bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential flow models of Rayleigh–Taylor and Richtmyer–Meshkov bubble fronts
Authors: Hecht Jacob , Alon Uri , Shvarts Dov
Published: July 2002

A potential flow model of Rayleigh–Taylor and Richtmyer–Meshkov bubbles on an interface between an incompressible fluid and a constant supporting pressure (Atwood number A=1) is presented. In the model, which extends the work of Layzer [Astrophys. J. 122, 1 (1955)], ordinary differential equations for the bubble heights and curvatures are obtained by considering the potential flow equations near the bubble tips. The model is applied to two-dimensional single-mode evolution as well as two-bubble competition, for both the Rayleigh–Taylor (RT) and the Richtmyer–Meshkov (RM) instabilities, the latter treated in an impulse approximation. The model predicts that the asymptotic velocity of a single-mode RM bubble of wavelength λ decays as λt−1, in contrast with the constant asymptotic velocity attained in the RT case. Bubble competition, which is believed to determine the multimode front evolution, is demonstrated for both the RT and RM instabilities. The capability of the model to predict bubble growth in a finite-thickness fluid layer is shown. Finally, the model is applied to the evolution of three-dimensional modes with an initial rectangular geometry. The model yields the aspect ratio dependence of the early nonlinear stages, in agreement with third-order perturbation theory. However, in the late nonlinear stage, the model predicts that the bubbles forget the initial geometry and attain the fastest growing shape, with a circular tip. The model results are in good agreement with full hydrodynamic simulations and analytic solutions, where available.
Timing of vortex formation from an oscillating cylinder
Authors: Gu W. , Chyu C. , Rockwell D.
Published: July 2002

The instantaneous structure of the near-wake of a cylinder subjected to forced oscillations is examined using particle imaging, which leads to representations of the streamline patterns and distributions of vorticity. As the frequency of excitation of the cylinder is increased relative to the inherent vortex formation frequency, the initially formed concentration of vorticity moves closer to the cylinder until a limiting position is reached; at this position, the vorticity concentration abruptly switches to the opposite side of the cylinder. This process induces abrupt changes of the topology of the corresponding streamline patterns; such topological patterns alone, however, do not properly suggest the existence and rearrangement of the vorticity concentrations. Moreover, this vorticity-switching concept persists to high values of Reynolds number, where the values of the mean base pressure coefficient and vortex formation length differ substantially from those at low Reynolds number. The switching mechanism is not significantly altered, either in an instantaneous or ensemble-averaged sense, by the presence of small-scale Kelvin–Helmholtz vortices that coexist with the large-scale (Kármán) vortices.
Transverse and longitudinal scaling laws in non-homogeneous low Re turbulence
Authors: Camussi R. , Barbagallo D. , Guj G. , Stella F.
Published: July 2002

The statistical properties of the velocity fluctuation components at moderate Reλ are studied in homogeneous and non-homogeneous turbulence by an experimental technique. Measurements are performed by means of a hot-wire anemometer with X-probe downstream of a screen for several positions, x/M=9 up to 109, where M is the screen mesh size, with Reλ ranging from 37 to 82. The homogeneity of the flow is analyzed by means of velocity measurements in different transverse positions and a direct evaluation of the local isotropy of the flow is performed by means of velocity spectra. The scaling properties of the statistical moments of the structure functions up to the order of six, are investigated in the extreme positions by means of the extended self-similarity (ESS) method and the intermittency exponents are detected for both homogeneous and non-homogeneous flow conditions. A comparison of the longitudinal and transverse intermittency exponents as functions of the position is then performed and discussed in addition to the analysis of the transition from the anomalous to a regular scaling for small spatial separation.
A paradox involving the second law of thermodynamics
Authors: Sheehan D. P.
Published: July 2002

A simple paradox is posed that appears to challenge the second law of thermodynamics in a blackbody plasma environment. Laboratory experiments approximating salient aspects of this system fail to resolve the paradox.
Observation of the bremsstrahlung generation in the process of the Rayleigh–Taylor instability development at gas puff implosion
Authors: Baksht R. B. , Fedunin A. V. , Labetsky A. Yu. , Rousskich A. G. , Shishlov A. V.
Published: July 2002

