bluuebunny/crossref_metadata_2025 huggingface dataset:
ELASTIC SURFACE WAVES IN THE PRESENCE OF FLUID LAYER
Authors: Das P. , Araghi M. N.
Published: February 2004

The presence of a liquid layer on the Rayleigh wave delay line is found to increase the original delay by a factor of 2 with loss of only a few percent in amplitude. Different modes with variable delay can be excited simultaneously and can be picked up by another transducer in contact with liquid layer. Possible uses of thin layer waves as an acoustic interferometer are discussed.
LOW-POWER QUASI-cw RAMAN OSCILLATOR
Authors: Ippen E. P.
Published: February 2004

Quasi-cw stimulated Raman emission in the visible has been obtained from an oscillator cavity with a pump-power input of less than 5W. This large reduction of oscillator threshold is achieved with the use of a liquid-core optical waveguide structure. Significant conversion of pump to Stokes light has been observed. Extension of the system to continuous operation and to the study of other nonlinear effects is suggested.
MAGNETOSTATIC SURFACE MODES IN AN AXIALLY MAGNETIZED ELLIPTICAL CYLINDER
Authors: De Wames R. E. , Wolfram T.
Published: February 2004

The spin-wave spectrum of a long axially magnetized strip of noncircular cross sections is examined. The results are related to the limiting cases of the (1) ferromagnetic slab and (2) the circular cylinder. It is found that the elliptical cylinder results can be identified with the infinite-film results of Damon and Eshbach when k, the propagation constant along the long axis of the ellipse, is identically equal to zero. This identification leads to a geometrical quantization of the wave vector in the infinite-film calculation. For k ≠ 0 no simple quantization scheme can be used to relate the elliptical cylinder results to the infinite film results.
MAGNETICALLY STABILIZED CROSS-FIELD CO2 LASER
Authors: Buczek C. J. , Wayne R. J. , Chenausky P. , Freiberg R. J.
Published: February 2004

A new concept of stabilizing an electrical CO2 laser discharge against a transverse gas flow is described. A cross magnetic field, mutually perpendicular to the discharge axial electric field and the gas flow velocity, is employed to maintain the electrical discharge column colinear with the optic axis. The cross-field laser is convectively cooled and characterized by low flow velocities, low pressure drops, and high operating efficiencies.
ELASTIC WAVE GENERATION BY A GUNN EFFECT OSCILLATOR COUPLED TO A PIEZOELECTRIC
Authors: Lee R. E. , White R. M.
Published: February 2004

In a Gunn effect oscillator operating in the transit-time mode, domains of high electric field are formed periodically and travel through the device. When such an oscillator is placed close to the surface of a piezoelectric solid the moving high-field domains produce moving local stress concentrations in the piezoelectric, causing the radiation of elastic waves into the body of the piezoelectric and along its surface. The domain velocity is much higher than the elastic wave velocities, so the situation is similar to that of Cherenkov radiation, and the waves are emitted at a large angle to the direction of domain motion. Results are given on the generation of bulk and surface waves at a fundamental frequency of 114 MHz in a lithium niobate crystal coupled to a gallium arsenide Gunn effect oscillator.
HIGH-RESOLUTION INFRARED SPECTROSCOPY WITH A TUNABLE DIODE LASER
Authors: Hinkley E. D.
Published: February 2004

A current-tunable Pb0.88Sn0.12 Te diode laser was used to obtain the absorption spectrum of SF6 near the P(16) and P(20) CO2 laser lines at 10.6μm by both direct and heterodyne techniques. Because of its narrow linewidth, the diode laser can perform high-resolution spectroscopy beyond the limits of conventional instruments moreover, its infrared frequency can be tuned continuously over a range much greater than attainable with a gas laser.
CdSnP2 EMISSION AND DETECTION OF NEAR-INFRARED RADIATION
Authors: Shay J. L. , Leheny R. F. , Buehler E. , Wernick J.
Published: February 2004

The spectral dependence of the photoluminescence and the time decay of the cathodoluminescence are reported for CdSnP2 single crystals for 1.7 <T<300°K. We find that the room-temperature band gap lies very close to the 1.06μ Nd:YAG laser emission indicating that CdSnP2 may be useful as a room-temperature detector or for other optical devices such as limiters or passive Q-switchers. We report photoconductivity and photovoltage measurements which support these suggestions.
LONG-WAVELENGTH THRESHOLD OF Cs2O-COATED PHOTOEMITTERS
Authors: James L. W. , Uebbing J. J.
Published: February 2004

