bluuebunny/crossref_metadata_2025 huggingface dataset:
Attainment of submillimeter periods and a 0.3-T peak field in a novel micropole undulator device
Authors: Tatchyn Roman , Csonka Paul L.
Published: July 2002

A micropole undulator is defined here as an undulator with a very short (submillimeter) period and correspondingly shorter poles. The availability of micropole undulators is expected to profoundly affect the development of x-ray sources. For example, with such devices x-ray beams comparable to those from conventional undulators will be attainable on storage rings of much lower energy (and emittance) than those presently in use. X-ray instrumentation will also be affected, e.g., micropole undulators of sufficient length can provide extremely monochromatic outputs, obviating the need for expensive and complex ultrahigh vacuum monochromator systems. Here we describe the design and practical construction of a prototype 35 period micropole undulator whose total length is 2.54 cm. The device is of a novel hybrid/electromagnetic bias design. We also present results of field profile measurements along the central plane of the new device.
Refractive index, relaxation times and the viscoelastic model in dry-grown SiO2 films on Si
Authors: Landsberger L. M. , Tiller W. A.
Published: July 2002

Inert thermal anneals were performed at various temperatures to determine annealing kinetics of dry thermally grown SiO2 films on Si. Two stages of relaxation are demonstrated. The film relaxes quickly to an intermediate level, and then progresses more slowly toward full relaxation. The relaxation times to attain the fully relaxed refractive index, 1.460, and full ≤3% swelling were found to fall below typical oxidation times at T≥1150 °C, in concurrence with the experimentally observed breakpoint in the refractive index versus growth temperature data. It is concluded that the linear viscoelastic model is sufficient to quantitatively explain the breakpoints in refractive index for both wet and dry thermally grown oxide.
Staggered hollow-bore CO2 waveguide laser array
Authors: Hart R. A. , Newman L. A. , Cantor A. J. , Kennedy J. T.
Published: July 2002

A modified hollow-bore ridge waveguide, utilizing a staggered ridge geometry, has been used successfully to obtain single-frequency operation from an array of CO2 waveguide lasers. The gain volume of the array is equivalent to six waveguide lasers of 37 cm length and 2.25×2.25 mm2 cross section. The output power from the sealed-off device reached 68 W cw. Stable single-frequency operation in the antisymmetric supermode was observed.
Electron mobility in p-type GaAs
Authors: Nathan M. I. , Dumke W. P. , Wrenner K. , Tiwari S. , Wright S. L. , Jenkins K. A.
Published: July 2002

The mobility of electrons in p-type GaAs, μPn has been determined by measuring the common emitter cutoff frequency fT of heterojunction bipolar transistors with a wide, uniformly doped base. At 295 K, μPn =1150 cm2/(V s) is found for a hole concentration of 3.6×1018 cm−3. At 77 K, μPn =6000 cm2/(V s). The room-temperature value is considerably smaller and the 77 K value considerably larger than the electron mobility in comparably doped n-type material.
Measurement of thermal diffusivity for polymer film by flash radiometry
Authors: Tsutsumi Naoto , Kiyotsukuri Tsuyoshi
Published: July 2002

A new method to determine the thermal diffusivity of polymer film by flash radiometry is presented. The transient infrared radiation from the sample surface was measured after the sample film was heated by a short-duration pulsed light. The thermal diffusivity was determined by fitting the transient infrared signal to the theoretical curve of a one-dimensional heat diffusion model. The transient infrared signal of polymethylmethacrylate (PMMA) film could be well fitted by the theoretical curve using appropriate thermal diffusivity in the wide temperature range between 100 K and near the softening point of PMMA. The temperature dependence of thermal diffusivity of PMMA film showed that the thermal diffusivity sensitively reflects the molecular motion of polymer chain.
Single-mode buried channel waveguide by single-step electromigration technique using silver film
Authors: Zhenguang H. , Srivastava R. , Ramaswamy R. V.
Published: July 2002

Single-mode buried channel waveguides with circularly symmetric mode field distribution compatible with conventional single-mode fibers at 1.3 μm wavelength were fabricated and characterized for the first time using a novel single-step ion exchange process from silver films. The technique is relatively simple and eliminates the gold electrode deposition used in previously reported fabrication methods.
Characterization of interfacial atomic steps in GaAs/AlAs superlattices by transmission electron microscopy
Authors: Nakamura T. , Ikeda M. , Muto S. , Umebu I.
Published: July 2002

