bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of an electron beam pumped KrF laser amplifier with an atmospheric-pressure Kr-rich mixture in a strongly saturated region
Authors: Suda Akira , Kumagai Hiroshi , Obara Minoru
Published: July 2002

Amplifier characteristics of a KrF laser were investigated as a function of Kr concentration for a wide range of input intensity. The experiments were performed with atmospheric-pressure mixtures excited by a 65-ns (full width at half-maximum), 160-A/cm2 electron beam. The extracted intensity (output intensity minus input intensity) from a single-pass (50 cm) amplifier was maximized at an input intensity around 5 MW/cm2 and then, the power efficiencies were 12.6, 13.7, 13.3, and 11.3% for Kr concentrations of 10, 20, 40, and 99.4%, respectively. The small-signal gain, absorption coefficient, and saturation intensity were determined for each mixture by using an analysis due to Rigrod [J. Appl. Phys. 36, 2487 (1965)]. The KrF* formation efficiency and the extraction efficiency were also obtained as a function of Kr concentration.
Ultrafast time response of optical nonlinearity in polysilane polymers
Authors: Yang Lina , Wang Q. Z. , Ho P. P. , Dorsinville R. , Alfano R. R. , Zou W. K. , Yang N. L.
Published: July 2002

Ultrafast relaxation kinetics of third-order nonlinear susceptibility χ(3) of polysilane polymers was measured using both the picosecond Kerr gate and forward degenerated four-wave mixing. The relative contributions from various nonlinear decay mechanisms were estimated from the time response. The third-order nonlinear response was measured to be faster than 3 ps arising from electronic mechanism. A slower component is assigned to the refractive index grating due to light pressure.
The GATA factor Serpent is required for the onset of the humoral immune response in
Drosophila
embryos
Authors: Tingvall Tove Önfelt , Roos Erik , Engström Ylva
Published: July 2002

Innate immunity in
Drosophila
is characterized by
the inducible expression of antimicrobial peptides. We have
investigated the development and regulation of immune responsiveness in
Drosophila
embryos after infection. Immune competence,
as monitored by the induction of
Cecropin
A1
-
lacZ
constructs, was observed first in the
embryonic yolk. This observation suggests that the yolk plays an
important role in the humoral immune response of the developing embryo
by synthesizing antimicrobial peptides. Around midembryogenesis, the
response in the yolk was diminished. Simultaneously,
Cecropin
expression became inducible in a large number
of cells in the epidermis, demonstrating that late-stage embryos can
synthesize their own antibiotics in the epidermis. This production
likely serves to provide the hatching larva with an active
antimicrobial barrier and protection against systemic infections.
Cecropin
expression in the yolk required the presence of
a GATA site in the promoter as well as the involvement of the
GATA-binding transcription factor Serpent (dGATAb). In contrast,
neither the GATA site nor Serpent were necessary for
Cecropin
expression in the epidermis. Thus, the
inducible immune responses in the yolk and in the epidermis can be
uncoupled and call for distinct sets of transcription factors. Our data
suggest that Serpent is involved in the distinction between a systemic
response in the yolk/fat body and a local immune response in
epithelial cells. In addition, the present study shows that signal
transduction pathways controlling innate and epithelial defense
reactions can be dissected genetically in
Drosophila
embryos.
An
N
-methyl-
d
-aspartate receptor channel blocker with neuroprotective activity
Authors: Tai Kwok-Keung , Blondelle Sylvie E. , Ostresh John M. , Houghten Richard A. , Montal Mauricio
Published: July 2002

Excitotoxicity, resulting from sustained activation of
glutamate receptors of the
N
-methyl-
d
-aspartate (NMDA) subtype, is
considered to play a causative role in the etiology of ischemic stroke
and several neurodegenerative diseases. The NMDA receptor is therefore
a target for the development of neuroprotective agents. Here, we
identify an N-benzylated triamine (denoted as NBTA) as a highly
selective and potent NMDA-receptor channel blocker selected by
screening a reduced dipeptidomimetic synthetic combinatorial library.
NBTA blocks recombinant NMDA receptors expressed in
Xenopus laevis
oocytes with a mean
IC
50
of 80 nM; in contrast, it does not block GluR1, a
glutamate receptor of the non-NMDA subtype. The blocking activity of
NBTA on NMDA receptors exhibits the characteristics of an open-channel
blocker: (
i
) no competition with agonists,
(
ii
) voltage dependence, and (
iii
) use
dependence. Significantly, NBTA protects rodent hippocampal neurons
from NMDA receptor, but not kainate receptor-mediated excitotoxic cell
death, in agreement with its selective action on the corresponding
recombinant receptors. Mutagenesis data indicate that the N site, a key
asparagine on the M2 transmembrane segment of the NR1 subunit, is the
main determinant of the blocker action. The results highlight the
potential of this compound as a neuroprotectant.
Translocation pathway of protein substrates in ClpAP protease
Authors: Ishikawa Takashi , Beuron Fabienne , Kessel Martin , Wickner Sue , Maurizi Michael R. , Steven Alasdair C.
Published: July 2002

