bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart
Authors: Gutkowska Jolanta , Jankowski Marek , Lambert Chantal , Mukaddam-Daher Suhayla , Zingg Hans H. , McCann Samuel M.
Published: July 2002

Previous studies indicated that the central nervous system induces release of the cardiac hormone atrial natriuretic peptide (ANP) by release of oxytocin from the neurohypophysis. The presence of specific transcripts for the oxytocin receptor was demonstrated in all chambers of the heart by amplification of cDNA by the PCR using specific oligonucleotide primers. Oxytocin receptor mRNA content in the heart is 10 times lower than in the uterus of female rats. Oxytocin receptor transcripts were demonstrated by
in situ
hybridization in atrial and ventricular sections and confirmed by competitive binding assay using frozen heart sections. Perfusion of female rat hearts for 25 min with Krebs–Henseleit buffer resulted in nearly constant release of ANP. Addition of oxytocin (10
−6
M) significantly stimulated ANP release, and an oxytocin receptor antagonist (10
−7
and 10
−6
M) caused dose-related inhibition of oxytocin-induced ANP release and in the last few minutes of perfusion decreased ANP release below that in control hearts, suggesting that intracardiac oxytocin stimulates ANP release. In contrast, brain natriuretic peptide release was unaltered by oxytocin. During perfusion, heart rate decreased gradually and it was further decreased significantly by oxytocin (10
−6
M). This decrease was totally reversed by the oxytocin antagonist (10
−6
M) indicating that oxytocin released ANP that directly slowed the heart, probably by release of cyclic GMP. The results indicate that oxytocin receptors mediate the action of oxytocin to release ANP, which slows the heart and reduces its force of contraction to produce a rapid reduction in circulating blood volume.
Evaluating treatment protocols to prevent antibiotic resistance
Authors: Bonhoeffer Sebastian , Lipsitch Marc , Levin Bruce R.
Published: July 2002

The spread of bacteria resistant to antimicrobial agents calls for population-wide treatment strategies to delay or reverse the trend toward antibiotic resistance. Here we propose new criteria for the evaluation of the population-wide effects of treatment protocols for directly transmitted bacterial infections and discuss different usage patterns for single and multiple antibiotic therapy. A mathematical model suggests that the long-term benefit of single drug treatment from introduction of the antibiotic until a high frequency of resistance precludes its use is almost independent of the pattern of antibiotic use. When more than one antibiotic is employed, sequential use of different antibiotics in the population (“cycling”) is always inferior to treatment strategies where, at any given time, equal fractions of the population receive different antibiotics. However, treatment of all patients with a combination of antibiotics is in most cases the optimal treatment strategy.
Cerebral cortical astroglia from the trisomy 16 mouse, a model for Down syndrome, produce neuronal cholinergic deficits in cell culture
Authors: Nelson P. G. , Fitzgerald S. , Rapoport S. I. , Neale E. A. , Galdzicki Z. , Dunlap V. , Bowers L. , v. Agoston D.
Published: July 2002

Trisomy 21 (Down syndrome) is associated with a high incidence of Alzheimer disease and with deficits in cholinergic function in humans. We used the trisomy 16 (Ts16) mouse model for Down syndrome to identify the cellular basis for the cholinergic dysfunction. Cholinergic neurons and cerebral cortical astroglia, obtained separately from Ts16 mouse fetuses and their euploid littermates, were cultured in various combinations. Choline acetyltransferase activity and cholinergic neuron number were both depressed in cultures in which both neurons and glia were derived from Ts16 fetuses. Cholinergic function of normal neurons was significantly down-regulated by coculture with Ts16 glia. Conversely, neurons from Ts16 animals could express normal cholinergic function when grown with normal glia. These observations indicate that astroglia may contribute strongly to the abnormal cholinergic function in the mouse Ts16 model for Down syndrome. The Ts16 glia could lack a cholinergic supporting factor present in normal glia or contain a factor that down-regulates cholinergic function.
Computer-based analysis of the binding steps in protein complex formation
Authors: Bray Dennis , Lay Steven
Published: July 2002

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and
K
d
s and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.
Direct phase determination in protein electron crystallography: The pseudo-atom approximation
Authors: Dorset Douglas L.
Published: July 2002