We have studied interfacial atomic steps in GaAs/AlAs superlattices using high-resolution transmission electron microscopy (HRTEM) and lattice image simulation. We find arrays of bright spots at the interface in the TEM image to be good indicators of the interface configuration. Doubling of the bright spot arrays and step-shaped arrays in TEM lattice images indicate a ‘‘type 1’’ monolayer step whose front is perpendicular to the direction of the electron beam and a ‘‘type 2’’ monolayer step whose front is parallel to the direction of the electron beam. Our HRTEM observations indicate that the atomic steps at GaAs and AlAs interfaces grown at 700 °C are denser than at interfaces grown at 500 °C.
Kinetics of the generation and annealing of deep defects and recombination centers in amorphous silicon
Authors: Shepard K. , Smith Z E. , Aljishi S. , Wagner S.
Published: July 2002

The number of deep defects (as measured by sub-band-gap optical absorption) and the number of recombination centers (as measured by steady-state photoconductivity) in high quality undoped hydrogenated amorphous silicon are monitored as the film undergoes light-induced defect generation and thermal annealing (the Staebler–Wronski effect). The kinetics of the growth in the density of deep defects agree well with the kinetics of spin centers reported by Stutzmann, Jackson, and Tsai. [Appl. Phys. Lett. 45, 1075 (1984)]. The density of recombination centers is directly related, though not simply proportional to, the density of deep defects; as samples are annealed, the recombination center density drops much more quickly than the total defect density. This behavior is shown to arise if the distribution of defect energy levels corresponds to a distribution of defect annealing energies.
Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE
Authors: Van Herreweghe Franky , Mao Jianqiang , Chaplen Frank W. R. , Grooten Johan , Gevaert Kris , Vandekerckhove Joël , Vancompernolle Katia
Published: July 2002

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 is a caspase-independent process that is characterized by increased production of reactive oxygen species (ROS) in the mitochondria. To elucidate this ROS-dependent cell death pathway, a comparative study of the phosphoproteins present in TNF-treated and control cells was performed. Here we report that TNF induces an increased phosphorylation of glyoxalase I that is mediated by protein kinase A and required for cell death. We also show that TNF induces a substantial increase in intracellular levels of methylglyoxal (MG) that leads to the formation of a specific MG-derived advanced glycation end product and that this formation occurs as a consequence of increased ROS production. These data indicate that MG modification of proteins is a targeted process and that MG may thus function as a signal molecule during the regulation of cell death. Furthermore, we provide evidence that the TNF-induced phosphorylation of glyoxalase I is not involved in detoxification of MG by means of the glyoxalase system, but that phosphorylated glyoxalase I is on the pathway leading to the formation of a specific MG-derived advanced glycation end product.
Self-generated DNA termini relax the specificity of
Sgr
AI restriction endonuclease
Authors: Bitinaite Jurate , Schildkraut Ira
Published: July 2002

The primary target of
Sgr
AI restriction endonuclease is a multiple sequence of the form 5′-CPu↓CCGGPyG. Previous work had indicated that
Sgr
AI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999)
J. Biol. Chem.
274, 36379–36386]. In the present study,
Sgr
AI is shown to cleave not only its canonical sequences, but also the sequences 5′-CPuCCGGPy(A,T,C) and 5′-CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7,
Sgr
AI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for
Sgr
AI assist in the cleavage of secondary sites. The
Sgr
AI-termini in cis with respect to secondary site are markedly preferred over those in trans. The
Sgr
AI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the
Sgr
AI-termini over substrate, the
Sgr
AI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which
Sgr
AI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the
Sgr
AI restriction endonuclease is proposed.
APH-1 is a multipass membrane protein essential for the Notch signaling pathway in
Caenorhabditis elegans
embryos
Authors: Goutte Caroline , Tsunozaki Makoto , Hale Valerie A. , Priess James R.
Published: July 2002

Early embryonic cells in
Caenorhabditis elegans
embryos interact through a signaling pathway closely related to the Notch signaling pathway in
Drosophila
and vertebrates.Components of this pathway include a ligand, receptor, the presenilin proteins, and a novel protein, APH-2, that is related to the Nicastrin protein in humans. Here we identify the
aph-1
gene as a new component of the Notch pathway in
Caenorhabditis elegans. aph-1
is predicted to encode a novel, highly conserved multipass membrane protein. We show that
aph-1
and the presenilin genes share a similar function in that they are both required for proper cell-surface localization of APH-2/Nicastrin.
Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions
Authors: Urbauer Jeffrey L. , Simeonov Mario F. , Urbauer Ramona J. Bieber , Adelman Karen , Gilmore Joshua M. , Brody Edward N.
Published: July 2002