Intracellular protein degradation, which must be tightly controlled
to protect normal proteins, is carried out by ATP-dependent proteases.
These multicomponent enzymes have chaperone-like ATPases that
recognize and unfold protein substrates and deliver them to the
proteinase components for digestion. In ClpAP, hexameric rings of the
ClpA ATPase stack axially on either face of the ClpP proteinase, which
consists of two apposed heptameric rings. We have used cryoelectron
microscopy to characterize interactions of ClpAP with the model
substrate, bacteriophage P1 protein, RepA. In complexes stabilized by
ATPγS, which bind but do not process substrate, RepA dimers are seen
at near-axial sites on the distal surface of ClpA. On ATP addition,
RepA is translocated through ≈150 Å into the digestion chamber
inside ClpP. Little change is observed in ClpAP, implying that
translocation proceeds without major reorganization of the ClpA
hexamer. When translocation is observed in complexes containing a ClpP
mutant whose digestion chamber is already occupied by unprocessed
propeptides, a small increase in density is observed within ClpP, and
RepA-associated density is also seen at other axial sites. These sites
appear to represent intermediate points on the translocation pathway,
at which segments of unfolded RepA subunits transiently accumulate en
route to the digestion chamber.
Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors
Authors: Wilkinson Robert W. , Ross Elizabeth L. , Poulsom Richard , Ilyas Mohammad , Straub Josef , Snary David , Bodmer Walter F. , Mather Stephen J.
Published: July 2002

Monoclonal antibodies (mAbs) have been used to treat malignancies in humans with varying degrees of success. Progress has been hindered by the lack of suitable animal models, which would ideally consist of immunocompetent animals that are tolerant to tumor-associated antigens. Suitable models would allow the study and optimization of anti-tumor immunotherapy. We describe a murine model for the study of immunotherapy in colorectal cancers. Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein that is expressed on normal human intestinal epithelium and that is overexpressed in intestinal tumors. Mice that are transgenic for the human CEA gene (CEA.Tg) were crossed with multiple intestinal neoplasia (MIN) mice. MIN mice carry a germline
APC
mutation and are prone to the development of intestinal adenomas. The offspring from the MIN × CEA.Tg cross developed intestinal adenomas that were shown by immunohistochemistry to overexpress CEA. Pharmacokinetic studies by using
125
I-labeled anti-CEA mAb PR1A3 showed rapid localization of antibody to tissues expressing CEA, especially the gastrointestinal tract. Macroscopic and microscopic radioautographic analysis of the gastrointestinal tracts from MIN/CEA.Tg mice indicated that PR1A3 targeted and was retained in tumors at levels higher than in areas of normal gut. These results demonstrate the utility of the MIN/CEA.Tg mouse as a model for the study of anti-CEA immunotherapy and, furthermore, demonstrate the efficiency of tumor localization by PR1A3.
Vaccination, prevention, and treatment of experimental autoimmune neuritis (EAN) by an oligomerized T cell epitope
Authors: Stienekemeier M. , Falk K. , Rötzschke O. , Weishaupt A. , Schneider C. , Toyka K. V. , Gold R. , Strominger J. L.
Published: July 2002

