2002

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.
A molecular level picture of the stabilization of A-DNA in mixed ethanol–water solutions
Authors: Cheatham Thomas E. , Crowley Michael F. , Fox Thomas , Kollman Peter A.
Published: July 2002

Advances in computer power, methodology, and empirical force fields now allow routine “stable” nanosecond-length molecular dynamics simulations of DNA in water. The accurate representation of environmental influences on structure remains a major, unresolved issue. In contrast to simulations of A-DNA in water (where an A-DNA to B-DNA transition is observed) and in pure ethanol (where disruption of the structure is observed), A-DNA in ≈85% ethanol solution remains in a canonical A-DNA geometry as expected. The stabilization of A-DNA by ethanol is likely due to disruption of the spine of hydration in the minor groove and the presence of ion-mediated interhelical bonds and extensive hydration across the major groove.
Expansion
in vitro
of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic–
scid/scid
mice
Authors: Conneally E. , Cashman J. , Petzer A. , Eaves C.
Published: July 2002

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic–
scid/scid
(NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34
−
CD19
+
(B-lymphoid) and CD34
+
(myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38
−
and CD38
+
subsets of CD34
+
CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34
+
CD38
−
human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5–8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant,
P
< 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.
Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1
Authors: Scheek Sigrid , Brown Michael S. , Goldstein Joseph L.
Published: July 2002

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH
2
-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.
Arabidopsis thaliana
mutants altered in homologous recombination
Authors: Masson Jean E. , Paszkowski Jerzy
Published: July 2002

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine
Arabidopsis thaliana
mutants hypersensitive to x-ray irradiation (
xrs
) and examined their recombination properties. For the three
xrs
loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMC
s
) and/or methyl methanesulfonate (MMS
s
) and alterations in homologous recombination. Mutant
xrs9
(Xray
s
, MMS
s
) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the
rad52
epistatic group.
xrs11
(Xray
s
, MMC
s
) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect.
xrs4
(Xray
s
, MMS
s
, MMC
s
) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.
Proteins on ribosome surface: Measurements of protein exposure by hot tritium bombardment technique
Authors: Agafonov Dmitry E. , Kolb Vyacheslav A. , Spirin Alexander S.
Published: July 2002

The hot tritium bombardment technique [Goldanskii, V. I., Kashirin, I. A., Shishkov, A. V., Baratova, L. A. & Grebenshchikov, N. I. (1988)
J. Mol. Biol.
201, 567–574] has been applied to measure the exposure of proteins on the ribosomal surface. The technique is based on replacement of hydrogen by high energy tritium atoms in thin surface layer of macromolecules. Quantitation of tritium radioactivity of each protein has revealed that proteins S1, S4, S5, S7, S18, S20, and S21 of the small subunit, and proteins L7/L12, L9, L10, L11, L16, L17, L24, and L27 of the large subunit are well exposed on the surface of the
Escherichia coli
70 S ribosome. Proteins S8, S10, S12, S16, S17, L14, L20, L29, L30, L31, L32, L33, and L34 have virtually no groups exposed on the ribosomal surface. The remaining proteins are found to be exposed to lesser degree than the well exposed ones. No additional ribosomal proteins was exposed upon dissociation of ribosomes into subunits, thus indicating the absence of proteins on intersubunit contacting surfaces.
Isotropic isotopy and symplectic null sets
Authors: Tokieda Tadashi F.
Published: July 2002

Capacity is an important numerical invariant of symplectic manifolds. This paper studies when a subset of a symplectic manifold is null, i.e., can be removed without affecting the ambient capacity. After examples of open null sets and codimension-2 non-null sets, geometric techniques are developed to perturb any isotopy of a loop to a hamiltonian flow; it follows that sets of dimension 0 and 1 are null. For isotropic sets of higher dimensions, obstructions to the perturbation are found in homotopy groups of the orthogonal groups.
Sequence of the
FRA3B
common fragile region: Implications for the mechanism of
FHIT
deletion
Authors: Inoue Hiroshi , Ishii Hideshi , Alder Hansjuerg , Snyder Eric , Druck Teresa , Huebner Kay , Croce Carlo M.
Published: July 2002