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of
Escherichia coli
, σ
70
. AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix–turn–helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to σ
70
. The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to σ
70
, where an AsiA protomer is displaced from the dimer via the interaction of σ
70
with the same residues in AsiA that constitute the dimer interface.
Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells
in
vitro
Authors: Parkkila Seppo , Rajaniemi Hannu , Parkkila Anna-Kaisa , Kivelä Jyrki , Waheed Abdul , Pastoreková Silvia , Pastorek Jaromir , Sly William S.
Published: July 2002

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 μM acetazolamide inhibited the relative invasion rate of these cell lines between 18–74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells
in vitro
, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least
in vitro
, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.
Replication ofCryptococcus neoformansin macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm
Authors: Tucker Stephanie C. , Casadevall Arturo
Published: July 2002

Cryptococcus neoformans(CN), an encapsulated, ubiquitous environmental yeast, is pathogenic for humans, primarily those with compromised immune function. CN is believed to be a facultative intracellular pathogen. Time-lapsed video microscopy revealed that yeast began to replicate and divide 2 hours after ingestion by J774.16 macrophage cells, with the average cell hosting 10–40 organisms of varying morphologies before ultimately lysing and releasing organisms, either singly or in clumps. Intracellular growth was accompanied by the accumulation of polysaccharide-filled vesicles in the macrophage. Studies with fluorescently labeled dextran revealed that the phagolysosomal compartment became leaky during the course of intracellular infection. Consistent with this observation, phagosomes containing CN had an increased pH relative to similar phagosomes containing inert magnetic beads, as indicated by a colorimetric change in the pH-sensitive Lysosensor dye. Immunocytochemistry revealed differences in the reactivity of polysaccharide elaborated by CN inside macrophages relative to that expressedin vitro. Taken together these results are suggestive of a novel mechanism of intracellular survival by an encapsulated organism, whereby ingestion is followed by damage to the phagosomal membrane resulting in continuity with the cytoplasm, accumulation of polysaccharide-containing vesicles, and possibly, production of a structurally different polysaccharide.
ClpA mediates directional translocation of substrate proteins into the ClpP protease
Authors: Reid Brian G. , Fenton Wayne A. , Horwich Arthur L. , Weber-Ban Eilika U.
Published: July 2002

The intracellular degradation of many proteins is mediated in an
ATP-dependent manner by large assemblies comprising a chaperone ring
complex associated coaxially with a proteolytic cylinder, e.g., ClpAP,
ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes.
Recent studies of the chaperone ClpA indicate that it mediates
ATP-dependent unfolding of substrate proteins and directs their
ATP-dependent translocation into the ClpP protease. Because the axial
passageway into the proteolytic chamber is narrow, it seems likely that
unfolded substrate proteins are threaded from the chaperone into the
protease, suggesting that translocation could be directional. We have
investigated directionality in the ClpA/ClpP-mediated reaction by
using two substrate proteins bearing the COOH-terminal ssrA recognition
element, each labeled near the NH
2
or COOH terminus with
fluorescent probes. Time-dependent changes in both fluorescence
anisotropy and fluorescence resonance energy transfer between donor
fluorophores in the ClpP cavity and the substrate probes as acceptors
were measured to monitor translocation of the substrates from ClpA into
ClpP. We observed for both substrates that energy transfer occurs 2–4
s sooner with the COOH-terminally labeled molecules than with the
NH
2
-terminally labeled ones, indicating that translocation
is indeed directional, with the COOH terminus of the substrate protein
entering ClpP first.
Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells
Authors: Chayama Kosuke , Papst Philip J. , Garrington Timothy P. , Pratt Joanne C. , Ishizuka Tamotsu , Webb Saiphone , Ganiatsas Soula , Zon Leonard I. , Sun Weiyong , Johnson Gary L. , Gelfand Erwin W.
Published: July 2002

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.
Antisense-mediated depletion of p300 in human cells leads to premature G
1
exit and up-regulation of c-
MYC
Authors: Kolli Sivanagarani , Buchmann Ann Marie , Williams Justin , Weitzman Sigmund , Thimmapaya Bayar
Published: July 2002