Using a polypeptide oligomer harboring 16 repeats of the neuritogenic epitope (aa 58–73) of myelin P2 protein separated by spacers, enhancement of the immune response to the P2 protein, an important neuritogenic autoantigen in experimental autoimmune neuritis (EAN), was attempted. In contrast to a previous study with PLP-16-mer antigen-specific response of T cells was attenuated at all doses examined to a variable degree. Treatment of Lewis rats with the P2-16-mer up to 2 months before immunization with P253–78(vaccination) or after immunization but before appearance of disease (prevention) had a strong tolerizing effect against the induction of EAN on immunization with P253–78. Moreover, rats injected with 200 μg of the P2-16-mer i.v. on day 11 after disease induction, at which time the initial signs of disease had appeared, were almost completely protected against progression of clinical disease, whereas animals treated with the same amount of monomeric control peptide developed severe disease (treatment). Similar results were obtained by i.v. treatment of adoptive-transfer EAN with the P2-16-mer. The lack of clinical signs of disease after 16-mer therapy could be correlated with a reduced proliferative response of P253–78-specific lymph node cells. The frequency of apoptotic T cells in sciatic nerve or in lymph node cells, however, was not increased by the 16-mer treatment, suggesting that induction of anergy or other forms of peripheral tolerance may be responsible for the effect. Thus, the oligomerized P2 peptide antigen was highly effective in all three treatment modalities examined in this specific autoreactive T cell-mediated immune response.
Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.
Authors: Bamberger A M , Bamberger C M , Gellersen B , Schulte H M
Published: July 2002

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.
Development of Valpha4+ NK T cells in the early stages of embryogenesis.
Authors: Makino Y , Kanno R , Koseki H , Taniguchi M
Published: July 2002

The majority of T lymphocytes start to develop at around day 15 of gestation (d15)-d17 in the thymus and comprise the peripheral repertoire characterized by the expression of polymorphic T-cell antigen receptors (TCRs). Contrary to these conventional T cells, a subset of T cells, called natural killer (NK) T cells (most of them expressing an invariant TCR encoded by the Valpha14Jalpha281 gene with a 1-nt N-region), preferentially differentiates extrathymically and dominates the peripheral T-cell population at a high frequency (5% in splenic T cells and 40% in bone marrow T cells). Here, we investigated the development of NK T cells and found that the invariant Valpha14+ TCR transcripts and the circular DNA created by Valpha14 and Jalpha281 gene rearrangements can be detected in the embryo body at d9.5 of gestation and in the yolk sac and the fetal liver at d11.5-d13.5 of gestation, but not in the thymus, whereas T cells with Valpha1+ TCR expression, a major population in the thymus, were not observed at these early stages of gestation. Fluorescence-activated cell sorter analysis also demonstrated that there exist CD3+ alpha beta+ T cells, almost all of which are Valpha14/Vbeta8+ NK+ T cells, during early embryogenesis. To our knowledge, this demonstrates for the first time that a T lymphocyte subset develops in extrathymic tissues during the early stages of embryogenesis.
Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.
Authors: Torres-Ramos C A , Yoder B L , Burgers P M , Prakash S , Prakash L
Published: July 2002

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.
Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.
Authors: Chen S T , Iida A , Guo L , Friedmann T , Yee J K
Published: July 2002

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.
Developmental expression of synthetic cis-regulatory systems composed of spatial control elements from two different genes
Authors: Kirchhamer Carmen V. , Bogarad Leonard D. , Davidson Eric H.
Published: July 2002

Synthetic cis-regulatory systems consisting of positively and
negatively acting cis-regulatory modules of the
Endo16
gene were combined with the lineage-specific regulatory element of the
SM50
gene associated with a reporter and injected into
eggs of sea urchins. We show here that synthetic cis-regulatory systems
consisting of the positive
Endo16
regulatory elements
linked with the
SM50
regulatory element are expressed
spatially exactly as the sum of the individual endodermal and
skeletogenic expression patterns. In combination, both lineage-specific
positive regulatory elements function autonomously. However, addition
of the
Endo16
regulatory module that represses ectopic
skeletogenic expression of
Endo16
receptor constructs
does not affect expression driven by the
SM50
regulatory
elements in the same skeletogenic cells. The repression function of
this element is thus dedicated to control of the positive spatial
output of the
Endo16
regulatory system.
An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression
Authors: Magis Wendy , Fiering Steven , Groudine Mark , Martin David I. K.
Published: July 2002