The crystal structure of halorhodopsin is determined directly in its centrosymmetric projection using 6.0-Å-resolution electron diffraction intensities, without including any previous phase information from the Fourier transform of electron micrographs. The potential distribution in the projection is assumed
a priori
to be an assembly of globular densities. By an appropriate dimensional re-scaling, these “globs” are then assumed to be pseudo-atoms for normalization of the observed structure factors. After this treatment, the structure is determined directly by conventional direct methods, followed by Fourier refinement, leading to a mean phase deviation of only 20° (from the values originally found from the image transform) for the 45 most intense reflections.
Structural basis for dual excitation and photoisomerization of the
Aequorea victoria
green fluorescent protein
Authors: Brejc Katjuša , Sixma Titia K. , Kitts Paul A. , Kain Steven R. , Tsien Roger Y. , Ormö Mats , Remington S. James
Published: July 2002

The 2.1-Å resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 → Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore’s excited state to Glu-222. Loss of the Ser-205–Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.
In vitro
evolution of terminal protein-containing genomes
Authors: Esteban José A. , Blanco Luis , Villar Laurentino , Salas Margarita
Published: July 2002

A new self-sustained terminal protein-primed DNA amplification system has been used to describe
in vitro
evolutionary changes affecting maintenance of the genome size of bacteriophage φ29. These changes involve generation and efficient amplification of short palindromic molecules containing an inverted duplication of one of the original DNA ends. A template-switching mechanism is proposed to account for the appearance of these molecules. After their formation, they would replicate by means of hairpin intermediates. Relevant kinetic information about this DNA replication system has been obtained from the competition between the input full-length φ29 DNA and its derived truncated versions. The physiological relevance of these molecules and the mechanisms to control their formation are discussed.
Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells
Authors: Cui Xiao-Lan , Douglas Janice G.
Published: July 2002

In kidney epithelial cells, arachidonic acid and other fatty acids are important signal transduction molecules for G protein-coupled receptors. We now demonstrate that arachidonic acid induced a time- and dose-dependent activation of JNK, a member of the mitogen-activated protein kinase family, as assessed by phosphorylation of the transcription factor ATF-2. Increments in JNK activity were detectable at 5 μM arachidonic acid and plateaued at 30 μM. Activation was specific to arachidonic acid and linoleic acid, since other fatty acids of then− 3 andn− 6 series and/or various degrees of saturation were without effect. Specific inhibitors of cyclooxygenase-, lipoxygenase-, and cytochrome P450-dependent metabolism did not affect arachidonic acid-induced JNK activity. We further demonstrated that the free radical scavengerN-acetylcysteine blocked arachidonic acid-induced JNK activation, while H2O2, a reactive oxidative molecule, activated JNK in a dose-dependent manner, providing additional support for a redox mechanism. Moreover, arachidonic acid activated NADPH oxidase (EC 1.6.-.-, EC 1.6.99.-) in a dose-dependent manner, and the potency of superoxide generation paralleled that of JNK activation by other fatty acids. We conclude that in kidney epithelial cells arachidonic acid activates JNK by means of NADPH oxidase and superoxide generation, independent of eicosanoid biosynthesis.
Gypsy retrotransposon as a tool for the
in vivo
analysis of the regulatory region of the
optomotor-blind
gene in
Drosophila
Authors: Tsai Shih-Feng , Jang Chuen-Chuen , Prikhod’ko Grigoriy G. , Bessarab Dmitri A. , Tang Chiou-Yang , Pflugfelder Gert O. , Sun Y. Henry
Published: July 2002

We report here a method for the
in vivo
dissection of the regulatory region of a gene in the
Drosophila
genome. Our system includes (
i
) the reporter genes
lacZ
and
white
to detect transcriptional enhancer and silencer activities in a target gene, (
ii
) an efficient way to induce integration of gypsy elements in the genome, and (
iii
) unidirectional blocking of regulatory activities by the gypsy element, which is dependent on the
su
(
Hw
) protein. The
optomotor-blind
(
omb
) gene was analyzed. In the
omb
P1
line, a P[
lacW
] construct is inserted about 1.4 kb upstream of the
omb
transcription start site. The
lacZ
reporter gene within P[
lacW
] exhibits the same expression pattern as
omb
. The
white
reporter gene is expressed in a “bipolar” pattern. We induced high frequency gypsy mobilization in
omb
P1
and identified two lines (D11 and D13–1) with altered eye pigmentation pattern, which is dependent on
su
(
Hw
) activity. A gypsy element was found inserted in the first intron of
omb
in D13–1 and in P[
lacW
] in D11. These results indicate that it is the blocking of regulatory activities by gypsy that caused the changes in the
white
reporter gene expression. The effect of these gypsy insertions on the expression patterns allowed us to predict several aspects of the organization of the regulatory elements in the
omb
locus.
The microwave background anisotropies: Observations
Authors: Wilkinson David
Published: July 2002