The electron magnetohydrodynamic model predicts the appearance of anode–cathode voltage in the process of Rayleigh–Taylor instability development during gas puff implosions. The appearance of the anode–cathode voltage should be accompanied by the accelerated electron flow and the generation of the bremsstrahlung radiation. Experiments with neon and krypton gas puffs were performed on the GIT-4 [S. P. Bugaev, et al., Plasma Sci. 18, 115 (1990)] generator (1.6 MA, 120 ns) to observe the bremsstrahlung radiation during the gas puff implosion. Two spikes of the bremsstrahlung radiation were observed in the experiments. The first spike is connected with the gas breakdown; the second one is connected with the final stage of the implosion. The development of the RT instabilities does not initiate the bremsstrahlung radiation, therefore, the absence of anode–cathode voltage is demonstrated.
Bremsstrahlung emission from ion–dust grain Coulomb scatterings in dusty plasmas
Authors: Jung Young-Dae , Tawara Hiro
Published: July 2002

Bremsstrahlung processes in ion–dust grain Coulomb scatterings in dusty plasmas are investigated using the classical trajectory method. Attractive interaction between the projectile ion and its own image charge inside the positively charged dust grain is included to obtain the total interaction potential between the projectile ion and the dust grain. The classical straight-line trajectory method is applied to represent the differential radiation cross section as a function of the scaled impact parameter, projectile energy, and photon energy. The result shows that the induced image charge enlarges the bremsstrahung cross section near the dust surface.
Comment “On the physics of Landau damping” [Phys. Plasmas 6, 2976 (1999)]
Authors: Puri Satish
Published: July 2002

“Resonant diffusion” rather than “thermal spread” [Stubbe and Sukhorukov, Phys. Plasmas 6, 2976 (1999)] correctly describes the physics of Landau damping.
Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer
in vivo
Authors: Stephen Antonia E. , Masiakos Peter T. , Segev Dorry L. , Vacanti Joseph P. , Donahoe Patricia K. , MacLaughlin David T.
Published: July 2002

Techniques of tissue engineering and cell and molecular biology
were used to create a biodegradable scaffold for transfected cells to
produce complex proteins. Mullerian Inhibiting Substance (MIS) causes
regression of Mullerian ducts in the mammalian embryo. MIS also causes
regression
in vitro
of ovarian tumor cell lines and
primary cells from ovarian carcinomas, which derive from Mullerian
structures. In a strategy to circumvent the complicated purification
protocols for MIS, Chinese hamster ovary cells transfected with the
human MIS gene were seeded onto biodegradable polymers of polyglycolic
acid fibers and secretion of MIS confirmed. The polymer-cell graft was
implanted into the right ovarian pedicle of severe combined
immunodeficient mice. Serum MIS in the mice rose to supraphysiologic
levels over time. One week after implantation of the polymer-cell
graft, IGROV-1 human tumors were implanted under the renal capsule of
the left kidney. Growth of the IGROV-1 tumors was significantly
inhibited in the animals with a polymer-cell graft of MIS-producing
cells, compared with controls. This novel MIS delivery system could
have broader applications for other inhibitory agents not amenable to
efficient purification and provides
in vivo
evidence for
a role of MIS in the treatment of ovarian cancer.
Migration of keratinocytes through tunnels of digested fibrin
Authors: Ronfard Vincent , Barrandon Yann
Published: July 2002

We report here a hitherto undescribed form of cell migration. When
a suspension of human keratinocytes is plated on a fibrin matrix,
single cells invade the matrix and progress through it as rounded cells
by dissolving the fibrin and thereby creating tunnels. These tunnels
are cylindrical or helical, the latter being the result of constant
change in the path of cellular advance around the helical axis. Helical
tunnel formation is strongly promoted by epidermal growth factor. The
rate of migration of the cell through the track of a helical tunnel (up
to 2.1 mm per day) is about 7-fold greater than through a cylindrical
tunnel. Pericellular fibrinolysis leading to tunnel formation depends
on the presence of plasminogen in the medium and its conversion to
plasmin by a cellular activator. Formation of tunnels requires that
plasminogen activator be localized on the advancing surface of the
keratinocyte; we propose that the tunnel is cylindrical when the site
of release of plasmin is located at a fixed point on the cell surface
and helical when the site of release precesses.
In vivo
detection of gene expression in liver by
31
P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene
Authors: Auricchio Alberto , Zhou Rong , Wilson James M. , Glickson Jerry D.
Published: July 2002

