bluuebunny/crossref_metadata_2025 huggingface dataset:
Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle.
Authors: Stiles J R , Van Helden D , Bartol T M , Salpeter E E , Salpeter M M
Published: July 2002

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20-80% rise time (tr) of approximately 80 micros at 22 degrees C, shorter than any previously reported values, and tr variability (SD) with an upper limit of 25-30 micros. Extracellular electrode pressure can increase tr and its variability by 2- to 3-fold. Using Monte Carlo simulations, we modeled passive acetylcholine diffusion through a vesicle fusion pore expanding radially at 25 nm x ms(-1) (rapid, from endplate omega figure appearance) or 0.275 nm x ms(-1) (slow, from mast cell exocytosis). Simulated mEPCs obtained with rapid expansion reproduced tr and the overall shape of our experimental mEPCs, and were similar to simulated mEPCs obtained with instant acetylcholine release. We conclude that passive transmitter diffusion, coupled with rapid expansion of the fusion pore, is sufficient to explain the time course of experimentally measured synaptic currents with trs of less than 100 micros.
Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors.
Authors: Xu R M , Carmel G , Kuret J , Cheng X
Published: July 2002

A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases. One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources. Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution. The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases. Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring. We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8. Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents.
Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.
Authors: Rettig J , Sheng Z H , Kim D K , Hodson C D , Snutch T P , Catterall W A
Published: July 2002

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.
Persistent confusion of total entropy and chemical system entropy in chemical thermodynamics.
Authors: Weber G
Published: July 2002

The change in free energy with temperature at constant pressure of a chemical reaction is determined by the sum (dS) of changes in entropy of the system of reagents, dS(i), and the additional entropy change of the surroundings, dS(H), that results from the enthalpy change, W. A faulty identification of the total entropy change on reaction with dS(i) has been responsible for the attribution of general validity to the expressions (d deltaG/dT)p = -deltaS(i) and d(deltaG/T)/d(1/T)= deltaH, which are found in most textbooks and in innumerable papers.
An interspecies hybrid RNA virus is significantly more virulent than either parental virus.
Authors: Ding S W , Shi B J , Li W X , Symons R H
Published: July 2002

Cucumber mosaic cucumovirus (CMV) infects a very wide range of plant species (>1000 species). We recently demonstrated that a previously undescribed gene (2b) encoded by RNA 2 of the tripartite RNA genome of CMV is required for systemic virus spread and disease induction in its hosts. Herein we report that when this CMV gene is replaced by its homologue from tomato aspermy cucumovirus (TAV), the resultant hybrid virus is significantly more virulent, induces earlier onset of systemic symptoms, and accumulates to a higher level in seven host species from three families than either of the parents. Our results indicate that CMV and the TAV 2b protein interact synergistically despite the fact that no synergism occurs in double infections with the two parental viruses. To our knowledge, this is the first example of an interspecific hybrid made from plant or animal RNA viruses that is more efficient in systemic infection of a number of hosts than the naturally occurring parents. As CMV and the hybrid virus accumulated to a similar level in the infected tobacco protoplasts, the observed synergistic responses most likely resulted from an increased efficacy of the hybrid virus in systemic spread in host plants provided by the TAV 2b protein. The relevance of our finding to the application of pathogen-derived resistance is discussed.
Formation of junctions involved in excitation-contraction coupling in skeletal and cardiac muscle.
Authors: Flucher B E , Franzini-Armstrong C
Published: July 2002

During excitation-contraction (e-c) coupling of striated muscle, depolarization of the surface membrane is converted into Ca2+ release from internal stores. This process occurs at intracellular junctions characterized by a specialized composition and structural organization of membrane proteins. The coordinated arrangement of the two key junctional components--the dihydropyridine receptor (DHPR) in the surface membrane and the ryanodine receptor (RyR) in the sarcoplasmic reticulum--is essential for their normal, tissue-specific function in e-c coupling. The mechanisms involved in the formation of the junctions and a potential participation of DHPRs and RyRs in this process have been subject of intensive studies over the past 5 years. In this review we discuss recent advances in understanding the organization of these molecules in skeletal and cardiac muscle, as well as their concurrent and independent assembly during development of normal and mutant muscle. From this information we derive a model for the assembly of the junctions and the establishment of the precise structural relationship between DHPRs and RyRs that underlies their interaction in e-c coupling.
Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.
Authors: Symes K , Mercola M
Published: July 2002