Most cosmologists now believe that we live in an evolving universe
that has been expanding and cooling since its origin about 15 billion
years ago. Strong evidence for this standard cosmological model comes
from studies of the cosmic microwave background radiation (CMBR), the
remnant heat from the initial fireball. The CMBR spectrum is blackbody,
as predicted from the hot Big Bang model before the discovery of the
remnant radiation in 1964. In 1992 the cosmic background explorer
(COBE) satellite finally detected the anisotropy of the
radiation—fingerprints left by tiny temperature fluctuations in the
initial bang. Careful design of the COBE satellite, and a bit of luck,
allowed the 30 μK fluctuations in the CMBR temperature (2.73 K) to be
pulled out of instrument noise and spurious foreground emissions.
Further advances in detector technology and experiment design are
allowing current CMBR experiments to search for predicted features in
the anisotropy power spectrum at angular scales of 1° and smaller. If
they exist, these features were formed at an important epoch in the
evolution of the universe—the decoupling of matter and radiation at a
temperature of about 4,000 K and a time about 300,000 years after the
bang. CMBR anisotropy measurements probe directly some detailed physics
of the early universe. Also, parameters of the cosmological model can
be measured because the anisotropy power spectrum depends on
constituent densities and the horizon scale at a known cosmological
epoch. As sophisticated experiments on the ground and on balloons
pursue these measurements, two CMBR anisotropy satellite missions are
being prepared for launch early in the next century.
Primordial nucleosynthesis
Authors: Schramm David N.
Published: July 2002

With the advent of the new extragalactic deuterium observations,
Big Bang nucleosynthesis (BBN) is on the verge of undergoing a
transformation. In the past, the emphasis has been on demonstrating the
concordance of the BBN model with the abundances of the light isotopes
extrapolated back to their primordial values by using stellar and
galactic evolution theories. As a direct measure of primordial
deuterium is converged upon, the nature of the field will shift to
using the much more precise primordial D/H to constrain the more
flexible stellar and galactic evolution models (although the question
of potential systematic error in
4
He abundance
determinations remains open). The remarkable success of the theory to
date in establishing the concordance has led to the very robust
conclusion of BBN regarding the baryon density. This robustness remains
even through major model variations such as an assumed first-order
quark-hadron phase transition. The BBN constraints on the cosmological
baryon density are reviewed and demonstrate that the bulk of the
baryons are dark and also that the bulk of the matter in the universe
is nonbaryonic. Comparison of baryonic density arguments from Lyman-α
clouds, x-ray gas in clusters, and the microwave anisotropy are made.
Calculated
11
B–
13
C NMR chemical shift relationship in hypercoordinate methonium and boronium ions
Authors: Rasul Golam , Prakash G. K. Surya , Olah George A.
Published: July 2002

The boronium-carbonium continuum was extended to include hypercoordinated protonated methanes and their boron analogs. The
11
B NMR chemical shifts of the hypercoordinated hydriodo boron compounds and the
13
C NMR chemical shifts of the corresponding isoelectronic and isostructural carbocations were calculated by using the GIAO-MP2 method. The data show good linear correlation between
11
B and
13
C NMR chemical shifts, which indicates that the same factors that determine the chemical shifts of the boron nuclei also govern the chemical shifts of carbon nuclei of these hypercoordinated hydriodo compounds.
Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia
Authors: Yan Shi-Fang , Zou Yu Shan , Gao Yun , Zhai Chao , Mackman Nigel , Lee Stephen L. , Milbrandt Jeffrey , Pinsky David , Kisiel Walter , Stern David
Published: July 2002