In vivo
assessment of gene expression is desirable
to obtain information on the extent and duration of transduction of
tissue after gene delivery. We have developed an
in
vivo
, potentially noninvasive, method for detecting virally
mediated gene transfer to the liver. The method employs an adenoviral
vector carrying the gene for the brain isozyme of murine creatine
kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and
brain but absent from liver, kidney, and pancreas. Gene expression was
monitored by
31
P magnetic resonance spectroscopy (MRS)
using the product of the CK enzymatic reaction, phosphocreatine, as an
indicator of transfection. The vector was administered into nude mice
by tail vein injection, and exogenous creatine was administered in the
drinking water and by i.p. injection of 2% creatine solution before
31
P MRS examination, which was performed on surgically
exposed livers. A phosphocreatine resonance was detected in livers of
mice injected with the vector and was absent from livers of control
animals. CK expression was confirmed in the injected animals by Western
blot analysis, enzymatic assays, and immunofluorescence measurements.
We conclude that the syngeneic enzyme CK can be used as a marker gene
for
in vivo
monitoring of gene expression after virally
mediated gene transfer to the liver.
Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF)
Authors: Visconti Richard P. , Richardson Charlene D. , Sato Thomas N.
Published: July 2002

Multiple classes of factors contribute to angiogenesis. In past years, the primary focus has been to understand the functions of individual classes of angiogenic factors. However, few studies have focused on the combinatorial roles of multiple classes of factors in angiogenesis. In this report, we have investigated the
in vivo
angiogenic processes regulated by two major classes of angiogenic factors, the angiopoietins and vascular endothelial growth factor (VEGF). Here we show that angiopoietin-1, a factor previously considered to be proangiogenic, can offset VEGF-induced angiogenesis
in vivo
. We also provide direct
in vivo
evidence for the synergistic effect of angiopoietin-2 and VEGF on the induction of angiogenesis. Furthermore, we show that these two classes of factors control the ratio of arterial and venous blood vessel types during angiogenesis. We believe that our study is a step toward understanding how multiple classes of factors harmonize angiogenesis and blood vessel types.
Parent–progeny recognition as a function of MHC odortype identity
Authors: Yamazaki K. , Beauchamp G. K. , Curran M. , Bard J. , Boyse E. A.
Published: July 2002

The several linked polymorphic genes of the MHC, which has been proposed as a prime determinant of sensed genetic individuality within species, is known to operate in mice by olfactory recognition in aspects of reproductive behavior that concern mate selection, thereby favoring outbreeding and heterozygosity, and also concern the maintenance of pregnancy. A single base-change can alter an individual MHC odortype, and the potential range of combinatorial MHC-determined odortypes is clearly vast. Following our findings that newborn mice already express their MHC odortype (which is detectable at 9 days of gestational age), we sought to determine whether MHC is involved in behavioral aspects of early development, such as rearing. In the studies presented herein, we report the ability and proclivity of mothers to recognize and preferentially retrieve syngeneic (genetically identical) pups from other pups differing only for MHC. Reciprocally, we report the ability of pups to recognize their familial environment, regardless of whether they had been nursed by their biological mothers or by foster mothers. Early learning experiences of the MHC environment are apparently a key element in survival, assuring maternal protection and promoting outbreeding.
Effect of spermidine on the
in vivo
degradation of ornithine decarboxylase in
Saccharomyces cerevisiae
Authors: Gupta Rashmi , Hamasaki-Katagiri Nobuko , Tabor Celia White , Tabor Herbert
Published: July 2002

As part of our studies on the regulation of polyamine biosynthesis in
Saccharomyces cerevisiae
, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in
S. cerevisiae
, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed
SPE1
strains is best seen in
spe2
mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that
S. cerevisiae
contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in
in vitro
experiments or in the
S. cerevisiae
genome.
A high-speed atomic force microscope for studying biological macromolecules
Authors: Ando Toshio , Kodera Noriyuki , Takai Eisuke , Maruyama Daisuke , Saito Kiwamu , Toda Akitoshi
Published: July 2002

The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450–650 kHz) and small spring constants (150–280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 × 100 pixel
2
image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see
http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm
).
Brain-specific expression of an exogenous gene after i.v. administration
Authors: Shi Ningya , Zhang Yun , Zhu Chunni , Boado Ruben J. , Pardridge William M.
Published: July 2002