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.
Schizophrenia and cognitive dysmetria: a positron-emission tomography study of dysfunctional prefrontal-thalamic-cerebellar circuitry.
Authors: Andreasen N C , O'Leary D S , Cizadlo T , Arndt S , Rezai K , Ponto L L , Watkins G L , Hichwa R D
Published: July 2002

Patients suffering from schizophrenia display subtle cognitive abnormalities that may reflect a difficulty in rapidly coordinating the steps that occur in a variety of mental activities. Working interactively with the prefrontal cortex, the cerebellum may play a role in coordinating both motor and cognitive performance. This positron-emission tomography study suggests the presence of a prefrontal-thalamic-cerebellar network that is activated when normal subjects recall complex narrative material, but is dysfunctional in schizophrenic patients when they perform the same task. These results support a role for the cerebellum in cognitive functions and suggest that patients with schizophrenia may suffer from a "cognitive dysmetria" due to dysfunctional prefrontal-thalamic-cerebellar circuitry.
Mismatch repair in Xenopus egg extracts: DNA strand breaks act as signals rather than excision points.
Authors: Varlet I , Canard B , Brooks P , Cerovic G , Radman M
Published: July 2002

In Xenopus egg extracts, DNA strand breaks (nicks) located 3' or 5' to a mismatch cause an overall 3-fold stimulation of the repair of the mismatch in circular heteroduplex DNA molecules. The increase in mismatch repair is almost entirely due to an increase in repair of the nicked strand, which is stimulated 5-fold. Repair synthesis is centered to the mismatch site, decreases symmetrically on both sides, and its position is not significantly altered by the presence of the nick. Therefore, it appears that in the Xenopus germ cells, the mismatch repair system utilizes nicks as signals for the induction and direction of mismatch repair, but not as the start or end point for excision and resynthesis.
Island colonization and evolution of the insular woody habit in Echium L. (Boraginaceae).
Authors: Böhle U R , Hilger H H , Martin W F
Published: July 2002

Numerous island-inhabiting species of predominantly herbaceous angiosperm genera are woody shrubs or trees. Such "insular woodiness" is strongly manifested in the genus Echium, in which the continental species of circummediterranean distribution are herbaceous, whereas endemic species of islands along the Atlantic coast of north Africa are woody perennial shrubs. The history of 37 Echium species was traced with 70 kb of noncoding DNA determined from both chloroplast and nuclear genomes. In all, 239 polymorphic positions with 137 informative sites, in addition to 27 informative indels, were found. Island-dwelling Echium species are shown to descend from herbaceous continental ancestors via a single island colonization event that occurred < 20 million years ago. Founding colonization appears to have taken place on the Canary Islands, from which the Madeira and Cape Verde archipelagos were invaded. Colonization of island habitats correlates with a recent origin of perennial woodiness from herbaceous habit and was furthermore accompanied by intense speciation, which brought forth remarkable diversity of forms among contemporary island endemics. We argue that the origin of insular woodiness involved response to counter-selection of inbreeding depression in founding island colonies.
A synaptic basis for memory storage in the cerebral cortex
Authors: Bear Mark F.
Published: July 2002

A cardinal feature of neurons in the cerebral cortex is stimulus selectivity, and experience-dependent shifts in selectivity are a common correlate of memory formation. We have used a theoretical “learning rule,” devised to account for experience-dependent shifts in neuronal selectivity, to guide experiments on the elementary mechanisms of synaptic plasticity in hippocampus and neocortex. These experiments reveal that many synapses in hippocampus and neocortex are bidirectionally modifiable, that the modifications persist long enough to contribute to long-term memory storage, and that key variables governing the sign of synaptic plasticity are the amount of NMDA receptor activation and the recent history of cortical activity.
A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the
Euprymna–Vibrio
mutualism
Authors: Weis Virginia M. , Small Andrea L. , McFall-Ngai Margaret J.
Published: July 2002