The mouse metallothionein-I (mMT-I) promoter is activated
by the metal response element-binding transcription factor (MTF), which
binds metal response elements (MREs) when stimulated with heavy metals.
We analyzed eight K562 erythroleukemia cell clones, each carrying a
single integrated copy of an mMT-I/β-geo construct, using
a system that can independently assess the level of β-geo expression
and the rate at which it is silenced. In these clones, basal expression
and rate of silencing vary widely and independently with integration
site. This implies that the rates of transcription and of silencing are
separate properties determined by interaction of the regulatory
elements of the transgene with the site of integration. Induction of
the mMT-I promoter with zinc both increases expression level and
strongly retards silencing of β-geo expression. At a given
integration site, expression level and silencing are affected
coordinately by induction. Taken together with earlier studies of
distant metal-responsive elements, these results suggest that distance
from the promoter may determine whether a factor can increase
transcription rate. Stimulation of an MRE can both increase
transcription and overcome repressive effects of chromatin; we suggest
that these functions are linked.
Modulation of the chaperone-like activity of bovine α-crystallin
Authors: Clark John I. , Huang Qing-ling
Published: July 2002

The effects of pantethine, glutathione, and selected chemical
reagents on the anti-aggregation activity of α-crystallin was
evaluated. Protein aggregation was monitored by light scattering of
solutions of denatured β
L
-crystallin or alcohol
dehydrogenase (ADH). The ratios of
β
L
-crystallin/α-crystallin and
ADH/α-crystallin were adjusted so that partial inhibition
of protein aggregation at 60°C or 37°C, respectively, was observed
and modulation of the chaperone action of α-crystallin could be
evaluated easily with selected endogenous metabolites.
Enhancement of the anti-aggregation activity in the
β
L
-crystallin assay was strongest with pantethine, which
appeared to interact with α-crystallin. Enhancement of the
anti-aggregation activity in the ADH assay was strongest with
glutathione which appeared to interact with ADH. The results indicated
that the products of common metabolic pathways can modulate the
chaperone-like effects of α-crystallin on protein aggregation.
Dynamic friction and the origin of the complexity of earthquake sources.
Authors: Madariaga R , Cochard A
Published: July 2002

We study a simple antiplane fault of finite length embedded in a homogeneous isotropic elastic solid to understand the origin of seismic source heterogeneity in the presence of nonlinear rate- and state-dependent friction. All the mechanical properties of the medium and friction are assumed homogeneous. Friction includes a characteristic length that is longer than the grid size so that our models have a well-defined continuum limit. Starting from a heterogeneous initial stress distribution, we apply a slowly increasing uniform stress load far from the fault and we simulate the seismicity for a few 1000 events. The style of seismicity produced by this model is determined by a control parameter associated with the degree of rate dependence of friction. For classical friction models with rate-independent friction, no complexity appears and seismicity is perfectly periodic. For weakly rate-dependent friction, large ruptures are still periodic, but small seismicity becomes increasingly nonstationary. When friction is highly rate-dependent, seismicity becomes nonperiodic and ruptures of all sizes occur inside the fault. Highly rate-dependent friction destabilizes the healing process producing premature healing of slip and partial stress drop. Partial stress drop produces large variations in the state of stress that in turn produce earthquakes of different sizes. Similar results have been found by other authors using the Burridge and Knopoff model. We conjecture that all models in which static stress drop is only a fraction of the dynamic stress drop produce stress heterogeneity.
Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.
Authors: Powers A M , Kamrud K I , Olson K E , Higgs S , Carlson J O , Beaty B J
Published: July 2002

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.
E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor β through E2F binding sites
Authors: Li Jian-Ming , Hu Patrick Pei-chih , Shen Xing , Yu Yong , Wang Xiao-Fan
Published: July 2002

Transforming growth factor β (TGF-β) causes growth arrest in most cell types. TGF-β induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G
1
. It is possible, however, that the E2F-RB complex induced by TGF-β may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-β treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of
E2F1
,
B-myb
, or
HsORC1
genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-β. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-β treatment. Consistent with their repression of TGF-β-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15
INK4B
gene effectively inhibited its induction by TGF-β. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-β induction of p15
INK4B
gene when tethered to p15
INK4B
promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-β. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-β treatment, which may constitute an integral part of the TGF-β-induced growth arrest program.
The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase
Authors: Arnez John G. , Augustine John G. , Moras Dino , Francklyn Christopher S.
Published: July 2002

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from
Escherichia coli
, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (
k
cat
/
K
m
) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl
2
reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.
Loss of the maternal
H19
gene induces changes in
Igf
2 methylation in both cis and trans
Authors: Forné T. , Oswald J. , Dean W. , Saam J. R. , Bailleul B. , Dandolo L. , Tilghman S. M. , Walter J. , Reik W.
Published: July 2002