The hypothesis that chromosomal fragile sites may be “weak links” that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the
FHIT
gene, which encompasses the common fragile site,
FRA3B
. Sequence analysis of 276 kb of the
FRA3B
/
FHIT
locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in
FHIT
gene internal deletions, probably as a result of carcinogen-induced damage at
FRA3B
fragile sites.
5-Methylcytosine is not a mutation hot spot in nondividing
Escherichia coli
Authors: Lieb Margaret , Rehmat Shehnaz
Published: July 2002

Spontaneous deamination of 5-methylcytosine (5meC) causes hot spots of C·G → T·A mutations in
Escherichia coli
and in human cells. In
E. coli
, the resulting T·G mispairs can be corrected to C·G by very short patch (VSP) repair, which requires the product of gene
vsr
. Mutation hot spots in genes of replicating
vsr
+
bacteria are attributable to low Vsr activity. To determine the rate of deamination of 5meC and the efficiency of VSP repair in nondividing bacteria, we used kanamycin-sensitive (Kan
S
) lysogens containing a λ
kan
−
prophage. Deamination of a 5meC in the
kan
−
gene resulted in mutation to kanamycin resistance (Kan
R
). Lysogens containing a single λ
kan
−
prophage per bacterial genome were grown in synthetic medium with limiting amino acids and stored at 15°C or 37°C. In the absence of VSP repair, Kan
R
mutants accumulated at the rate of approximately 1.3 × 10
−7
per bacterium per day at 37°C. This is similar to the 5meC → T mutation rate reported for DNA in solution. In
vsr
+
bacteria, the Kan
R
accumulation rate was 3 × 10
−9
per bacterium per day, which is not significantly higher than the rate observed when the target cytosine was unmethylated. The increase in Kan
R
mutants was barely detectable in
vsr
+
cultures stored at 15°C for 4 months. It is likely that mutation hot spots at 5meC in rapidly dividing cells are attributable to insufficient time for T·G correction in the interval between deamination of 5meC and subsequent DNA replication. DNA synthesis occurred in bacteria starved for amino acids and this synthesis was not highly mutagenic.
Binding of an arm repeat protein to the kinase domain of the
S
-locus receptor kinase
Authors: Gu Tiesheng , Mazzurco Maria , Sulaman Waheeda , Matias Dinah D. , Goring Daphne R.
Published: July 2002

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an
S
-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (
A
rm
R
epeat
C
ontaining). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and
in vitro
binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different
Arabidopsis
receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.
A cobalt complex that selectively disrupts the structure and function of zinc fingers
Authors: Louie A. Y. , Meade T. J.
Published: July 2002

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes.
1
H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.
Clines of nuclear DNA markers suggest a largely Neolithic ancestry of the European gene pool
Authors: Chikhi Lounès , Destro-Bisol Giovanni , Bertorelle Giorgio , Pascali Vincenzo , Barbujani Guido
Published: July 2002

Comparisons between archaeological findings and allele frequencies at protein loci suggest that most genes of current Europeans descend from populations that have been expanding in Europe in the last 10,000 years, in the Neolithic period. Recent mitochondrial data have been interpreted as indicating a much older, Paleolithic ancestry. In a spatial autocorrelation study at seven hypervariable loci in Europe (four microsatellites, two larger, tandem-repeat loci, and a sequence polymorphism) broad clinal patterns of DNA variation were recognized. The observed clines closely match those described at the protein level, in agreement with a possible Near Eastern origin for the ancestral population. Separation times between populations were estimated on the basis of a stepwise mutation model. Even assuming low mutation rates and long generation times, we found no evidence for population splits older than 10,000 years, with the predictable exception of Saami (Lapps). The simplest interpretation of these results is that the current nuclear gene pool largely reflects the westward and northward expansion of a Neolithic group. This conclusion is now supported by purely genetic evidence on the levels and patterns of microsatellite diversity, rather than by correlations of biological and nonbiological data. We argue that many mitochondrial lineages whose origin has been traced back to the Paleolithic period probably reached Europe at a later time.
cAMP-dependent protein kinase phosphorylates and activates nuclear Ca
2+
-ATPase
Authors: Rogue Patrick J. , Humbert Jean-Paul , Meyer Alphonse , Freyermuth Solange , Krady Marie-Marthe , Malviya Anant N.
Published: July 2002