While Cs2O-coated III-V semiconductors can have Kelvin work functions as low as 0.7 eV, the long-wavelength threshold for efficient photoemission is determined by a significantly higher barrier at the heterojunction between the Cs2O and the semiconductor. This barrier is 1.23±0.03 eV for Cs2O on GaSb. For Cs2O on silver, the barrier height is 1.0 eV.
Improved sperm cryopreservation using cold cryoprotectant
Authors: Clarke G. N. , Liu D. Y. , Baker H. W. G.
Published: February 2004

It has generally been assumed that very rapid cooling above freezing point would be deleterious to human sperm because it would result in cold shock. Consequently, most routine cryopreservation protocols involve the use of warm (20–30°C) cryoprotectant and slow cooling above the freezing point in order to minimise the risk of cold shock. In order to test this assumption, we added an equal volume of cold (4°C) cryoprotectant in a single aliquot to warm (20, 30 or 37°C) semen to induce rapid cooling. The results of this procedure were compared with those obtained using warm cryoprotectant or with the routine cryopreservation protocol used in this laboratory. The use of cold cryoprotectant resulted in a significant (P = 0.016) improvement (mean 63%, range 42%–79%) in post-thaw motility recovery compared with a standard procedure(mean 47%, range 35%–67%) and a significant (P = 0.016) improvement in post-thaw sperm velocity. A cold glycerol/egg yolk/citrate (GEYC) mixture also gave significantly higher motility recovery than GEYC equilibrated to either room temperature (20°C) or body temperature (37°C). Sperm frozen using the cold cryoprotectant protocol were as efficient at binding to and penetrating the human zona pellucida as sperm frozen with a standard protocol.The modified cryopreservation procedure may lead to improved pregnancy rates in donor insemination and in vitro fertilisation. Further investigation is required to determine how the cold cryoprotectant improves the cryopreservation outcome.
Distribution patterns of leucocyte subpopulations expressing different cell markers in the cumulus - oocyte complexes of pregnant and pseudopregnant mice
Authors: Akkoyunlu Gökhan , Korgun Emin Türkay , Çelik-Özenci Çiler , Seval Yasemin , Demir Ramazan , Üstünel İsmail
Published: February 2004

The nature of leucocyte subpopulations expressing different cell markers around the cumulus–oocyte complex (COC) of pregnant and pseudopregnant mice was investigated in the present study. Immunolabelling for CD4, CD8, CD14, CD45 and CD163 and transmission electron microscopy were used to determine whether leucocytes differ between pregnant and pseudopregnant mice. Sexually mature female BALB/c mice (n = 36; 18 pregnant, 18 pseudopregnant) were stimulated to superovulate with pregnant mare’s serum gonadotropin and human chorionic gonadotrophin, then mated with either fertile or vasectomised males. Postovulatory oocytes were collected after mating. The cumulus cell masses of the pregnant group contained spermatozoa between cells and were more variable than COCs of the pseudopregnant group. Streptavidin–biotin–peroxidase immunohistochemical labelling of the cell markers CD4, CD8, CD14, CD45 and CD163 showed that there were fewer leucocytes in the COCs of the pseudopregnant group compared with the pregnant group. Transmission electron microscopy revealed that often there were macrophage-like cells containing spermiophagic bodies between the cumulus cells. These observations suggest that, together with other cumulus cells and oviducal cells, these macrophage-like cells may be involved in removing unsuitable or excess spermatozoa and, therefore, in maintaining a suitable microenvironment for normal fertilisation.
Birth of a cloned calf derived from a vitrified hand-made cloned embryo
Authors: Tecirlioglu R. T. , French A. J. , Lewis I. M. , Vajta G. , Korfiatis N. A. , Hall V. J. , Ruddock N. T. , Cooney M. A. , Trounson A. O.
Published: February 2004

The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. The technique involves manual bisection of zona-free oocytes, selection of cytoplasts by Hoechst staining and fusion of a single somatic cell and two cytoplasts. In this proof-of-principle experiment, the objective was to examine the developmental competence of HMC embryos following embryo transfer. Modifications to the original method include not selecting of matured oocytes and simultaneous fusion of cytoplasts and karyoplast. Blastocyst rates for embryos cultured in the glass oviduct system as singles (10.5%; 24/228) or in pairs (16.1%; 36/224) did not differ significantly. Fresh and vitrified–thawed blastocysts were transferred to 16 synchronised recipients (three to four embryos per recipient). Ultrasound examination on Days 35–45 showed an initial pregnancy rate of 43.8% (7/16) and a pregnancy rate >8 months of 12.5% (2/16). A male cloned calf (42 kg) derived from a vitrified HMC blastocyst was delivered by Caesarean section on Day 271. The birth and ongoing survival (15 months, 243 kg) of a healthy and apparently normal calf, combining both HMC and vitrification technologies, provides a ‘proof of principle’ of the technology and a promising alternative to traditional nuclear-transfer techniques.
Transgenic zebrafish produced by retroviral infection of
in vitro
-cultured sperm
Authors: Kurita Kayoko , Burgess Shawn M. , Sakai Noriyoshi
Published: February 2004