The cAMP-response element-binding protein (CREB)-binding protein
and p300 are two highly conserved transcriptional coactivators and
histone acetyltransferases that integrate signals from diverse signal
transduction pathways in the nucleus and also link chromatin remodeling
with transcription. In this report, we have examined the role of p300
in the control of the G
1
phase of the cell cycle in
nontransformed immortalized human breast epithelial cells (MCF10A) and
fibroblasts (MSU) by using adenovirus vectors expressing p300-specific
antisense sequences. Quiescent MCF10A and MSU cells expressing
p300-specific antisense sequences synthesized p300 at much reduced
levels and exited G
1
phase without serum stimulation. These
cells also showed an increase in cyclin A and cyclin A- and
E-associated kinase activities characteristic of S phase induction.
Further analysis of the p300-depleted quiescent MCF10A cells revealed a
5-fold induction of c
-
MYC and a 2-fold induction of
c
-
JUN. A direct target of c
-MYC, CAD
,
which is required for DNA synthesis, was also found to be up-regulated,
indicating that up-regulation of c
-MYC
functionally
contributed to DNA synthesis. Furthermore, S phase induction in
p300-depleted cells was reversed when antisense c-
MYC
was expressed in these cells, indicating that up-regulation of c-MYC
may directly contribute to S phase induction. Adenovirus E1A also
induced DNA synthesis and increased the levels of c-
MYC
and c-
JUN
in serum-starved MCF10A cells in a
p300-dependent manner. Our results suggest an important role of p300 in
cell cycle regulation at G
1
and raise the possibility that
p300 may negatively regulate early response genes, including
c-
MYC
and c-
JUN
, thereby preventing DNA
synthesis in quiescent cells.
Even-odd alternation in mass spectrum of thymine and uracil clusters: Evidence of intracluster photodimerization
Authors: Kim Nam Joon , Kang Hyuk , Jeong Gawoon , Kim Yung Sam , Lee Kang Taek , Kim Seong Keun
Published: July 2002

Multiphoton ionization of thymine and uracil clusters generated by
a supersonic molecular beam gave rise to a remarkable alternation of
mass spectral intensities between even- and odd-numbered clusters. Such
alternation was observed in clusters of up to 30 molecules. Excitation
to the two lowest electronically excited states seemed to be a strong
prerequisite. In view of the well known photodimerization reaction of
thymine and uracil in the bulk phase, it is proposed that such
alternation in the mass spectral intensity resulted from formation of
photodimer units within the cluster on intense UV irradiation. Several
analogues of thymine with no known propensity for photodimerization in
the bulk phase did not exhibit any sign of such alternation in the
cluster mass spectrum. The intrinsic UV window for photodimerization,
and hence photoinduced mammalian mutagenesis, was estimated to be
approximately 210–280 nm, significantly narrower than the previously
reported bulk values of 150–300 nm.
An essential intermediate in the folding of dihydrofolate reductase
Authors: Heidary David K. , O'Neill John C. , Roy Melinda , Jennings Patricia A.
Published: July 2002

The folding of
Escherichia coli
dihydrofolate reductase was examined at pH 7.8 and 15°C by using stopped-flow fluorescence and absorbance spectroscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow absorbance spectroscopy using methotrexate binding assays shows a distinct lag phase during these time frames for the native state. The lag in absorbance kinetics and the lack of fast-track folding events indicate that the formation of this ensemble of intermediates is an obligatory step in the folding reaction.
Rates of nucleotide substitution in sexual and anciently asexual rotifers
Authors: Welch David B. Mark , Meselson Matthew S.
Published: July 2002

The class Bdelloidea of the phylum Rotifera is the largest well studied eukaryotic taxon in which males and meiosis are unknown, and the only one for which these indications of ancient asexuality are supported by cytological and molecular genetic evidence. We estimated the rates of synonymous and nonsynonymous substitutions in thehsp82heat shock gene in bdelloids and in facultatively sexual rotifers of the class Monogononta, employing distance based and maximum likelihood methods. Relative-rate tests, using acanthocephalan rotifers as an outgroup, showed slightly higher rates of nonsynonymous substitution and slightly lower rates of synonymous substitution in bdelloids as compared with monogononts. The opposite trend, however, was seen in intraclass pairwise comparisons. If, as it seems, bdelloids have evolved asexually, an equality of bdelloid and monogonont substitution rates would suggest that the maintenance of sexual reproduction in monogononts is not attributable to an effect of sexual reproduction in limiting the load of deleterious nucleotide substitutions.
Induced instability of two
Arabidopsis
constitutive pathogen-response alleles
Authors: Stokes Trevor L. , Richards Eric J.
Published: July 2002