The treatment of brain diseases with gene therapy requires the gene to be expressed throughout the central nervous system, and this is possible by using gene targeting technology that delivers the gene across the blood–brain barrier after i.v. administration of a nonviral formulation of the gene. The plasmid DNA is targeted to brain with pegylated immunoliposomes (PILs) using a targeting ligand such as a peptidomimetic mAb, which binds to a transporting receptor on the blood–brain barrier. The present studies adapt the PIL gene targeting technology to the mouse by using the rat 8D3 mAb to the mouse transferrin receptor. Tissue-specific expression in brain and peripheral organs of different exogenous genes (β-galactosidase, luciferase) is examined at 1–3 days after i.v. injection in adult mice of the exogenous gene packaged in the interior of 8D3-PIL. The expression plasmid is driven either by a broadly expressed promoter, simian virus 40, or by a brain-specific promoter taken from the 5′ flanking sequence of the human glial fibrillary acidic protein (GFAP) gene. The transgene is expressed in both brain and peripheral tissues when the simian virus 40 promoter is used, but the expression of the exogenous gene is confined to the brain when the transgene is under the influence of the brain-specific GFAP promoter. Confocal microscopy colocalizes immunoreactive bacterial β-galactosidase with immunoreactive GFAP in brain astrocytes. These studies indicate that tissue-specific gene expression in brain is possible after the i.v. administration of a nonviral vector with the combined use of gene targeting technology and tissue-specific gene promoters.
Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage.
Authors: Heller R , Brown K E , Burgtorf C , Brown W R
Published: July 2002

We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.
Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.
Authors: Takahashi A , Alnemri E S , Lazebnik Y A , Fernandes-Alnemri T , Litwack G , Moir R D , Goldman R D , Poirier G G , Kaufmann S H , Earnshaw W C
Published: July 2002

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.
Molecular phylogeny analysis of fiddler crabs: test of the hypothesis of increasing behavioral complexity in evolution.
Authors: Sturmbauer C , Levinton J S , Christy J
Published: July 2002

The current phylogenetic hypothesis for the evolution and biogeography of fiddler crabs relies on the assumption that complex behavioral traits are assumed to also be evolutionary derived. Indo-west Pacific fiddler crabs have simpler reproductive social behavior and are more marine and were thought to be ancestral to the more behaviorally complex and more terrestrial American species. It was also hypothesized that the evolution of more complex social and reproductive behavior was associated with the colonization of the higher intertidal zones. Our phylogenetic analysis, based upon a set of independent molecular characters, however, demonstrates how widely entrenched ideas about evolution and biogeography led to a reasonable, but apparently incorrect, conclusion about the evolutionary trends within this pantropical group of crustaceans. Species bearing the set of "derived traits" are phylogenetically ancestral, suggesting an alternative evolutionary scenario: the evolution of reproductive behavioral complexity in fiddler crabs may have arisen multiple times during their evolution. The evolution of behavioral complexity may have arisen by coopting of a series of other adaptations for high intertidal living and antipredator escape. A calibration of rates of molecular evolution from populations on either side of the Isthmus of Panama suggest a sequence divergence rate for 16S rRNA of 0.9% per million years. The divergence between the ancestral clade and derived forms is estimated to be approximately 22 million years ago, whereas the divergence between the American and Indo-west Pacific is estimated to be approximately 17 million years ago.
Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.
Authors: Mumm J S , Shou J , Calof A L
Published: July 2002

The mammalian olfactory epithelium (OE) supports continual neurogenesis throughout life, suggesting that a neuronal stem cell exists in this system. In tissue culture, however, the capacity of the OE for neurogenesis ceases after a few days. In an attempt to identify conditions that support the survival of neuronal stem cells, a population of neuronal progenitors was isolated from embryonic mouse OE and cultured in defined serum-free medium. The vast majority of cells rapidly gave rise to neurons, which died shortly thereafter. However, when purified progenitors were co-cultured with cells derived from the stroma underlying the OE, a small subpopulation (0.07-0.1%) gave rise to proliferative colonies. A morphologically identifiable subset of these colonies generated new neurons as late as 7 days in vitro. Interestingly, development of these neuronal colonies was specifically inhibited when purified progenitors were plated onto stromal feeder cells in the presence of a large excess of differentiated OE neurons. These results indicate that a rare cell type, with the potential to undergo prolonged neurogenesis, can be isolated from mammalian OE and that stroma-derived factors are important in supporting neurogenesis by this cell. The data further suggest that differentiated neurons provide a signal that feeds back to inhibit production of new neurons by their own progenitors.
Definition of HLA-DQ as a transplantation antigen
Authors: Petersdorf Effie W. , Longton Gary M. , Anasetti Claudio , Mickelson Eric M. , Smith Anajane G. , Martin Paul J. , Hansen John A.
Published: July 2002