Transgenic modification of sperm before fertilization has distinct advantages over conventional transgenic methods. The primary advantage is that the mosaicism inherent in those other techniques is avoided. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation from spermatogonia to functional sperm can occur
in vitro
, provides the opportunity for genetic modification of sperm
in vitro
. Here, we report the production of transgenic zebrafish from cultured sperm. The sperm were differentiated from premeiotic germ cells infected with a pseudotyped retrovirus
in vitro
. The collected sperm were used to perform successful
in vitro
fertilizations, and transgenic embryos were identified. The transgenic fish transmitted the proviral integration to the next generation in a Mendelian fashion. We report the generation of a transgenic animal by cultured sperm and open the door to many exciting possibilities for the rapid generation of transgenic lines in model organisms such as zebrafish or other animal systems that are otherwise intractable to transgenesis.
Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism
Authors: Schief William R. , Clark Rutilio H. , Crevenna Alvaro H. , Howard Jonathon
Published: February 2004

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in
in vitro
motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis–Menten equation. In the absence of products, the
K
M
(the [ATP] required for half-maximal speed) was 28 ± 1 μM, and the maximum speed was 904 nm/s. P
i
behaved as a competitive inhibitor with
K
I
= 9 ± 1 mM. ADP behaved approximately as a competitive inhibitor with
K
I
= 35 ± 2 μM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P
i
is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P
i
before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P
i
release from the other.
Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of α-helical content and compactness
Authors: Uzawa Takanori , Akiyama Shuji , Kimura Tetsunari , Takahashi Satoshi , Ishimori Koichiro , Morishima Isao , Fujisawa Tetsuro
Published: February 2004

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A–E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (
f
H
) and radius of gyration (
R
g
), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to ≈50% of the overall change in
R
g
from the unfolded to native conformation, was observed within 300 μs after the pH jump. The collapsed intermediate has a
f
H
of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
Giant plasmid-encoded polyketide synthases produce the macrolide toxin of
Mycobacterium ulcerans
Authors: Stinear Timothy P. , Mve-Obiang Armand , Small Pamela L. C. , Frigui Wafa , Pryor Melinda J. , Brosch Roland , Jenkin Grant A. , Johnson Paul D. R. , Davies John K. , Lee Richard E. , Adusumilli Sarojini , Garnier Thierry , Haydock Stephen F. , Leadlay Peter F. , Cole Stewart T.
Published: February 2004

Mycobacterium ulcerans
(MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a
Mycobacterium
, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.
Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis
Authors: Puthenveedu Manojkumar A. , Linstedt Adam D.
Published: February 2004

Functional characterization of protein interactions in mammalian systems has been hindered by the inability to perform complementation analyses
in vivo
. Here, we use functional replacement of the vesicle docking protein p115 to separate its essential from its nonessential interactions. p115 is required for biogenesis of the Golgi apparatus, but it is unclear whether its mechanism of action requires its golgin and/or SNARE interactions. Short interfering RNA-mediated knockdown of p115 induced extensive Golgi fragmentation and impaired secretory traffic. Reassembly of a structurally and functionally normal Golgi occurred on expression of a p115 homologue not recognized by the short interfering RNA. Strikingly, versions of p115 lacking its phosphorylation site and the golgin-binding domains also restored the Golgi apparatus in cells lacking endogenous p115. In contrast, the p115 SNARE-interacting domain was required for Golgi biogenesis. This suggests that p115 acts directly, rather than via a tether, to catalyze trans-SNARE complex formation preceding membrane fusion.
Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis
Authors: Greif Daniel M. , Sacks David B. , Michel Thomas
Published: February 2004

The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [
32
P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2
in vitro
reduces the
V
max
of immunopurified eNOS by a factor of 2 but has no effect on the
K
A
for CaM or calcium. Additionally, [
32
P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM (“phosphonull” S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the “phosphomimetic” Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using
Escherichia coli
-expressed and phenyl-Sepharose-purified CaM mutants revealed that the S81A mutation abrogates
in vitro
CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.
Costimulatory ligand 4-1BBL (CD137L) as an efficient adjuvant for human antiviral cytotoxic T cell responses
Authors: Bukczynski Jacob , Wen Tao , Ellefsen Kim , Gauldie Jack , Watts Tania H.
Published: February 2004