Paramutation is an example of a non-Mendelian-directed allelic interaction that results in the epigenetic alteration of one allele. We describe a paramutation-like interaction between two alleles,
bal
and
cpr1–1
(
constitutive expressor of PR genes 1
), which map to a complex
R
-like gene cluster on
Arabidopsis
chromosome 4. Both alleles cause dwarfing and constitutive defense responses, similar to another dwarf variant,
ssi1
(
suppressor of SA-insensitivity 1
). Previous work has demonstrated that the
bal
and
ssi1
phenotypes are caused by overexpression of an
R
-like gene from the cluster, which activates an salicylic acid-dependent defense pathway. Here, we show that the
cpr1–1
variant does not alter gene expression from the
R
-like gene cluster. The
bal
and
cpr1–1
alleles did not complement each other in F
1
hybrids, but F
2
populations that segregated
bal
and
cpr1–1
alleles contained plants with normal morphology at a frequency of 20%. By using molecularly marked
bal
and
cpr1–1
lines, we found that the majority of the normal phenotypes were correlated with inheritance of an altered
cpr1–1
allele. Our observation that
cpr1–1
is a metastable allele suggests that
cpr1–1
is an epigenetic allele. The
cpr1–1
allele is the third candidate epigenetic allele originating from this
R
-like gene cluster, making the region a possible hotspot of epigenetic variation.
Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors
Authors: Pesquero João B. , Araujo Ronaldo C. , Heppenstall Paul A. , Stucky Cheryl L. , Silva José A. , Walther Thomas , Oliveira Suzana M. , Pesquero Jorge L. , Paiva Antonio C. M. , Calixto João B. , Lewin Gary R. , Bader Michael
Published: July 2002

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using anin vitropreparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.
Regulation of protein kinase CβI by two protein-tyrosine kinases, Btk and Syk
Authors: Kawakami Yuko , Kitaura Jiro , Hartman Stephen E. , Lowell Clifford A. , Siraganian Reuben P. , Kawakami Toshiaki
Published: July 2002

Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcɛRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcɛRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCβI among the conventional PKC isoforms (α, βI, and βII) expressed in mast cells. Syk/Btk-mediated PKCβI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor α genes through the JNK pathway induced by FcɛRI stimulation. Accordingly, FcɛRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCβ. Specific regulation of PKCβI by Btk is consistent with the selective association of Btk with PKCβI. Components of this signaling pathway may represent an attractive set of potential targets of pharmaceutical interference for the treatment of allergic and other immunologic diseases.
Tolerance of human
MSH2
+/−
lymphoblastoid cells to the methylating agent temozolomide
Authors: Marra Giancarlo , D'Atri Stefania , Corti Chantal , Bonmassar Laura , Cattaruzza Maria Sofia , Schweizer Pascal , Heinimann Karl , Bartosova Zdena , Nyström-Lahti Minna , Jiricny Josef
Published: July 2002

Members of hereditary nonpolyposis colon cancer (HNPCC) families harboring heterozygous germline mutations in the DNA mismatch repair genes
hMSH2
or
hMLH1
present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation.
hMSH2
+/−
lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in
hMSH2
+/−
cell lines. In contrast,
hMLH1
+/−
heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in
hMSH2
or
hMLH1
cannot be distinguished clinically, the early stages of the carcinogenic process in
hMSH2
and
hMLH1
mutation carriers may be different. Should
hMSH2
+/−
colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.
Crystal structure of conserved hypothetical protein Aq1575 from
Aquifex
aeolicus
Authors: Shin Dong Hae , Yokota Hisao , Kim Rosalind , Kim Sung-Hou
Published: July 2002

The crystal structure of a conserved hypothetical protein, Aq1575, from
Aquifex aeolicus
has been determined by using x-ray crystallography. The protein belongs to the domain of unknown function DUF28 in the Pfam and PALI databases for which there was no structural information available until now. A structural homology search with the DALI algorithm indicates that this protein has a new fold with no obvious similarity to those of other proteins of known three-dimensional structure. The protein reveals a monomer consisting of three domains arranged along a pseudo threefold symmetry axis. There is a large cleft with approximate dimensions of 10 Å × 10 Å × 20 Å in the center of the three domains along the symmetry axis. Two possible active sites are suggested based on the structure and multiple sequence alignment. There are several highly conserved residues in these putative active sites. The structure based molecular properties and thermostability of the protein are discussed.
Coreceptor restriction within the HLA-DQ locus for Epstein–Barr virus infection
Authors: Haan Keith M. , Longnecker Richard
Published: July 2002