Recent studies have demonstrated the importance of recipient HLA-DRB1 allele disparity in the development of acute graft-versus-host disease (GVHD) after unrelated donor marrow transplantation. The role of HLA-DQB1 allele disparity in this clinical setting is unknown. To elucidate the biological importance of HLA-DQB1, we conducted a retrospective analysis of 449 HLA-A, -B, and -DR serologically matched unrelated donor transplants. Molecular typing of HLA-DRB1 and HLA-DQB1 alleles revealed 335 DRB1 and DQB1 matched pairs; 41 DRB1 matched and DQB1 mismatched pairs; 48 DRB1 mismatched and DQB1 matched pairs; and 25 DRB1 and DQB1 mismatched pairs. The conditional probabilities of grades III-IV acute GVHD were 0.42, 0.61, 0.55, and 0.71, respectively. The relative risk of acute GVHD associated with a single locus HLA-DQB1 mismatch was 1.8 (1.1, 2.7;P= 0.01), and the risk associated with any HLA-DQB1 and/or HLA-DRB1 mismatch was 1.6 (1.2, 2.2;P= 0.003). These results provide evidence that HLA-DQ is a transplant antigen and suggest that evaluation of both HLA-DQB1 and HLA-DRB1 is necessary in selecting potential donors.
Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.
Authors: Jucovic M , Hartley R W
Published: July 2002

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.
Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.
Authors: Zisman E , Katz-Levy Y , Dayan M , Kirshner S L , Paas-Rozner M , Karni A , Abramsky O , Brautbar C , Fridkin M , Sela M , Mozes E
Published: July 2002

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.
Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype
Authors: Goldbaum Fernando A. , Velikovsky C. Alejandro , Dall’Acqua William , Fossati Carlos A. , Fields Barry A. , Braden Bradford C. , Poljak Roberto J. , Mariuzza Roy A.
Published: July 2002

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109M−1and 2.4 × 107M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106M−1and 3.8 × 106M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.
Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis
Authors: Hoek Robert M. , van Kesteren Ronald E. , Smit August B. , de Jong-Brink Marijke , Geraerts Wijnand P. M.
Published: July 2002

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome
Trichobilharzia ocellata
on peptidergic centers in the brain of the intermediate snail host
Lymnaea stagnalis
. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized
Lymnaea
was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.
Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates
Authors: Pasquinelli Amy E. , Powers Maureen A. , Lund Elsebet , Forbes Douglass , Dahlberg James E.
Published: July 2002

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.
Adenovirus-mediated gene transfer and expression of human β-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice
Authors: Ohashi Toya , Watabe Kazuhiko , Uehara Keiko , Sly William S. , Vogler Carole , Eto Yoshikatsu
Published: July 2002

Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme β-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human β-glucuronidase and administered this recombinant adenovirus to β-glucuronidase-deficient mice intravenously. The β-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the β-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the β-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of β-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology.
Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase
Authors: Kwon Young-Guen , Lee Soo Young , Choi Yongwon , Greengard Paul , Nairn Angus C.
Published: July 2002

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression.
In vitro
, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1Cα phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.
Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase
Authors: Ohya Ken-ichi , Kajigaya Sachiko , Kitanaka Akira , Yoshida Koji , Miyazato Akira , Yamashita Yoshihiro , Yamanaka Takeo , Ikeda Uichi , Shimada Kazuyuki , Ozawa Keiya , Mano Hiroyuki
Published: July 2002

Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (
B
C
R d
ownstream signalin
g
1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly
in vitro
. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling.
Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease
Authors: Daly Thomas M. , Vogler Carole , Levy Beth , Haskins Mark E. , Sands Mark S.
Published: July 2002

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 × 10
6
infectious units of recombinant adeno-associated virus encoding the human β-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.
Genes for calcineurin B-like proteins in
Arabidopsis
are differentially regulated by stress signals
Authors: Kudla Jörg , Xu Qiang , Harter Klaus , Gruissem Wilhelm , Luan Sheng
Published: July 2002