Effective adjuvants capable of inducing strong cytotoxic T cell responses in humans are lacking. In this study, we tested 4-1BBL as an adjuvant for activation of human memory antiviral CD8 T cell responsesex vivo. A recombinant replication-defective 4-1BBL adenovirus was used to convert autologous monocytes into efficient antigen-presenting cells after overnight incubation, bypassing the need to generate dendritic cells. Together with viral peptides, 4-1BBL led to robust memory responses of human Epstein–Barr virus- and influenza virus-specific cytotoxic T cells, with expansion of peptide-specific CD8 effector cells; up-regulation of Bcl-xL, granzyme A, and perforin; enhanced cytotoxic activity; and increased cytokine production. The response was significant even at a 100-fold lower peptide dose, compared with responses obtained with control adenovirus. Adenovirus-delivered B7.1 also expanded and activated virus-specific CD8 T cells, but 4-1BBL was more effective in driving the T cells toward a more fully differentiated CD27–effector state. Thus, 4-1BBL is a promising adjuvant for human memory CD8 T cells and will likely be most effective in the boost phase of a prime-boost strategy.
A role for p300/CREB binding protein genes in promoting cancer progression in colon cancer cell lines with microsatellite instability
Authors: Ionov Yurij , Matsui Sei-Ichi , Cowell John K.
Published: February 2004

Our manipulation of the nonsense-mediated decay pathway in microsatellite unstable colon cancer cell lines identified the
p300
gene as a potential tumor suppressor in this subtype of cancer. Here, we have demonstrated that not only the
p300
gene but also the highly homologous cAMP-response element-binding protein (CREB) binding protein (
CBP
) gene together are mutated in >85% of microsatellite instability (MSI)+ colon cancer cell lines. A limited survey of primary tumors with MSI+ shows that
p300
is also frequently mutated in these cancers, demonstrating that these mutations are not consequences of
in vitro
growth. The mutations in both genes occur frequently in mononucleotide repeats that generate premature stop codons. Reintroduction of
p300
into MSI colon cancer cells could only be supported in the presence of an inactivated
CBP
gene, suggesting the idea that one or the other function must be inactivated for cancer cell viability. p300 is known to acetylate p53 in response to DNA damage, and when MSI+ cells null for p300 activity are forced to reexpress exogenous
p300
cells show slower growth and a flatter morphology. p53 acetylation is increased upon reexpression of p300, suggesting that MSI+ cells constitutively activate the DNA damage response pathway in the absence of DNA-damaging agents. In support of this hypothesis, c-ABL kinase, which is also activated in response to DNA damage, shows higher levels of basal kinase activity in MSI+ cells. These observations suggest that there is a selective growth/survival advantage to mutational inactivation of
p300
/
CBP
in cells with inactivated mismatch repair capabilities.
Molecular dissection of water and glycerol permeability of the aquaglyceroporin from
Plasmodium falciparum
by mutational analysis
Authors: Beitz Eric , Pavlovic-Djuranovic Slavica , Yasui Masato , Agre Peter , Schultz Joachim E.
Published: February 2004

The selectivity of aquaporins for water and solutes is determined by pore diameter. Paradoxically, the wider pores of glycerol facilitators restrict water passage by an unknown mechanism. Earlier we characterized an aquaglyceroporin from
Plasmodium falciparum
with high permeability for both glycerol and water. We use point mutations to demonstrate that amino acids directly lining the pore are not responsible for the excellent water permeability of the
Plasmodium
aquaglyceroporin but affect permeability of pentitols. Within a conserved WET triad in the extracellular C-loop we identified a
Plasmodium
aquaglyceroporin-specific glutamate (E125) located in proximity to a conserved arginine (R196) at the pore mouth. Mutation of E125 to serine largely abolished water permeability. Concomitantly, the activation energy for water permeation was increased by 4 kcal/mol. Mutation of the adjacent tryptophan to cysteine led to irreversible inhibition of water passage by Hg
2+
. This unequivocally proves the proximity of the couple W124/E125 close to the pore mouth. We conclude that in the
Plasmodium
aquaglyceroporin the electrostatic environment at the extracellular pore entry regulates water permeability.
Analysis of
Pseudomonas aeruginosa
4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication
Authors: Déziel Eric , Lépine François , Milot Sylvain , He Jianxin , Mindrinos Michael N. , Tompkins Ronald G. , Rahme Laurence G.
Published: February 2004