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both
in vitro
and
in vivo
, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO
2
≈13 torr) had increased levels of tissue factor transcripts (≈18-fold) and an increased rate of transcription (≈15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; −111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using
32
P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.
The evolution of interspecific mutualisms
Authors: Doebeli Michael , Knowlton Nancy
Published: July 2002

Interspecific mutualisms are widespread, but how they evolve is not clear. The Iterated Prisoner’s Dilemma is the main theoretical tool to study cooperation, but this model ignores ecological differences between partners and assumes that amounts exchanged cannot themselves evolve. A more realistic model incorporating these features shows that strategies that succeed with fixed exchanges (e.g., Tit-for-Tat) cannot explain mutualism when exchanges vary because the amount exchanged evolves to 0. For mutualism to evolve, increased investments in a partner must yield increased returns, and spatial structure in competitive interactions is required. Under these biologically plausible assumptions, mutualism evolves with surprising ease. This suggests that, contrary to the basic premise of past theoretical analyses, overcoming a potential host’s initial defenses may be a bigger obstacle for mutualism than the subsequent recurrence and spread of noncooperative mutants.
Knockout of the abetalipoproteinemia gene in mice: Reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes
Authors: Raabe Martin , Flynn Laura M. , Zlot Constance H. , Wong Jinny S. , Véniant Murielle M. , Hamilton Robert L. , Young Stephen G.
Published: July 2002

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (
Mttp
). In heterozygous knockout mice (
Mttp
+/− ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (
Mttp
+/+), chow-fed
Mttp
+/− mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the
Mttp
+/− mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in
Mttp
+/+ mice. Both the livers of adult
Mttp
+/− mice and the visceral endoderm of the yolk sacs from
Mttp
+/− embryos manifested an accumulation of cytosolic fat. All homozygous embryos (
Mttp
−/−) died during embryonic development. In the visceral endoderm of
Mttp
−/− yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.
Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: Caveolin-2 is up-regulated in response to cell injury
Authors: Galbiati Ferruccio , Volonté Daniela , Gil Orlando , Zanazzi George , Salzer James L. , Sargiacomo Massimo , Scherer Philipp E. , Engelman Jeffrey A. , Schlegel Amnon , Parenti Marco , Okamoto Takashi , Lisanti Michael P.
Published: July 2002

Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether caveolins are expressed within neuronal cells. Here we demonstrate the expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express caveolins.
Immediate and long-term effects of neonatal MK-801 treatment on nonspatial learning
Authors: Griesbach Grace S. , Amsel Abram
Published: July 2002

These experiments observed the immediate and long-term effects of neonatal treatment with MK-801 on patterned single alternation (PSA), a form of nonspatial, memory-based learning. Rat pups were injected daily on postnatal days (PND) 7–19, with MK-801 (MK
+
) or the less active isomer of MK-801 (MK
−
) (0.25 mg/kg), and trained at either PND 22 or 60. Rats treated with MK
+
or MK
−
and trained on PND 22 were significantly impaired in PSA when compared with the saline control. Beyond the learning impairment, MK
+
rats showed an overall decreased running speed during training. They also presented an array of abnormal behaviors and significant weight loss. These nonassociative variables were determined for several doses (0.025, 0.05, 0.1, 0.15, and 0.20 mg/kg) through PND days 22–25. Rats that received the threshold dose for secondary effects (0.025 mg/kg) also showed an overall decrease in running speed, but failed to show a significant nonspatial learning impairment on PSA. The PSA learning impairment was found to be not long lasting; rats trained at PND 60, after neonatally receiving the original high dose of MK-801, did not differ from controls.
A novel multicomponent regulatory system mediates H
2
sensing in
Alcaligenes eutrophus
Authors: Lenz Oliver , Friedrich Bärbel
Published: July 2002

Oxidation of molecular hydrogen catalyzed by [NiFe] hydrogenases is a widespread mechanism of energy generation among prokaryotes. Biosynthesis of the H
2
-oxidizing enzymes is a complex process subject to positive control by H
2
and negative control by organic energy sources. In this report we describe a novel signal transduction system regulating hydrogenase gene (
hox
) expression in the proteobacterium
Alcaligenes eutrophus
. This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA. HoxB and HoxC share characteristic features of dimeric [NiFe] hydrogenases and form the putative H
2
receptor that interacts directly or indirectly with the histidine protein kinase HoxJ*. A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H
2
-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H
2
. Whereas deletions in
hoxB
or
hoxC
abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level
hox
gene expression, indicating that the active sensor kinase exerts a negative effect on
hox
gene expression in the absence of H
2
. Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H
2
signal-transduction chain. Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation. The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.
Insulin-like growth factor I is a growth-promoting factor forLeishmaniapromastigotes and amastigotes
Authors: Goto H. , Gomes C. M. C. , Corbett C. E. P. , Monteiro H. P. , Gidlund M.
Published: July 2002