Many animal–bacteria cooperative associations occur in highly
modified host organs that create a unique environment for housing and
maintaining the symbionts. It has been assumed that these specialized
organs develop through a program of symbiosis-specific or -enhanced
gene expression in one or both partners, but a clear example of this
process has been lacking. In this study, we provide evidence for the
enhanced production of an enzyme in the symbiotic organ of the squid
Euprymna scolopes
, which harbors a culture of the
luminous bacterium
Vibrio fischeri
. Our data show that
this enzyme has a striking biochemical similarity to mammalian
myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine
peroxidase that occurs in neutrophils. MPO and the squid peroxidase
catalyze the same reaction, have similar apparent subunit molecular
masses, and a polyclonal antibody to native human MPO specifically
localized a peroxidase-like protein to the bacteria-containing regions
of the symbiotic organ. We also provide evidence that a previously
described squid cDNA encodes the protein (LO4) that is responsible for
the observed dianisidine peroxidase activity. An antibody made against
a fragment of LO4 immunoprecipitated dianisidine peroxidase activity
from extracts of the symbiotic organ, and reacted against these
extracts and human MPO in Western blot analysis. These data suggest
that related biochemical mechanisms for the control of bacterial number
and growth operate in associations that are as functionally diverse as
pathogenesis and mutualism, and as phylogenetically distant as molluscs
and mammals.
Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene
Authors: Grether-Beck Susanne , Olaizola-Horn Sylvia , Schmitt Heidi , Grewe Markus , Jahnke Andreas , Johnson Judith P. , Briviba Karlis , Sies Helmut , Krutmann Jean
Published: July 2002

UVA radiation is the major component of the UV solar spectrum that
reaches the earth, and the therapeutic application of UVA radiation is
increasing in medicine. Analysis of the cellular effects of UVA
radiation has revealed that exposure of human cells to UVA radiation at
physiological doses leads to increased gene expression and that this
UVA response is primarily mediated through the generation of singlet
oxygen. In this study, the mechanisms by which UVA radiation induces
transcriptional activation of the human intercellular adhesion molecule
1 (ICAM-1) were examined. UVA radiation was capable of inducing
activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and
protein expression. These UVA radiation effects were inhibited by
singlet oxygen quenchers, augmented by enhancement of singlet oxygen
life-time, and mimicked in unirradiated cells by a singlet
oxygen-generating system. UVA radiation as well as singlet
oxygen-induced ICAM-1 promoter activation required activation of the
transcription factor AP-2. Accordingly, both stimuli activated AP-2,
and deletion of the putative AP-2-binding site abrogated ICAM-1
promoter activation in this system. This study identified the AP-2 site
as the UVA radiation- and singlet oxygen-responsive element of the
human ICAM-1 gene. The capacity of UVA radiation and/or singlet
oxygen to induce human gene expression through activation of AP-2
indicates a previously unrecognized role of this transcription factor
in the mammalian stress response.
A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.
Authors: Cho J W , Partin J S , Lennarz W J
Published: July 2002

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCo3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.
Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody-interleukin 2 fusion proteins.
Authors: Becker J C , Pancook J D , Gillies S D , Mendelsohn J , Reisfeld R A
Published: July 2002

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.
Occurrence of poly(alpha2,8-deaminoneuraminic acid) in mammalian tissues: widespread and developmentally regulated but highly selective expression on glycoproteins.
Authors: Ziak M , Qu B , Zuo X , Zuber C , Kanamori A , Kitajima K , Inoue S , Inoue Y , Roth J
Published: July 2002