Bacterial communities use “quorum sensing” (QS) to coordinate their population behavior through the action of extracellular signal molecules, such as the
N
-acyl-
l
-homoserine lactones (AHLs). The versatile and ubiquitous opportunistic pathogen
Pseudomonas aeruginosa
is a well-studied model for AHL-mediated QS. This species also produces an intercellular signal distinct from AHLs, 3,4-dihydroxy-2-heptylquinoline (PQS), which belongs to a family of poorly characterized 4-hydroxy-2-alkylquinolines (HAQs) previously identified for their antimicrobial activity. Here we use liquid chromatography (LC)/MS, genetics, and whole-genome expression to investigate the structure, biosynthesis, regulation, and activity of HAQs. We show that the
pqsA-E
operon encodes enzymes that catalyze the biosynthesis of five distinct classes of HAQs, and establish the sequence of synthesis of these compounds, which include potent cytochrome inhibitors and antibiotics active against human commensal and pathogenic bacteria. We find that anthranilic acid, the product of the PhnAB synthase, is the primary precursor of HAQs and that the HAQ congener 4-hydroxy-2-heptylquinoline (HHQ) is the direct precursor of the PQS signaling molecule. Significantly, whereas
phnAB
and
pqsA-E
are positively regulated by the virulence-associated transcription factor MvfR, which is also required for the expression of several QS-regulated genes, the conversion of HHQ to PQS is instead controlled by LasR. Finally, our results reveal that HHQ is itself both released from, and taken up by, bacterial cells where it is converted into PQS, suggesting that it functions as a messenger molecule in a cell-to-cell communication pathway. HAQ signaling represents a potential target for the pharmacological intervention of
P. aeruginosa
-mediated infections.
Histone deacetylase (HDAC) inhibitor activation of p21
WAF1
involves changes in promoter-associated proteins, including HDAC1
Authors: Gui C.-Y. , Ngo L. , Xu W. S. , Richon V. M. , Marks P. A.
Published: February 2004

Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis
in vivo
and
in vitro
. The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21
WAF1
is one of the most commonly induced. SAHA does not alter the expression of p27
KIPI
, an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21
WAF1
promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21
WAF1
promoter. These changes did not occur in the p27
KIPI
or ε-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21
WAF1
promoter. Thus, this study identifies effects of SAHA on p21
WAF1
-associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.
Coat protein regulates formation of replication complexes during tobacco mosaic virus infection
Authors: Asurmendi S. , Berg R. H. , Koo J. C. , Beachy R. N.
Published: February 2004

The genome of tobacco mosaic virus (TMV) encodes replicase protein(s), movement protein (MP), and capsid protein (CP). On infection, one or more viral proteins direct the assembly of virus replication complexes (VRCs), in association with host-derived membranes. The impact of CP-mediated resistance on the structures of the replication complexes was examined in nontransgenic and transgenic BY-2 cell lines that produce wild-type CP, mutant CP
T42W
, and Ds-Red, which was targeted to endoplasmic reticulum by using immunofluorescence and 3D microscopy. We developed a model of VRCs that shows a clear association of MP with and surrounding the endoplasmic reticulum. Replicase is located within the MP bodies, as well as isolated sites throughout the cell. CP surrounds the VRCs. CP enhances the production of MP and increases the size of the VRC; however, the mutant CP
T42W
reduces the amount of MP and interferes with the formation of VRCs. We propose a regulatory role of the CP in the establishment of the VRC. We suggest that the lack of formation of VRCs restricts the efficiency of virus replication and the formation of virus movement complexes, resulting in restriction of cell–cell spread of infection. This results in higher levels of plant CP-mediated protection provided by CP
T42W
.
Cerebellar granule cell precursors can differentiate into astroglial cells
Authors: Okano-Uchida Takayuki , Himi Toshiyuki , Komiya Yoshiaki , Ishizaki Yasuki
Published: February 2004

During CNS development, multipotent neural stem cells give rise first to various kinds of specified precursor cells, which proliferate extensively before terminally differentiating into either neurons or glial cells. It is still not clear, however, whether the specified precursor cells are irreversibly determined to differentiate into their particular cell types. In this study, we show that isolated mouse cerebellar granule cell precursors from the outermost, proliferative zone of the external germinal layer can differentiate into astroglial cells when exposed to sonic hedgehog (Shh) and bone morphogenetic proteins. These induced cells initially expressed both glial fibrillary acidic protein and neuronal markers, but they then lost their neuronal markers and acquired S100-β, a marker of differentiated astroglial cells. These results indicate that at least some granule cell precursors are not irreversibly committed to neuronal development but can be induced to differentiate into astroglial cells by appropriate extracellular signals.
Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: A mechanism for generation of soluble cytokine receptors
Authors: Hawari Feras I. , Rouhani Farshid N. , Cui Xinle , Yu Zu-Xi , Buckley Caitriona , Kaler Maryann , Levine Stewart J.
Published: February 2004