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (
Igf2
) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of
Igf2
, and the
H19
gene (lying 90 kb downstream of
Igf2
gene), which upon inactivation leads to relaxation of the
Igf2
imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of
Igf2
imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the
Igf2
gene in two strains of mouse with distinct deletions of the
H19
gene. The results show that maternal transmission of
H19
gene deletions leads the maternal allele of
Igf2
to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the
H19
gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the
Igf2
gene is affected in trans by deletions of the active maternal allele of the
H19
gene. Selection during development for the appropriate expression of
Igf2,
dosage-dependent factors that bind to the
Igf2
gene, or methylation transfer between the parental alleles could be involved in this trans effect.
The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex
Authors: Lin Paul , Sherman Fred
Published: July 2002

The structural and functional organization of the Cct complex was addressed by genetic analyses of subunit interactions and catalytic cooperativity among five of the eight different essential subunits, Cct1p–Cct8p, in the yeast
Saccharomyces cerevisiae
. The
cct1–1
,
cct2–3
, and
cct3–1
alleles, containing mutations at the conserved putative ATP-binding motif, GDGTT, are cold-sensitive, whereas single and multiple replacements of the corresponding motif in Cct6p are well tolerated by the cell. We demonstrated herein that
cct6–3
(L19S), but not the parolog
cct1–5
(R26I), specifically suppresses the
cct1–1
,
cct2–3
, and
cct3–1
alleles, and that this suppression can be modulated by mutations in a putative phosphorylation motif, RXS, and the putative ATP-binding pocket of Cct6p. Our results suggest that the Cct ring is comprised of a single hetero-oligomer containing eight subunits of differential functional hierarchy, in which catalytic cooperativity of ATP-binding/hydrolysis takes place in a sequential manner different from the concerted cooperativity proposed for GroEL.
Structural features of the binding site for ribosomal protein S8 in
Escherichia coli
16S rRNA defined using NMR spectroscopy
Authors: Kalurachchi K. , Uma K. , Zimmermann R. A. , Nikonowicz E. P.
Published: July 2002

Ribosomal protein S8 of
Escherichia coli
plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with
spc
operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597·C643 and A596·U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595·(A596· U644). The NMR data suggest that S8–RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598·A640 base pair and appears to stabilize the G597·C643 and A596·U644 base pairs.
The universe at
z
> 5: When and how did the “dark age” end?
Authors: Rees Martin J.
Published: July 2002

This paper considers how the first subgalactic structures produced
the UV radiation that ionized the intergalactic medium before
z
= 5 and the “feedback” effects of the UV
radiation on structure formation. The first “pregalaxies” may
eventually be detectable by their direct UV emission, with
characteristic spectral features at Lyman α; high-
z
supernovae may also be detectable. Other probes of the intergalactic
medium beyond
z
= 5, and of the epochs of reheating
and reionization, are discussed, along with possible links between the
diffusion of pregalactic metals and the origin of magnetic fields.
Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences
Authors: Britten Roy J.
Published: July 2002

The terminal regions (last 20 kb) of
Saccharomyces
cerevisiae
chromosomes universally contain blocks of precise
sequence similarity to other chromosome terminal regions. The left and
right terminal regions are distinct in the sense that the sequence
similarities between them are reverse complements. Direct sequence
similarity occurs between the left terminal regions and also between
the right terminal regions, but not between any left ends and right
ends. With minor exceptions the relationships range from 80% to 100%
match within blocks. The regions of similarity are composites of
familiar and unfamiliar repeated sequences as well as what could be
considered “single-copy” (or better “two-copy”) sequences.
All terminal regions were compared with all other chromosomes, forward
and reverse complement, and 768 comparisons are diagrammed. It appears
there has been an extensive history of sequence exchange or copying
between terminal regions. The subtelomeric sequences fall into two
classes. Seventeen of the chromosome ends terminate with the Y′ repeat,
while 15 end with the 800-nt “X2” repeats just adjacent to the
telomerase simple repeats. The just-subterminal repeats are very
similar to each other except that chromosome 1 right end is more
divergent.
Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement
Authors: Wang Gene Jin , Collinge Mark , Blasi Franscesco , Pardi Ruggero , Bender Jeffrey R.
Published: July 2002

As an adhesion receptor, the β2integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5,6-dichloro-1-β-d-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3′-untranslated region of the uPAR cDNA into a serum-inducible rabbit β-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3′-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3′ AU-rich elements.
A yeast model for the study of Batten disease
Authors: Pearce David A. , Sherman Fred
Published: July 2002