An important effector of Ca
2+
signaling in animals and yeast is the Ca
2+
/calmodulin-dependent protein phosphatase calcineurin. However, the biochemical identity of plant calcineurin remained elusive. Here we report the molecular characterization of AtCBL (

A

rabidopsis

t

haliana
c
alcineurin
B
-
l
ike protein) from
Arabidopsis
. The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase. AtCBL also shows significant similarity with another Ca
2+
-binding protein, the neuronal calcium sensor in animals. It contains typical EF-hand motifs with Ca
2+
-binding capability, as confirmed by
in vitro
Ca
2+
-binding assays, and it interacts
in vivo
with rat calcineurin A in the yeast two-hybrid system. Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant. Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in
Arabidopsis
. Genes for three isoforms were identified in this study.
AtCBL1
mRNA was preferentially expressed in stems and roots and its mRNA levels strongly increased in response to specific stress signals such as drought, cold, and wounding. In contrast,
AtCBL2
and
AtCBL3
are constitutively expressed under all conditions investigated. Our data suggest that AtCBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions.
Comparative T cell receptor repertoire selection by antigen after adoptive transfer: A glimpse at an antigen-specific preimmune repertoire
Authors: Attuil Valérie , Bucher Philipp , Rossi Mauricette , Mutin Mireille , Maryanski Janet L.
Published: July 2002

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.
Altered HOX and WNT7A expression in human lung cancer
Authors: Calvo Roser , West James , Franklin Wilbur , Erickson Paul , Bemis Lynne , Li Efang , Helfrich Barbara , Bunn Paul , Roche Joelle , Brambilla Elisabeth , Rosell Rafael , Gemmill Robert M. , Drabkin Harry A.
Published: July 2002

HOX genes encode transcription factors that control
patterning and cell fates. Alterations in HOX expression have been
clearly implicated in leukemia, but their role in most other malignant
diseases remains unknown. By using degenerate reverse
transcription–PCR and subsequent real-time quantitative assays, we
examined HOX expression in lung cancer cell lines, direct tumor-control
pairs, and bronchial epithelial cultures. As in leukemia, genes of the
HOX9 paralogous group and HOXA10 were frequently overexpressed. For
HOXB9, we confirmed that elevated RNA was associated with protein
overexpression. In some cases, marked HOX overexpression was associated
with elevated FGF10 and FGF17. During development, the WNT pathway
affects cell fate, polarity, and proliferation, and WNT7a has been
implicated in the maintenance of HOX expression. In contrast to normal
lung and mortal short-term bronchial epithelial cultures, WNT7a was
frequently reduced or absent in lung cancers. In immortalized bronchial
epithelial cells, WNT7a was lost concomitantly with HOXA1, and a
statistically significant correlation between the expression of both
genes was observed in lung cancer cell lines. Furthermore, we
identified a homozygous deletion of β-catenin in the mesothelioma,
NCI-H28, associated with reduced WNT7a and the lowest overall cell line
expression of HOXA1, HOXA7, HOXA9, and HOXA10, whereas HOXB9 levels
were unaffected. Of note, both WNT7a and β-catenin are encoded on
chromosome 3p, which undergoes frequent loss of heterozygosity in these
tumors. Our results suggest that alterations in regulatory circuits
involving HOX, WNT, and possibly fibroblast growth factor pathways
occur frequently in lung cancer.
Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task
Authors: Karlstrom Amelie , Zhong Guofu , Rader Christoph , Larsen Nicholas A. , Heine Andreas , Fuller Roberta , List Benjamin , Tanaka Fujie , Wilson Ian A. , Barbas Carlos F. , Lerner Richard A.
Published: July 2002

Catalytic aldolase antibodies generated by immunization with two different, but structurally related, β-diketone haptens were cloned and sequenced to study similarities and differences between independently evolved catalysts. Kinetic and sequence analysis coupled with mutagenesis, structural, and modeling studies reveal that the defining event in the evolution of these catalysts was a somatic mutation that placed a lysine residue in a deep, yet otherwise unrefined, hydrophobic pocket. We suggest that covalent chemistries may be as readily selected from the immune repertoire as the traditional noncovalent interactions that have formed the basis of immunochemistry until this time. Further, we believe that these experiments recapitulate the defining events in the evolution of nature's enzymes, particularly as they relate to chemical mechanism, catalytic promiscuity, and gene duplication.