A Ca
2+
-pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca
2+
-ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca
2+
-ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated/mol of partially purified enzyme. Measurement of ATP-dependent
45
Ca
2+
uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca
2+
-pumping activity of NCA. We show that PKA phosphorylation of Ca
2+
-ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP/PKA- and Ca
2+
-dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope.
OB-Rb gene transfer to leptin-resistant islets reverses diabetogenic phenotype
Authors: Wang May-Yun , Koyama Kazunori , Shimabukuro Michio , Newgard Christopher B. , Unger Roger H.
Published: July 2002

In obese Zucker diabetic fatty (ZDF) rats with mutant leptin receptors, pancreatic islets have an ≈50-fold increase in fat (TG), overproduce nitric oxide (NO), and lack a normal proinsulin mRNA response to fatty acids. We overexpressed the wild-type full-length “b” isoform of the leptin receptor (OB-Rb) in ZDF islets by perfusing ZDF pancreata with recombinant adenovirus containing the cDNA encoding OB-Rb. In cultured islets isolated from these animals, leptin lowered islet TG by 87% and completely blocked TG formation from free fatty acids. Overproduction of NO was reduced, and the preproinsulin mRNA response to free fatty acids was restored. This establishes defective leptin action as the proximate cause of lipotoxic diabetes in ZDF rats.
Roles of SecG in ATP- and SecA-dependent protein translocation
Authors: Matsumoto Gen , Mori Hiroyuki , Ito Koreaki
Published: July 2002

SecA, the translocation ATPase in
Escherichia coli
, undergoes cycles of conformational changes (insertion/deinsertion) in response to ATP and a preprotein. The membrane-embedded portion of protein translocase, SecYEG, has crucial roles in the SecA-driven preprotein translocation reaction. We previously identified a
secY
mutation (
secY205
) that did not allow an ATP- and preprotein-dependent (productive) insertion of SecA as well as
secA
mutations that suppressed the
secY205
translocation defect. One of the suppressor mutations,
secA36
, also suppressed the cold-sensitive phenotype of the
secG
deletion mutant.
In vitro
experiments at 20°C showed that inverted membrane vesicles lacking SecG were almost inactive in combination with the wild-type SecA protein in translocation of proOmpA as well as in the accompanying ATP hydrolysis. In contrast, the SecA36 mutant protein was found to be able to execute the translocation activity fully at this temperature, even in the absence of SecG. A SecG requirement and its alleviation by the SecA36 alteration also were shown for the SecA insertion reaction. The finding that the SecA36 protein no longer requires assistance from SecG in its insertion and in its catalysis of protein translocation agrees with the idea that SecG normally assists in the functioning of SecA. In agreement with this notion, when the intrinsic SecA function was compromised by a lowered ATP concentration, SecG became essential even at 37°C and even for the SecA36 protein. We propose that in the normal translocase, SecG cooperates with SecA to facilitate efficient movement of preprotein in each catalytic cycle of SecA.
Over one-half billion years of head conservation? Expression of an
ems
class gene in
Hydractinia symbiolongicarpus
(Cnidaria: Hydrozoa)
Authors: Mokady Ofer , Dick Matthew H. , Lackschewitz Dagmar , Schierwater Bernd , Buss Leo W.
Published: July 2002

We report the isolation of an
empty spiracles
class homeodomain-containing gene,
Cn-ems
, from the hydrozoan
Hydractinia symbiolongicarpus,
the first gene of this class characterized in a lower metazoan.
Cn-ems
was found to be expressed in the head of gastrozooids, specifically in endodermal epithelial cells of the taeniolae of the hypostome.
Cn-ems
is not expressed in gonozooids, which lack taeniolae. Experimental conversion of the posterior region of the planula larva into head structures up-regulates expression of the gene. These findings establish that the association of
ems
-class genes with head structures preceded the evolution of bilateral symmetry.