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus that causes infectious mononucleosis and is etiologically associated with malignancies of multiple origins. EBV enters cells through a cascade of interactions between its envelope glycoprotein gp350 and the gp42-gH-gL complex with cellular receptors. Membrane fusion is catalyzed by the binding of gp42, a member of the C type lectin family, to HLA class II molecule HLA-DR, -DP, or -DQ. Here we demonstrate that only a subset of HLA-DQ alleles mediates EBV entry, indicating that individuals expressing these alleles may offer unique sites for EBV infection and subsequent sequelae. Additionally, the specific site within HLA-DQ determined to be essential for EBV entry is homologous to a site within MHC class I shown by structural studies to bind to the C type-lectin-like natural killer receptor, providing insight into the biochemical nature of the gp42–HLA class II interaction.
Avicins, a family of triterpenoid saponins fromAcacia victoriae(Bentham), inhibit activation of nuclear factor-κB by inhibiting both its nuclear localization and ability to bind DNA
Authors: Haridas Valsala , Arntzen Charles J. , Gutterman Jordan U.
Published: July 2002

Triterpenoid saponins, which are present in leguminous plants and some marine animals, possess a broad range of biological actions. We have earlier reported the extraction of avicins, a family of triterpenoid saponins obtained from the Australian desert treeAcacia victoriae(Leguminosae: Mimosoideae) that inhibit tumor cell growth and induce apoptosis, in part, by perturbing mitochondrial function. These saponins have also been found to prevent chemical-induced carcinogenesis in mice. This study examines the effect of a triterpene mixture (F094) and a single molecular species (avicin G) isolated from the mixture on tumor necrosis factor (TNF)-induced activation of nuclear transcription factor-κB (NF-κB) in Jurkat cells (human T cell leukemia). Both F094 and avicin G were found to be potent inhibitors of TNF-induced NF-κB. Treatment of Jurkat cells with avicin G resulted in a much slower accumulation of the p65 subunit of NF-κB into the nucleus whereas the degradation of IκBα was unaffected. Avicin G also impaired the binding of NF-κB to DNA inin vitrobinding assays. Treatment of cells with DTT totally reversed the avicin G-induced inhibition of NF-κB activity, suggesting that sulfhydryl groups critical for NF-κB activation were being affected. Avicin G treatment resulted in decreased expression of NF-κB-regulated proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2). Thus, the avicins may prove important for reducing both oxidative and nitrosative cellular stress and thereby suppressing the development of malignancies and related diseases.
SV40 replication in human mesothelial cells induces HGF/Met receptor activation: A model for viral-related carcinogenesis of human malignant mesothelioma
Authors: Cacciotti Paola , Libener Roberta , Betta Piergiacomo , Martini Fernanda , Porta Camillo , Procopio Antonio , Strizzi Luigi , Penengo Lorenza , Tognon Mauro , Mutti Luciano , Gaudino Giovanni
Published: July 2002

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.
ABA activates multiple Ca
2+
fluxes in stomatal guard cells, triggering vacuolar K
+
(Rb
+
) release
Authors: MacRobbie Enid A. C.
Published: July 2002

The mechanisms by which abscisic acid (ABA) activates the release
of K
+
(Rb
+
) from the vacuole of stomatal guard
cells, a process essential for ABA-induced stomatal closure, have been
investigated by tracer flux measurements. The form and timing of the
ABA-induced efflux transient could be manipulated by treatments that
alter three potential Ca
2+
fluxes into the cytoplasm, the
influx from the outside and two pathways of internal release, those
dependent on phospholipase C (inhibited by
U73122
) and cyclic
ADP-ribose (inhibited by nicotinamide). Ba
2+
, acting as a
competitive inhibitor of Ca
2+
influx but also as an
inhibitor of internal release, was an effective inhibitor of the
transient. The results suggest that a threshold level of cytoplasmic
Ca
2+
is required for the initiation of the minimal efflux
transient after a lag period and with a low rate of rise. As conditions
improve for the generation of an efflux transient (higher ABA or
reduced Ba
2+
), a second threshold is crossed, generating a
transient with zero lag and rapid rate of rise. This may reflect
different Ca
2+
levels required for activation of different
tonoplast K
+
channels. In this state, at high ABA, the
transient is inhibited by removal of external Ca
2+
,
suggesting Ca
2+
influx makes a major contribution to
increase in cytoplasmic Ca
2+
. By contrast, at low ABA, the
transient is not inhibited by removal of external Ca
2+
but
is sensitive to either
U73122
or nicotinamide, suggesting internal
release makes the major contribution, involving both pathways. ABA
appears to activate all three processes, and their relative importance
depends on conditions.
γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus
Authors: Do-Rego J. L. , Mensah-Nyagan G. A. , Beaujean D. , Vaudry D. , Sieghart W. , Luu-The V. , Pelletier G. , Vaudry H.
Published: July 2002