In tissues of higher organisms homopolymers of alpha2,8-linked N-acetylneuraminic acid can be found as a posttranslational modification on selected proteins. We report here the discovery of homopolymers of alpha2,8-linked deaminoneuraminic acid [poly(alpha2,8-KDN)] in various tissues derived from all three germ layers in vertebrates including mammals. The monoclonal antibody kdn8kdn in conjunction with a bacterial KDNase permitted the detection of poly(alpha2,8-KDN) by immunohistochemistry and immunoblotting. Further evidence for the existence of poly(alpha2,8-KDN) was obtained by gas/liquid chromatography. The poly(alpha2,8-KDN) glycan was detectable in all tissues studied with the exception of mucus-producing cells present in various organs, the extracellular matrix, and basement membranes. However, in certain organs such as muscle, kidney, lung, and brain its expression was developmentally regulated. Despite its widespread tissue distribution, the poly(alpha2,8-KDN) glycan was detected on a single 150-kDa glycoprotein except for a single >350-kDa glycoprotein in kidney, which makes it most distinctive among polysialic acids. The ubiquitous yet selective expression may be indicative of a general function of the poly(alpha2,8-KDN)-bearing glycoproteins.
Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation.
Authors: Cullinan E B , Abbondanzo S J , Anderson P S , Pollard J W , Lessey B A , Stewart C L
Published: July 2002

The uterine expression of leukemia inhibitory factor (LIF) is essential for embryo implantation in the mouse. Here, we describe the expression of LIF, related members of this group of cytokines, oncostatin M and ciliary neurotrophic factor, and the LIF receptor beta and glycoprotein gp130 in normal human tissues and in the endometrium of fertile women. Our results show that LIF is the only one of these factors expressed at detectable levels in the endometrium of women of proven fertility. LIF expression is restricted to the endometrial glands during the secretory/postovulatory phase but is not present in the endometrium during the proliferative/preovulatory phase. The LIF receptor beta is expressed during the proliferative and secretory phases of the cycle and is restricted to the luminal epithelium. The associated signal-transducing component of the LIF receptor, gp130, is also expressed in both the luminal and glandular epithelium throughout the cycle. These results suggest that uterine expression of LIF in humans, like mice, may have a role in regulating embryo implantation, possibly through an autocrine/paracrine interaction between LIF and its receptor at the luminal epithelium.
The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.
Authors: Lin N S , Lee Y S , Lin B Y , Lee C W , Hsu Y H
Published: July 2002

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.
Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2.
Authors: Molina H , Holers V M , Li B , Fung Y , Mariathasan S , Goellner J , Strauss-Schoenberger J , Karr R W , Chaplin D D
Published: July 2002

Complement receptor 1 (CR1, CD35) and complement receptor 2 (CR2, CD21) have been implicated as regulators of B-cell activation. We explored the role of these receptors in the development of humoral immunity by generating CR1- and CR2-deficient mice using gene-targeting techniques. These mice have normal basal levels of IgM and of IgG isotypes. B- and T-cell development are overtly normal. Nevertheless, B-cell responses to low and high doses of a T-cell-dependent antigen are impaired with decreased titers of antigen-specific IgM and IgG isotypes. This defect is not complete because there is still partial activation of B lymphocytes during the primary immune response, with generation of splenic germinal centers and a detectable, although reduced, secondary antibody response. These data suggest that certain T-dependent antigens manifest an absolute dependence on complement receptors for the initiation of a normally robust immune response.
N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.
Authors: Hell J W , Westenbroek R E , Breeze L J , Wang K K , Chavkin C , Catterall W A
Published: July 2002

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.
The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia
Authors: Pantaleo Giuseppe , Demarest James F. , Schacker Timothy , Vaccarezza Mauro , Cohen Oren J. , Daucher Marybeth , Graziosi Cecilia , Schnittman Steven S. , Quinn Thomas C. , Shaw George M. , Perrin Luc , Tambussi Giuseppe , Lazzarin Adriano , Sekaly Rafick P. , Soudeyns Hugo , Corey Lawrence , Fauci Anthony S.
Published: July 2002

Following infection of the host with a virus, the delicate balance between virus replication/spread and the immune response to the virus determines the outcome of infection, i.e., persistence versus elimination of the virus. It is unclear, however, what relative roles immunologic and virologic factors play during primary viral infection in determining the subsequent clinical outcome. By studying a cohort of subjects with primary HIV infection, it has been demonstrated that qualitative differences in the primary immune response to HIV, but not quantitative differences in the initial levels of viremia are associated with different clinical outcomes.
Mechanism of protein remodeling by ClpA chaperone
Authors: Pak Marie , Wickner Sue
Published: July 2002