Leishmaniases are diseases caused by protozoa of the genusLeishmaniathat affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. TheLeishmaniaparasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth ofLeishmaniapromastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response inLeishmania (Leishmania) mexicanaamastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes ofL. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivatedLeishmania (Viannia) panamensisis increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in theLeishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.
Derepression of human embryonic ζ-globin promoter by a locus-control region sequence
Authors: Huang B.-L. , Fan-Chiang I. R. , Wen S. C. , Koo H.-C. , Kao W. Y. , Gavva N. R. , Shen C.-K. James
Published: July 2002

A multiple protein–DNA complex formed at a human α-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic ζ-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human ζ-globin promoter in fetal and adult transgenic mice. Furthermore, ζ-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human ζ-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.
The effects of health changes on projections of health service needs for the elderly population of the United States
Authors: Singer Burton H. , Manton Kenneth G.
Published: July 2002

The 1982–1994 National Long-Term Care Surveys indicate an accelerating decline in disability among the U.S. elderly population, suggesting that a 1.5% annual decline in chronic disability for elderly persons is achievable. Furthermore, many risk factors for chronic diseases show improvements, many linked to education, from 1910 to the present. Projections indicate the proportion of persons aged 85–89 with less than 8 years of education will decline from 65% in 1980 to 15% in 2015. Health and socioeconomic status trends are not directly represented in Medicare Trust Fund and Social Security Administration beneficiary projections. Thus, they may have different economic implications from projections directly accounting for health trends. A 1.5% annual disability decline keeps the support ratio (ratio of economically active persons aged 20–64 to the number of chronically disabled persons aged 65+) above its 1994 value, 22:1, when the Hospital Insurance Trust Fund was in fiscal balance, to 2070. With no changes in disability, projections indicate a support ratio in 2070 of 8:1—63% below a cash flow balance.
A nucleated assembly mechanism of Alzheimer paired helical filaments
Authors: Friedhoff P. , von Bergen M. , Mandelkow E.-M. , Davies P. , Mandelkow E.
Published: July 2002

Alzheimer’s disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the Aβ-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism. The PHF nucleus comprises about 8–14 tau monomers. A prerequisite for nucleation is the dimerization of tau because tau dimers act as effective building blocks. PHF assembly can be seeded by preformed filaments (made either
in vitro
or isolated from Alzheimer brain tissue). These results suggest that dimerization and nucleation are the rate-limiting steps for PHF formation
in vivo
.
Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/P
i
cotransporter
Authors: Pfister Markus F. , Ruf Isabelle , Stange Gerti , Ziegler Urs , Lederer Eleanor , Biber Jürg , Murer Heini
Published: July 2002

We have studied the involvement of proteolytic pathways in the regulation of the Na/P
i
cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine,
l
-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (
i
) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/P
i
cotransport and (
ii
) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/P
i
cotransport. Further analysis by subcellular fractionation and morphological techniques showed (
i
) that the Na/P
i
cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (
ii
) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.
Amine binding and oxidation at the catalytic site for photosynthetic water oxidation
Authors: Ouellette Anthony J. A. , Anderson Lorraine B. , Barry Bridgette A.
Published: July 2002