Most of the actions of neurosteroids on the central nervous system
are mediated through allosteric modulation of the γ-aminobutyric acid
type A (GABA
A
) receptor, but a direct effect of GABA on the
regulation of neurosteroid biosynthesis has never been investigated. In
the present report, we have attempted to determine whether
3β-hydroxysteroid dehydrogenase (3β-HSD)-containing neurons, which
secrete neurosteroids in the frog hypothalamus, also express the
GABA
A
receptor, and we have investigated the effect of GABA
on neurosteroid biosynthesis by frog hypothalamic explants. Double
immunohistochemical labeling revealed that most 3β-HSD-positive
neurons also contain GABA
A
receptor α
3
and
β
2
/β
3
subunit-like
immunoreactivities. Pulse-chase experiments showed that GABA inhibited
in a dose-dependent manner the conversion of tritiated pregnenolone
into radioactive steroids, including 17-hydroxy-pregnenolone,
progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and
dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis
was mimicked by the GABA
A
receptor agonist muscimol but was
not affected by the GABA
B
receptor agonist baclofen. The
selective GABA
A
receptor antagonists bicuculline and
SR95531 reversed the inhibitory effect of GABA on neurosteroid
formation. The present results indicate that steroid-producing neurons
of the frog hypothalamus express the GABA
A
receptor
α
3
and β
2
/β
3
subunits. Our data also demonstrate that GABA, acting on
GABA
A
receptors at the hypothalamic level, inhibits the
activity of several key steroidogenic enzymes, including 3β-HSD and
cytochrome P450
C17
(17α-hydroxylase).
Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer
Authors: Baneyx Gretchen , Baugh Loren , Vogel Viola
Published: July 2002

Fluorescence resonance energy transfer (FRET) between
fluorophores attached to single proteins provides a tool to study the
conformation of proteins in solution and in cell culture. As a protein
unfolds, nanometer-scale increases in distance between donor and
acceptor fluorophores cause decreases in FRET. Here we demonstrate
the application of FRET to imaging coexisting conformations of
fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa
extracellular matrix protein, with functional sites that are regulated
by unfolding events. Fn was labeled with multiple donor and acceptor
fluorophores such that intramolecular FRET could be used to distinguish
a range of Fn conformations. The sensitivity of FRET to unfolding was
tested by progressively denaturing labeled Fn using guanidium chloride.
To investigate Fn conformation changes during cell binding and matrix
assembly, we added labeled Fn to the culture medium of NIH 3T3
fibroblasts. Coexisting conformations of Fn were visualized using
fluorescence microscopy, and spectra from specific features were
measured with an attached spectrometer. Using FRET as an indicator of
Fn conformation, Fn diffusely bound to cells was in a compact state,
whereas Fn in matrix fibrils was highly extended. Matrix fibrils
exhibited a range of FRET that suggested some degree of unfolding of
Fn's globular modules. Fn in cell-associated clusters that preceded
fibril formation appeared more extended than diffuse cell-bound Fn but
less extended than fibrillar Fn, suggesting that Fn undergoes extension
after cell binding and before polymerization. FRET thus provides an
approach to gain insight into the integrin-mediated pathway of Fn
fibrillogenesis.
Gαi3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins
Authors: Weiss Thomas S. , Chamberlain Chester E. , Takeda Tetsuro , Lin Ping , Hahn Klaus M. , Farquhar Marilyn Gist
Published: July 2002