Soluble tumor necrosis factor receptors (TNFRs) are important modulators of TNF bioactivity. Proteolytic cleavage of the 28-kDa ectodomain of TNFR1 has been recognized as the mechanism by which soluble TNFR is shed. We now describe the release of exosome-like vesicles as a mechanism for the generation of soluble, full-length 55-kDa TNFR1. We found unexpectedly that the predominant form of soluble TNFR1 in human serum and lung epithelial lining fluid is a full-length 55-kDa protein. Furthermore, supernatants from human vascular endothelial cells contain only full-length 55-kDa TNFR1 that can be sedimented by high-speed centrifugation, floated on sucrose gradients at a density of 1.1 g/ml, and associated with vesicles that range in diameter from 20 nm to 50 nm. We conclude that the release of TNFR1 exosome-like vesicles represents a previously unrecognized mechanism by which constitutive production of soluble cytokine receptors may be regulated, independent of ectodomain cleavage by receptor sheddases.
Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis
Authors: Kurt-Jones Evelyn A. , Chan Melvin , Zhou Shenghua , Wang Jennifer , Reed George , Bronson Roderick , Arnold Michelle M. , Knipe David M. , Finberg Robert W.
Published: February 2004

Human neonates infected with herpes simplex virus 1 (HSV-1) develop one of three distinct patterns of infection: (i) infection limited to the skin, eye or mouth; (ii) infection of the CNS; or (iii) disseminated infection. The disseminated form usually involves the liver, adrenal gland, and lung, and resembles the clinical picture of bacterial sepsis. This spectrum of symptoms in HSV-1-infected neonates suggests that inflammatory cytokines play a significant role in the pathogenesis of the disease. Recent studies suggest that the Toll-like receptors (TLRs) may play an important role in the induction of inflammatory cytokines in response to viruses. TLRs are mammalian homologues of Toll, aDrosophilaprotein that is essential for host defense against infection. Engagement of TLRs by bacterial, viral, or fungal components leads to the production and release of cytokines and other antimicrobial products. Here, we demonstrate that TLR2 mediates the inflammatory cytokine response to HSV-1 by using both transfected cell lines and knockout mice. Studies of infected mice revealed that HSV-1 induced a blunted cytokine response in TLR2–/–mice. Brain levels of monocyte chemoattractant protein 1 chemokine were significantly lower in TLR2–/–mice than in either wild-type or TLR4–/–mice. TLR2–/–mice had reduced mortality compared with wild-type mice. The differences between TLR2–/–mice and both wild-type and TLR4–/–mice in the induction of monocyte chemoattractant protein 1, brain inflammation, or mortality could not be accounted for on the basis of virus levels. Thus, these studies suggest the TLR2-mediated cytokine response to HSV-1 is detrimental to the host.
Neanderthal taxonomy reconsidered: Implications of 3D primate models of intra- and interspecific differences
Authors: Harvati Katerina , Frost Stephen R. , McNulty Kieran P.
Published: February 2004

The taxonomic status of Neanderthals lies at the center of the modern human origins debate. Proponents of the single-origin model often view this group as a distinct species with little or no contribution to the evolution of modern humans. Adherents to the regional continuity model consider Neanderthals a subspecies or population of
Homo sapiens
, which contributed significantly to the evolution of early modern Europeans. Paleontologists generally agree that fossil species should be equivalent to extant ones in the amount of their morphological variation. Recognition of fossil species therefore hinges on analogy to living species. A previous study by one of the authors and recent work by other researchers [Schillachi, M. A. & Froelich, J. W. (2001)
Am. J. Phys. Anthropol.
115, 157–166] have supported specific status for Neanderthals based on analogy to chimpanzees and Sulawesi macaques, respectively. However, these taxa may not be the most appropriate models for Pleistocene humans. Here we test the hypothesis that Neanderthals represent a subspecies of
H. sapiens
by comparing the degree of their morphological differentiation from modern humans to that found within and between 12 species of extant primates. The model taxa comprised >1,000 specimens, including phylogenetic (modern humans and African apes) and ecological (eight papionin taxa) models for Pleistocene humans. Morphological distances between model taxon pairs were compared to the distances between Neanderthals and modern humans obtained by using a randomization technique. Results strongly support a specific distinction for Neanderthals.
Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors
Authors: Granville David J. , Tashakkor Babak , Takeuchi Cindy , Gustafsson Åsa B. , Huang Chengqun , Sayen M. Richard , Wentworth Paul , Yeager Mark , Gottlieb Roberta A.
Published: February 2004

Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia/reperfusion injury.
Method for analyzing signaling networks in complex cellular systems
Authors: Plavec Ivan , Sirenko Oksana , Privat Sylvie , Wang Yuker , Dajee Maya , Melrose Jennifer , Nakao Brian , Hytopoulos Evangelos , Berg Ellen L. , Butcher Eugene C.
Published: February 2004

Now that the human genome has been sequenced, the challenge of assigning function to human genes has become acute. Existing approaches using microarrays or proteomics frequently generate very large volumes of data not directly related to biological function, making interpretation difficult. Here, we describe a technique for integrative systems biology in which: (i) primary cells are cultured under biologically meaningful conditions; (ii) a limited number of biologically meaningful readouts are measured; and (iii) the results obtained under several different conditions are combined for analysis. Studies of human endothelial cells overexpressing different signaling molecules under multiple inflammatory conditions show that this system can capture a remarkable range of functions by a relatively small number of simple measurements. In particular, measurement of seven different protein levels by ELISA under four different conditions is capable of reconstructing pathway associations of 25 different proteins representing four known signaling pathways, implicating additional participants in the NF-κBorRAS/mitogen-activated protein kinase pathways and defining additional interactions between these pathways.
Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome
Authors: Fouchier Ron A. M. , Schneeberger Peter M. , Rozendaal Frans W. , Broekman Jan M. , Kemink Stiena A. G. , Munster Vincent , Kuiken Thijs , Rimmelzwaan Guus F. , Schutten Martin , van Doornum Gerard J. J. , Koch Guus , Bosman Arnold , Koopmans Marion , Osterhaus Albert D. M. E.
Published: February 2004

Highly pathogenic avian influenza A viruses of subtypes H5 and H7 are the causative agents of fowl plague in poultry. Influenza A viruses of subtype H5N1 also caused severe respiratory disease in humans in Hong Kong in 1997 and 2003, including at least seven fatal cases, posing a serious human pandemic threat. Between the end of February and the end of May 2003, a fowl plague outbreak occurred in The Netherlands. A highly pathogenic avian influenza A virus of subtype H7N7, closely related to low pathogenic virus isolates obtained from wild ducks, was isolated from chickens. The same virus was detected subsequently in 86 humans who handled affected poultry and in three of their family members. Of these 89 patients, 78 presented with conjunctivitis, 5 presented with conjunctivitis and influenza-like illness, 2 presented with influenza-like illness, and 4 did not fit the case definitions. Influenza-like illnesses were generally mild, but a fatal case of pneumonia in combination with acute respiratory distress syndrome occurred also. Most virus isolates obtained from humans, including probable secondary cases, had not accumulated significant mutations. However, the virus isolated from the fatal case displayed 14 amino acid substitutions, some of which may be associated with enhanced disease in this case. Because H7N7 viruses have caused disease in mammals, including horses, seals, and humans, on several occasions in the past, they may be unusual in their zoonotic potential and, thus, form a pandemic threat to humans.
Patterns of Evolutionary Constraints in Intronic and Intergenic DNA of Drosophila
Authors: Halligan Daniel L. , Eyre-Walker Adam , Andolfatto Peter , Keightley Peter D.
Published: February 2004

We develop methods to infer levels of evolutionary constraints in the genome by comparing rates of nucleotide substitution in noncoding DNA with rates predicted from rates of synonymous site evolution in adjacent genes or other putatively neutrally evolving sites, while accounting for differences in base composition. We apply the methods to estimate levels of constraint in noncoding DNA of Drosophila. In introns, constraint (the estimated fraction of mutations that are selectively eliminated) is absolute at the 5′ and 3′ splice junction dinucleotides, and averages 72% in base pairs 3-6 at the 5′-end. Constraint at the 5′ base pairs 3-6 is significantly lower in the lineage leading to Drosophila melanogaster than in Drosophila simulans, a finding that agrees with other features of genome evolution in Drosophila and indicates that the effect of selection on intron function has been weaker in the melanogaster lineage. Elsewhere in intron sequences, the rate of nucleotide substitution is significantly higher than at synonymous sites. By using intronic sites outside splice control regions as a putative neutrally evolving standard, constraint in the 500 bp of intergenic DNA upstream and downstream regions of protein-coding genes averages ∼44%. Although the estimated level of constraint in intergenic regions close to genes is only about one-half of that of amino acid sites, selection against single-nucleotide mutations in intergenic DNA makes a substantial contribution to the mutation load in Drosophila.
A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q
Authors: Yamada Yoichi , Watanabe Hidemi , Miura Fumihito , Soejima Hidenobu , Uchiyama Michiko , Iwasaka Tsuyoshi , Mukai Tsunehiro , Sakaki Yoshiyuki , Ito Takashi
Published: February 2004