Photosynthetic water oxidation occurs at the Mn-containing catalytic site of photosystem II (PSII). By the use of
14
C-labeled amines and SDS-denaturing PAGE, covalent adducts derived from primary amines and the PSII subunits, CP47, D2/D1, and the Mn-stabilizing protein, can be observed. When PSII contains the 18- and 24-kDa extrinsic proteins, which restrict access to the active site, no
14
C labeling is obtained. NaCl, but not Na
2
SO
4
, competes with
14
C labeling in Mn-containing PSII preparations, and the concentration dependence of this competition parallels the activation of oxygen evolution. Formation of
14
C-labeled adducts is observed in the presence or in the absence of a functional manganese cluster. However, no significant Cl
−
effect on
14
C labeling is observed in the absence of the Mn cluster. Isolation and quantitation of the
14
C-labeled aldehyde product, produced from [
14
C]benzylamine, gives yields of 1.8 ± 0.3 mol/mol PSII and 2.9 ± 0.2 mol/mol in Mn-containing and Mn-depleted PSII, respectively. The corresponding specific activities are 0.40 ± 0.07 μmol(μmol PSII-hr)
−1
and 0.64 ± 0.04 μmol(μmol PSII-hr)
−1
. Cl
−
suppresses the production of [
14
C]benzaldehyde in Mn-containing PSII, but does not suppress the production in Mn-depleted preparations. Control experiments show that these oxidation reactions do not involve the redox-active tyrosines, D and Z. Our results suggest the presence of one or more activated carbonyl groups in protein subunits that form the active site of PSII.
Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity
Authors: Ritchie Sian , Gilroy Simon
Published: July 2002

The plant hormones abscisic acid (ABA) and gibberellic acid (GA) are important regulators of the dormancy and germination of seeds. In cereals, GA enhances the synthesis and secretion of enzymes (principally α-amylases) in the aleurone cells of the endosperm, which then mobilize the storage reserves that fuel germination. ABA inhibits this enhanced secretory activity and delays germination. Despite the central role of ABA in regulating germination, the signal transduction events leading to altered gene expression and cellular activity are essentially unknown. We report that the application of ABA to aleurone protoplasts increased the activity of the enzyme phospholipase D (PLD) 10 min after treatment. The product of PLD activity, phosphatidic acid (PPA), also increased transiently at this time. The application of PPA to aleurone protoplasts led to an ABA-like inhibition of α-amylase production, and induction of the ABA up-regulated proteins ASI (
a
mylase
s
ubtilisin
i
nhibitor) and RAB (
r
esponsive to
AB
A). Inhibition of PLD activity by 0.1% 1-butanol during the initial 20 min of ABA treatment resulted in inhibition of ABA-regulated processes. This inhibition coincided with the timing of PLD activation by ABA and was overcome by simultaneous addition of PPA. These results suggest that ABA activates the enzyme PLD to produce PPA that is involved in triggering the subsequent ABA responses of the aleurone cell.
Proliferation and phenotype regulation in the subventricular zone during experimental allergic encephalomyelitis:
In vivo
evidence of a role for nerve growth factor
Authors: Calzà Laura , Giardino Luciana , Pozza Monica , Bettelli Carla , Micera Alessandra , Aloe Luigi
Published: July 2002

Proliferating cells in the subventricular zone (SVZ) of adult rat brain could provide a source of cells for repair attempts during degenerative diseases. However, very few reports dealt with the spontaneous regulation of this cell population during experimental conditions. In this paper, we describe an increase in the proliferation activity in the SVZ during experimental allergic encephalomyelitis, a demyelinating disease widely used as an experimental model for human multiple sclerosis. Moreover, p75
LNGFR
-immunoreactive elements in the SVZ were larger in experimental allergic encephalomyelitis compared with control groups, and they also showed multiple and branched elongations. Finally, a selective uptake of
125
I-nerve growth factor was observed in the SVZ in neonatal rats, and positive elements migrated in the corpus callosum within a few days. These data indicate that cell populations in the SVZ are regulated during inflammatory conditions and degenerative diseases involving oligodendrocytes and neurotrophins, including nerve growth factor, could participate in these phenomena.
Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of
Acanthamoeba
myosin I heavy chain kinase
Authors: Szczepanowska Joanna , Ramachandran Umamaheswari , Herring Christopher J. , Gruschus James M. , Qin Jun , Korn Edward D. , Brzeska Hanna
Published: July 2002

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower
k
cat
and higher
K
m
for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.
Role of a critical water in scytalone dehydratase-catalyzed reaction
Authors: Zheng Ya-Jun , Bruice Thomas C.
Published: July 2002