ClpA, a newly discovered ATP-dependent molecular chaperone, remodels bacteriophage P1 RepA dimers into monomers, thereby activating the latent specific DNA binding activity of RepA. We investigated the mechanism of the chaperone activity of ClpA by dissociating the reaction into several steps and determining the role of nucleotide in each step. In the presence of ATP or a nonhydrolyzable ATP analog, the initial step is the self-assembly of ClpA and its association with inactive RepA dimers. ClpA-RepA complexes form rapidly and at 0°C but are relatively unstable. The next step is the conversion of unstable ClpA-RepA complexes into stable complexes in a time- and temperature-dependent reaction. The transition to stable ClpA-RepA complexes requires binding of ATP, but not ATP hydrolysis, because nonhydrolyzable ATP analogs satisfy the nucleotide requirement. The stable complexes contain approximately 1 mol of RepA dimer per mol of ClpA hexamer and are committed to activating RepA. In the last step of the reaction, active RepA is released upon exchange of ATP with the nonhydrolyzable ATP analog and ATP hydrolysis. Importantly, we discovered that one cycle of RepA binding to ClpA followed by ATP-dependent release is sufficient to convert inactive RepA to its active form.
Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1
Authors: Sun Li Jing , Peterson Blake R. , Verdine Gregory L.
Published: July 2002

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended throughin vitrobinding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.
Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation
Authors: Lee Robert K. K. , Araki Wataru , Wurtman Richard J.
Published: July 2002

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.
Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1
Authors: Yusibov Vidadi , Modelska Anna , Steplewski Klaudia , Agadjanyan Michail , Weiner David , Hooper D. Craig , Koprowski Hilary
Published: July 2002

The coat protein (CP) of alfalfa mosaic virus was used as a carrier molecule to express antigenic peptides from rabies virus and HIV. The antigens were separately cloned into the reading frame of alfalfa mosaic virus CP and placed under the control of the subgenomic promoter of tobacco mosaic virus CP in the 30BRz vector. The
in vitro
transcripts of recombinant virus with sequences encoding the antigenic peptides were synthesized from DNA constructs and used to inoculate tobacco plants. The plant-produced protein (virus particles) was purified and used for immunization of mice. Both antigens elicited specific virus-neutralizing antibodies in immunized mice.
The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive T cellsin situ
Authors: Chesney Jason , Bacher Michael , Bender Armin , Bucala Richard
Published: July 2002

Recent studies have identified a novel population of blood-borne cells, termed fibrocytes, that have a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and synthesize connective tissue matrix molecules. We found by flow cytometry that purified human fibrocytes express each of the known surface components that are required for antigen presentation, including class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR), the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. Human fibrocytes induced antigen-presenting cell-dependent T cell proliferation when cultured with specific antigen and this proliferative activity was significantly higher than that induced by monocytes and nearly as high as that induced by purified dendritic cells. Mouse fibrocytes also were found to express the surface components required for antigen presentation and to function as potent APCsin vitro. Mouse fibrocytes pulsedin vitrowith the HIV-proteins p24 or gp120 and delivered to a site of cutaneous injury were found to migrate to proximal lymph nodes and to specifically prime naive T cells. These data suggest that fibrocytes play an early and important role in the initiation of antigen-specific immunity.
Construction and characterization of a reovirus double temperature-sensitive mutant
Authors: Roner Michael R. , Nepliouev Igor , Sherry Barbara , Joklik Wolfgang K.
Published: July 2002

The infectious reovirus RNA system was used to construct a mutant with two temperature-sensitive (ts) lesions in genome segments M2 and S2, respectively. The double mutant is about 300 times more ts than either of its parents, which are about 1,500 and 170 times more ts than their wild-type parent reovirus ST3 strain Dearing. At 39°C the double mutant is essentially unable to multiply. In spite of its striking temperature sensitivity, the double mutant elicits the formation of significant amounts of neutralizing antibodies in newborn mice. Possible mechanisms responsible for this are discussed, as is the significance of this double ts mutant in relation to current searches for safe and efficient vaccine strains.
Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma
Authors: Helin Kristian , Holm Karin , Niebuhr Anita , Eiberg Hans , Tommerup Niels , Hougaard Susanne , Poulsen Hans Skovgaard , Spang-Thomsen Mogens , Nørgaard Peter
Published: July 2002