Approximately half of all human genes have CpG islands (CGIs)around their promoter regions. Although CGIs usually escape methylation, those on Chromosome X in females and those in the vicinity of imprinted genes are exceptions: They have both methylated and unmethylated alleles to display a “composite” pattern in methylation analysis. In addition, aberrant methylation of CGIs is known to often occur in cancer cells. Here we developed a simple HpaII-McrBC PCR method for discrimination of full, null, incomplete, and composite methylation patterns, and applied it to all computationally identified CGIs on human Chromosome 21q. This comprehensive analysis revealed that, although most CGIs (103 out of 149)escape methylation, a sizable fraction (31 out of 149)are fully methylated even in normal peripheral blood cells. Furthermore, we identified seven CGIs showing the composite methylation, and demonstrated that three of them are indeed methylated monoallelically. Further analyses using informative pedigrees revealed that two of the three are subject to maternal allele-specific methylation. Intriguingly, the other CGI is methylated in an allele-specific but parental-origin-independent manner. Thus, the cell seems to have a broader repertoire of methylating CGIs than previously thought, and our approach may contribute to uncover novel modes of allelic methylation.
Comparative Genomics of Gene Expression in the Parasitic and Free-Living Nematodes Strongyloides stercoralis and Caenorhabditis elegans
Authors: Mitreva Makedonka , McCarter James P. , Martin John , Dante Mike , Wylie Todd , Chiapelli Brandi , Pape Deana , Clifton Sandra W. , Nutman Thomas B. , Waterston Robert H.
Published: February 2004

Although developmental timing of gene expression is used to infer potential gene function, studies have yet to correlate this information between species. We analyzed 10,921 ESTs in 3311 clusters from first- and infective third-stage larva (L1, L3i) of the parasitic nematode Strongyloides stercoralis and compared the results to Caenorhabditis elegans, a species that has an L3i-like dauer stage. In the comparison of S. stercoralis clusters with stage-specific expression to C. elegans homologs expressed in either dauer or nondauer stages, matches between S. stercoralis L1 and C. elegans nondauer-expressed genes dominated, suggesting conservation in the repertoire of genes expressed during growth in nutrient-rich conditions. For example, S. stercoralis collagen transcripts were abundant in L1 but not L3i, a pattern consistent with C. elegans collagens. Although a greater proportion of S. stercoralis L3i than L1 genes have homologs among the C. elegans dauer-specific transcripts, we did not uncover evidence of a robust conserved L3i/dauer `expression signature.' Strikingly, in comparisons of S. stercoralis clusters to C. elegans homologs with RNAi knockouts, those with significant L1-specific expression were more than twice as likely as L3i-specific clusters to match genes with phenotypes. We also provide functional classifications of S. stercoralis clusters.
Association between Helicobacter pylori and recurrent aphthous stomatitis in children and adolescents
Authors: Fritscher A. M. G. , Cherubini K. , Chies J. , Dias A. C. O.
Published: February 2004

BACKGROUND:  The recurrent aphthous stomatitis (RAS) is a common disease with unknown etiology. Helicobacter pylori has been shown to be the causative factor in peptic ulcers. Considering the similarities of histologic features between gastric and oral ulceration, we studied the possible involvement of H. pylori in the development of RAS.

METHODS:  A total of 105 children and adolescents were investigated – 53 patients with RAS (case group) and 52 patients without lesions (control group). Specimens obtained by swabbing RAS lesions, intact oral mucosa, and dental plaque were submitted to a polymerase chain reaction (PCR)‐based assay.

RESULTS:  Helicobacter pylori was present in six patients of the case group (11.3%) and in three of the control group (5.8%). When the site of infection was studied, 9.4% of the RAS lesions were PCR positives. In the case group and control group, 5.7 and 1.9% of the specimens from dental plaque, respectively, and 5.7 and 3.8% of the specimens from the intact oral mucosa, respectively, were PCR positives.

CONCLUSION:  There was no association between RAS lesions and infection of the oral cavity by H. pylori in children and adolescents (P = 0.254).