Gαi3 is found both on the plasma membrane and on Golgi
membranes. Calnuc, an EF hand protein, binds both Gαi3 and
Ca
2+
and is found both in the Golgi lumen and in the
cytoplasm. To investigate whether Gαi3 binds calnuc in living cells
and where this interaction takes place we performed fluorescence
resonance energy transfer (FRET) analysis between Gαi3 and calnuc in
COS-7 cells expressing Gαi3-yellow fluorescent protein (YFP) and
calnuc-cyan fluorescent protein (CFP). The tagged proteins have the
same localization as the endogenous, nontagged proteins.
When Gαi3-YFP and calnuc-CFP are coexpressed, a FRET signal is
detected in the Golgi region, but no FRET signal is detected on the
plasma membrane. FRET is also seen within the Golgi region when
Gαi3 is coexpressed with cytosolic calnuc(ΔN2–25)-CFP lacking its
signal sequence. No FRET signal is detected when Gαi3(ΔC12)-YFP
lacking the calnuc-binding region is coexpressed with calnuc-CFP or
when Gαi3-YFP and calnuc(ΔEF-1,2)-CFP, which is unable to bind
Gαi3, are coexpressed. Gαi3(G2AC3A)-YFP lacking its lipid anchors
is localized in the cytoplasm, and no FRET signal is detected when it
is coexpressed with wild-type calnuc-CFP. These results indicate
that cytosolic calnuc binds to Gαi3 on Golgi membranes in living
cells and that Gαi3 must be anchored to the cytosolic surface of
Golgi membranes via lipid anchors for the interaction to occur. Calnuc
has the properties of a Ca
2+
sensor protein capable of
binding to and potentially regulating interactions of Gαi3 on Golgi
membranes.
Development and characterization of a rodent model of immune-mediated cholangitis.
Authors: Ueno Y , Phillips J O , Ludwig J , Lichtman S N , LaRusso N F
Published: July 2002

The cholangiopathies are a group of hepatobiliary diseases in which intrahepatic bile duct epithelial cells, or cholangiocytes, are the target for a variety of destructive processes, including immune-mediated damage. We tested the hypothesis that cholangitis could be induced in rodents by immunization with highly purified cholangiocytes. Inbred Wistar rats were immunized with purified hyperplastic cholangiocytes isolated after bile duct ligation from either syngeneic Wistar or allogeneic Fischer 344 rats; control rats were immunized with bovine serum albumin (BSA) or hepatocytes. After immunization with cholangiocytes, recipient animals developed histologic evidence of nonsuppurative cholangitis without inflammation in other organs; groups immunized with BSA or hepatocytes showed no cholangitis. Immunohistochemical studies revealed that portal tract infiltrates around bile ducts consisted of CD3-positive lymphocytes, some of which expressed major histocompatibility complex class II antigen; B cells and exogenous monocytes/macrophages were essentially absent. Transfer of unfractionated ConA-stimulated spleen cells from cholangiocyte-immunized (but not BSA-immunized) rats into recipients also caused nonsuppurative cholangitis. Moreover, these splenocytes from cholangiocyte-immunized (but not BSA-immunized) rats were cytotoxic in vitro for cultured rodent cholangiocytes; no cytotoxicity was observed against a rat hepatocyte cell line. Also, a specific antibody response in sera of cholangiocyte-immunized rats was demonstrated by immunoblots against cholangiocyte proteins. Finally, cholangiograms in cholangiocyte-immunized rats showed distortion and tortuosity of the entire intrahepatic biliary ductal system. This unique rodent model of experimental cholangitis demonstrates the importance of immune mechanisms in the pathogenesis of cholangitis and will prove useful in exploring the mechanisms by which the immune system targets and damages cholangiocytes.
Viral dynamics in vivo: limitations on estimates of intracellular delay and virus decay.
Authors: Herz A V , Bonhoeffer S , Anderson R M , May R M , Nowak M A
Published: July 2002

Anti-viral drug treatment of human immunodeficiency virus type I (HIV-1) and hepatitis B virus (HBV) infections causes rapid reduction in plasma virus load. Viral decline occurs in several phases and provides information on important kinetic constants of virus replication in vivo and pharmacodynamical properties. We develop a mathematical model that takes into account the intracellular phase of the viral life-cycle, defined as the time between infection of a cell and production of new virus particles. We derive analytic solutions for the dynamics following treatment with reverse transcriptase inhibitors, protease inhibitors, or a combination of both. For HIV-1, our results show that the phase of rapid decay in plasma virus (days 2-7) allows precise estimates for the turnover rate of productively infected cells. The initial quasi-stationary phase (days 0-1) and the transition phase (days 1-2) are explained by the combined effects of pharmacological and intracellular delays, the clearance of free virus particles, and the decay of infected cells. Reliable estimates of the first three quantities are not possible from data on virus load only; such estimates require additional measurements. In contrast with HIV-1, for HBV our model predicts that frequent early sampling of plasma virus will lead to reliable estimates of the free virus half-life and the pharmacological properties of the administered drug. On the other hand, for HBV the half-life of infected cells cannot be estimated from plasma virus decay.