Although the
CLN3
gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive. We previously cloned the
Saccharomyces cerevisiae
homologue to the human
CLN3
gene, designated
BTN1
, which is not essential and whose product is 39% identical and 59% similar to Cln3p. We report that
btn1
-Δ deletion yeast strains are more resistant to
d
-(−)-threo-2-amino-1-[
p
-nitrophenyl]-1,3-propanediol (denoted ANP), a phenotype that is complemented in yeast by the human
CLN3
gene. Furthermore, the severity of Batten disease in humans and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.
Direct effects of metabotropic glutamate receptor compounds on native and recombinant
N
-methyl-
d
-aspartate receptors
Authors: Contractor Anis , Gereau Robert W. , Green Tim , Heinemann Stephen F.
Published: July 2002

The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of
N
-methyl-
d
-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.
Targeting of GroEL to SecA on the cytoplasmic membrane of
Escherichia coli
Authors: Bochkareva Elena S. , Solovieva Maria E. , Girshovich Alexander S.
Published: July 2002

Chaperonin GroEL has been found to interact with isolated cytoplasmic membrane of
Escherichia coli
. Interaction requires Mg ions, whereas MgATP inhibits, and inhibition is stronger in the presence of co-chaperonin GroES. “Heat-shock” of the membrane at 45°C destroys irreversibly its ability to bind GroEL. The binding of GroEL is characterized by saturation with a maximum of about 100 pmol GroEL bound per mg of total membrane protein, indicating a limited capacity and specificity of the membrane to bind GroEL. According to results of immunoblotting analysis and cleavable photoactivable cross-linking, a membrane target of GroEL is SecA, a protein known as a central component of the translocation machinery. Moreover, in some cases GroEL could modulate a cycle of association of SecA with the membrane by stimulating release of SecA from the membrane. A physiological role of targeting of GroEL in or close to the protein-conducting membrane apparatus is discussed.
Trans-chromosomal recombination within the Ig heavy chain switch region in B lymphocytes
Authors: Kingzette Mae , Spieker-Polet Helga , Yam Pi-Chen , Zhai Shi-Kang , Knight Katherine L.
Published: July 2002

Somatic DNA rearrangements in B lymphocytes, including V(D)J gene rearrangements and isotype switching, generally occur in cis, i. e., intrachromosomally. We showed previously, however, that 3 to 7% of IgA heavy chains have the VHand Cα regions encoded in trans. To determine whether the trans-association of VHand Cα occurred by trans-chromosomal recombination, by trans-splicing, or by trans-chromosomal gene conversion, we generated and analyzed eight IgA-secreting rabbit hybridomas with trans-associated VHand Cα heavy chains. By ELISA and by nucleotide sequence analysis we found that the VHand Cα regions were encoded by genes that were in trans in the germline. We cloned the rearranged VDJ-Cα gene from a fosmid library of one hybridoma and found that the expressed VHand Cα genes were juxtaposed. Moreover, the juxtaposed VHand Cα genes originated from different IgH alleles. From the same hybridoma, we also identified a fosmid clone with the other expected product of a trans-chromosomal recombination. The recombination breakpoint occurred within the Sμ/Sα region, indicating that the trans-association of VHand Cα genes occurred by trans-chromosomal recombination during isotype switching. We conclude that trans-chromosomal recombination occurs at an unexpectedly high frequency (7%) within the IgH locus of B lymphocytes in normal animals, which may explain the high incidence of B-cell tumors that arise from oncogene translocation into the IgH locus.
Plant genomics: More than food for thought
Authors: Briggs Steven P.
Published: July 2002

In all but the poorest countries of South Asia and Africa, the
supply and quality of food will rise to meet the demand. Biotechnology,
accelerated by genomics, will create wealth for both producers and
consumers by reducing the cost and increasing the quality of food.
Famine and malnutrition in the poorest countries may be alleviated by
applying genomics or other tools of biotechnology to improving
subsistence crops. The role of the public sector and the impact of
patent law both could be great, but government policies on these issues
are still unclear.
Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants
Authors: Kim Jin-Soo , Pabo Carl O.
Published: July 2002

Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and specificity. Our studies focused on a fusion in which the three-finger Zif268 peptide was linked to a designed three-finger peptide (designated “NRE”) that specifically recognizes a nuclear hormone response element. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in the femtomolar range. We find that the slightly longer linkers used in this fusion protein provide a dramatic improvement in DNA-binding affinity, working much better than the canonical “TGEKP” linkers that have been used in previous studies. Tissue culture transfection experiments also show that the 268//NRE peptide is an extremely effective repressor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking peptides selected via phage display, should allow the design of novel DNA-binding proteins—with extraordinary affinity and specificity—for use in biological research and gene therapy.
Self-assembly of fibronectin into fibrillar networks underneath dipalmitoyl phosphatidylcholine monolayers: Role of lipid matrix and tensile forces
Authors: Baneyx Gretchen , Vogel Viola
Published: July 2002

The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized
in situ
by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways.
Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage
Authors: Shih I-hung , Been Michael D.
Published: July 2002

Ribozymes of hepatitis delta virus have been proposed to use
an active-site cytosine as an acid-base catalyst in the self-cleavage
reaction. In this study, we have examined the role of cytosine in more
detail with the antigenomic ribozyme. Evidence that proton transfer in
the rate-determining step involved cytosine 76 (C76) was obtained from
examining cleavage activity of the wild-type and imidazole
buffer-rescued C76-deleted (C76Δ) ribozymes in D
2
O and
H
2
O. In both reactions, a similar kinetic isotope effect
and shift in the apparent pKa indicate that the buffer is functionally
substituting for the side chain in proton transfer. Proton inventory of
the wild-type reaction supported a mechanism of a single proton
transfer at the transition state. This proton transfer step was further
characterized by exogenous base rescue of a C76Δ mutant with cytosine
and imidazole analogues. For the imidazole analogues that rescued
activity, the apparent pKa of the rescue reaction, measured under
k
cat
/
K
M
conditions, correlated with the pKa of the base. From these data a
Brønsted coefficient (β) of 0.51 was determined for the base-rescued
reaction of C76Δ. This value is consistent with that expected for
proton transfer in the transition state. Together, these data provide
strong support for a mechanism where an RNA side chain participates
directly in general acid or general base catalysis of the wild-type
ribozyme to facilitate RNA cleavage.
The Myth of a Finno-Ugrian Community in Practice
Authors: Saarinen Sirkka
Published: July 2002

Scholars had already suggested the relationship of some Finno-Ugrian languages in the sixteenth and seventeenth centuries, but the linguistic affinity of the Finno-Ugrian languages was proven only at the end of the eighteenth century by two Hungarian pioneers of comparative linguistics, János Sajnovics and Sámuel Gyarmathi. Henrik Gabriel Porthan (1739-1804), professor of rhetoric and a great humanist, brought their ideas to Finland. He was strongly influenced by August Ludwig Schlözer, author of a comprehensive and critical survey of the history of the Finno-Ugrian peoples. Having studied Sajnovics' work in Göttingen, Porthan published an extensive account of it in a Finnish newspaper in 1779, demolishing previous ideas about the kinship between Finnish and Hebrew. Porthan urged Finnish scholars to investigate the Finno-Ugrian languages of Russia, of which very little was then known (some lists of words and short grammars were all that was available).
Units, mg/l or % CDT for detection of harmful alcohol consumption?
Authors: GRIMSRUD KJERSTI , SUNDREHAGEN ERLING
Published: July 2002

Abstract Recent studies have demonstrated that the diagnostic performance of CDT measurements is influenced by variations in total transferrin concentration. Improved specificity is obtained with relative values, either %CDT or units CDT per total transferrin concentration. Whether relative or absolute values are used, confirmatory methods by HPLC or IEF should be available, and definition of the CDT values and correlation to HPLC or IEF should be given.
Generation of Reactive Oxygen Species by Polymorphonuclear Leukocytes and Its Modulation by Calcium Ions During Tumor Growth
Authors: Pustovidko A. V. , Potselueva M. M. , Evtodienko Yu. V.
Published: July 2002

The generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) and the role of Ca2+ in regulating their activity during Zajdela hepatoma growth in the animal peritoneal cavity were studied. We found a marked increase in the ROS‐generating activity of PMNL in circulating blood, the result of increases in both the specific activity of leukocytes and total number of PMNL in circulating blood. The ROS‐generating activity of PMNL was substantially activated by Ca2+ ions and a calcium ionophore (ionomycin), but this effect virtually completely disappeared during tumor growth. Perhaps the high ROS‐generating activity of PMNL and the lack of the sensitivity to extracellular Ca2+ during tumor growth in the organism are due to an accumulation of intracellular Ca2+ ions.