The retinoblastoma gene family consists of the tumor suppressor protein pRB and its two relatives p107 and p130. These proteins have been implicated in the regulation of cell cycle progression, in part, through inactivation of members of the E2F transcription factor family. Overexpression of pRB, p107, or p130 leads to growth arrest in the G
1
phase of the cell cycle, and this arrest is abolished by complex formation with the adenovirus E1A, human papilloma virus E7, or simian virus 40 T oncoproteins. Inactivation of pRB by gross structural alterations or point mutations in the
RB-1
gene has been described in a variety of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas. Despite the structural and functional similarity between pRB, p107, and p130, alterations in the latter two proteins have not been identified in human tumors. We have screened a panel of 17 small cell lung carcinoma cell lines for the presence of functional p107 and p130 by evaluating their ability to form complexes with E1A
in vitro
. In the GLC2 small cell lung carcinoma cells no p130 protein was detected. The loss of the p130 protein is the result of a single point mutation within a splice acceptor sequence in the GLC2 genomic DNA. This mutation eliminates exon 2, leading to an in-frame stop codon, and no detectable protein is produced. These data are, to our knowledge, the first to describe the loss of p130 as a consequence of a genetic alteration, suggesting that not only pRB but also the other members of the family may contribute to tumorigenesis, providing a rationale for the observation that the DNA tumor viruses selectively target all the members of the retinoblastoma protein family.
Chromatin assembly in a yeast whole-cell extract
Authors: Schultz Michael C. , Hockman Darren J. , Harkness Troy A. A. , Garinther Wendy I. , Altheim Brent A.
Published: July 2002

A simple
in vitro
system that supports chromatin assembly was developed for
Saccharomyces cerevisiae
. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.
Translation initiation factor eIF4G mediates
in vitro
poly(A) tail-dependent translation
Authors: Tarun Salvador Z. , Wells Sandra E. , Deardorff Julie A. , Sachs Alan B.
Published: July 2002

The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the
in vitro
translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation
in vitro
, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function
in vivo
.
Human argininosuccinate lyase: A structural basis for intragenic complementation
Authors: Turner Mary A. , Simpson Alan , McInnes Roderick R. , Howell P. Lynne
Published: July 2002

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits enzymatic activity that is greater than that found in the oligomeric proteins produced by each mutant allele alone. The mutations involved in the most successful complementation event observed in ASL deficiency were found to be an aspartate to glycine mutation at codon 87 of one allele (D87G) coupled with a glutamine to arginine mutation at codon 286 of the other (Q286R). To understand the structural basis of the Q286R:D87G intragenic complementation event at the ASL locus, we have determined the x-ray crystal structure of recombinant human ASL at 4.0 Å resolution. The structure has been refined to an
R
factor of 18.8%. Two monomers related by a noncrystallographic 2-fold axis comprise the asymmetric unit, and a crystallographic 2-fold axis of space group P3
1
21 completes the tetramer. Each of the four active sites is composed of residues from three monomers. Structural mapping of the Q286R and D87G mutations indicate that both are near the active site and each is contributed by a different monomer. Thus when mutant monomers combine randomly such that one active site contains both mutations, it is required by molecular symmetry that another active site exists with no mutations. These “native” active sites give rise to the observed partial recovery of enzymatic activity.
Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination
Authors: Hirose Sachiko , Yan Kwangseok , Abe Masaaki , Jiang Yi , Hamano Yoshitomo , Tsurui Hiromichi , Shirai Toshikazu
Published: July 2002

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F1mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly,in vitrostimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fashighand highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Faslowand apoptosis-resistant. The Faslowcells were included in the CD5+B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F1mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fasin vivoand were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.
The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin
Authors: Gately Stephen , Twardowski Przemyslaw , Stack M. Sharon , Cundiff Deborah L. , Grella Davida , Castellino Francis J. , Enghild Jan , Kwaan Hau C. , Lee Francis , Kramer Robert A. , Volpert Olga , Bouck Noel , Soff Gerald A.
Published: July 2002

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (
N
-acetyl-
l
-cysteine,
d
-penicillamine, captopril,
l
-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis
in vitro
and
in vivo
and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.