Scytalone dehydratase (EC
4.2.1.94
) catalyzes the dehydration of two important intermediates in the biosynthesis of melanin, and it functions without metal ions or any cofactors. Using molecular orbital theory, we have examined the role of a critical water molecule in the mechanism of scytalone dehydratase. The water, together with an internal hydrogen bonding, contributes significantly to the stabilization of the transition state (or the enolate intermediate). The role of two active site tyrosines (Tyr-50 and Tyr-30) is (
i
) to hold the critical water in place so that it may stabilize the transition state without much structural rearrangement during the catalytic reaction, and (
ii
) to polarize the water, making it a better general acid. The stereochemistry of the scytalone dehydratase-catalyzed dehydration is also discussed.
Manipulation of the membrane binding site of vitamin K-dependent proteins: Enhanced biological function of human factor VII
Authors: Shah Amit M. , Kisiel Walter , Foster Donald C. , Nelsestuen Gary L.
Published: July 2002

Recent studies suggested that modification of the membrane contact site of vitamin K-dependent proteins may enhance the membrane affinity and function of members of this protein family. The properties of a factor VII mutant, factor VII-Q10E32, relative to wild-type factor VII (VII, containing P10K32), have been compared. Membrane affinity of VII-Q10E32 was about 20-fold higher than that of wild-type factor VII. The rate of autoactivation VII-Q10E32 with soluble tissue factor was 100-fold faster than wild-type VII and its rate of activation by factor Xa was 30 times greater than that of wild-type factor VII. When combined with soluble tissue factor and phospholipid, activated factor VII-Q10E32 displayed increased activation of factor X. Its coagulant activity was enhanced in all types of plasma and with all sources of tissue factor tested. This difference in activity (maximum 50-fold) was greatest when coagulation conditions were minimal, such as limiting levels of tissue factor and/or phospholipid. Because of its enhanced activity, factor VII-Q10E32 and its derivatives may provide important reagents for research and may be more effective in treatment of bleeding and/or clotting disorders.
The theoretical limits of DNA sequence discrimination by linked polyamides
Authors: Walker Wynn L. , Landaw Elliot M. , Dickerson Richard E. , Goodsell David S.
Published: July 2002

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (
i
) the optimal choice of base-binding specificity for each ring and (
ii
) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific “placeholder” ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.
β1- and α6-integrin are surface markers on mouse spermatogonial stem cells
Authors: Shinohara Takashi , Avarbock Mary R. , Brinster Ralph L.
Published: July 2002

Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to our understanding of the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. We made use of the known association of stem cells with basement membranes and our spermatogonial transplantation assay system to identify specific molecular markers on the stem cell surface. Selection of mouse testis cells with anti-β1- or anti-α6-integrin antibody, but not anti-c-kit antibody, produced cell populations with a significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. We demonstrate spermatogonial stem cell-associated antigens by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells suggests that these cells share elements of a common molecular machinery with stem cells in other tissues.
Bruton’s tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein
Authors: Yamadori Tomoki , Baba Yoshihiro , Matsushita Masato , Hashimoto Shoji , Kurosaki Mari , Kurosaki Tomohiro , Kishimoto Tadamitsu , Tsukada Satoshi
Published: July 2002

Bruton’s tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentiallyassociates withBtk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1,4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway.
Cortical auditory signal processing in poor readers
Authors: Nagarajan Srikantan , Mahncke Henry , Salz Talya , Tallal Paula , Roberts Timothy , Merzenich Michael M.
Published: July 2002

Magnetoencephalographic responses recorded from auditory cortex evoked by brief and rapidly successive stimuli differed between adults with poor vs. good reading abilities in four important ways. First, the response amplitude evoked by short-duration acoustic stimuli was stronger in the post-stimulus time range of 150–200 ms in poor readers than in normal readers. Second, response amplitude to rapidly successive and brief stimuli that were identical or that differed significantly in frequency were substantially weaker in poor readers compared with controls, for interstimulus intervals of 100 or 200 ms, but not for an interstimulus interval of 500 ms. Third, this neurological deficit closely paralleled subjects’ ability to distinguish between and to reconstruct the order of presentation of those stimulus sequences. Fourth, the average distributed response coherence evoked by rapidly successive stimuli was significantly weaker in the β- and γ-band frequency ranges (20–60 Hz) in poor readers, compared with controls. These results provide direct electrophysiological evidence supporting the hypothesis that reading disabilities are correlated with the abnormal neural representation of brief and rapidly successive sensory inputs, manifested in this study at the entry level of the cortical auditory/aural speech representational system(s).