bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel expression mechanism for synaptic potentiation: Alignment of presynaptic release site and postsynaptic receptor
Authors: Xie Xiaping , Liaw Jim-Shih , Baudry Michel , Berger Theodore W.
Published: July 2002

A combination of experimental and modeling approaches was used to study cellular–molecular mechanisms underlying the expression of short-term potentiation (STP) and long-term potentiation (LTP) of glutamatergic synaptic transmission in the hippocampal slice. Electrophysiological recordings from dentate granule cells revealed that high-frequency stimulation of perforant path afferents induced a robust STP and LTP of both (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and
N
-methyl-
d
-aspartic acid (NMDA) receptor-mediated synaptic responses. However, the decay time constant for STP of the AMPA receptor-mediated excitatory postsynaptic potential was approximately 6 min, whereas the decay time constant for STP of the NMDA receptor-mediated excitatory postsynaptic potential was only 1 min. In addition, focal application of agonists during the expression of STP revealed that the magnitude of conductance change elicited by NMDA application was significantly enhanced, whereas the magnitude of conductance change elicited by application of AMPA remained constant. These findings are most consistent with a postsynaptic mechanism of STP and LTP. Different putative mechanisms were evaluated formally using a computational model that included diffusion of glutamate within the synaptic cleft, different kinetic properties of AMPA and NMDA receptor/channels, and geometric relations between presynaptic release sites and postsynaptic receptor/channels. Simulation results revealed that the only hypothesis consistent with experimental data is that STP and LTP reflect a relocation of AMPA receptor/channels in the postsynaptic membrane such that they become more closely “aligned” with presynaptic release sites. The same mechanism cannot account for STP or LTP of NMDA receptor-mediated responses; instead, potentiation of the NMDA receptor subtype is most consistent with an increase in receptor sensitivity or number.
Identification of immunodominant T cell epitopes of human glutamic acid decarboxylase 65 by using HLA-DR(α1*0101,β1*0401) transgenic mice
Authors: Patel Salil D. , Cope Andrew P. , Congia Mauro , Chen Teddy T. , Kim Eugene , Fugger Lars , Wherrett Diane , Sonderstrup-McDevitt Grete
Published: July 2002

Glutamic acid decarboxylase isoform 2 (GAD65; EC
4.1.1.15
) has been identified as a key target autoantigen of insulin-dependent diabetes mellitus (IDDM). IDDM is genetically associated with the major histocompatibility complex (MHC), and particular alleles from the HLA-DQ and HLA-DR loci contribute to disease. Among DR4 subtypes, HLA-DRB1*0401, HLA-DRB1*0402, and HLA-DRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confers protection. We have utilized HLA-DR(α1*0101,β1*0401) (hereafter referred to as DR0401), human CD4, murine class II null triple transgenic mice and recombinant GAD65 to generate T cell hybridomas, and we have used overlapping sets of peptides to map the immunodominant epitopes of this autoantigen. We have identified 10 immunogenic regions for GAD65, of which 6 are recognized by multiple hybridomas. These epitopes are also generated by human antigen-presenting cells and their presentation is DR0401 restricted, as shown by the use of typed human lymphoblastoid cell lines and antibody blocking experiments. Immunodominant GAD65 epitopes defined in transgenic mice correspond to GAD65 regions previously shown to elicit T cell responses specifically in DR0401 IDDM patients, underscoring the validity of this approach. Interestingly, although the major epitopes contain DR0401 binding motifs, one of the epitopes contains a DR0405 motif.
Macrophages lacking scavenger receptor A show a decrease in binding and uptake of acetylated low-density lipoprotein and of apoptotic thymocytes, but not of oxidatively damaged red blood cells
Authors: Terpstra Valeska , Kondratenko Nonna , Steinberg Daniel
Published: July 2002

Macrophage binding of oxidatively damaged red blood cells (OxRBC) and apoptotic thymocytes correlates in many instances with a loss of phospholipid bilayer asymmetry, i.e., with an increase in expression of phosphatidylserine on the outer leaflet of the plasma membrane. Oxidatively modified LDL (OxLDL) can compete for the binding of these ligands to macrophages. However, the receptor(s) responsible remains to be identified. The present studies show that mouse peritoneal macrophages totally lacking scavenger receptor A (SRA) bound OxRBC just as effectively as wild-type macrophages, whereas their binding and uptake of acetyl LDL was reduced by more than 80%. Binding of apoptotic thymocytes and binding of OxLDL were also reduced, but only by 20–30%. We conclude that SRA is not involved in the recognition of phosphatidylserine-rich membranes but contributes to the binding of OxLDL and apoptotic thymocytes. The binding of OxRBC was almost totally calcium-dependent, whereas the binding of apoptotic thymocytes was not, suggesting that the mechanisms involved in their uptake by macrophages under these conditions were different.
Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2
Authors: Kong Wuyi , McConalogue Karen , Khitin Lev M. , Hollenberg Morley D. , Payan Donald G. , Böhm Stephan K. , Bunnett Nigel W.
Published: July 2002

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E
2
and F
1α
from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E
2
secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.
Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform
Authors: James Thomas L. , Liu He , Ulyanov Nikolai B. , Farr-Jones Shauna , Zhang Hong , Donne David G. , Kaneko Kiyotoshi , Groth Darlene , Mehlhorn Ingrid , Prusiner Stanley B. , Cohen Fred E.
Published: July 2002

The scrapie prion protein (PrP
Sc
) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrP
C
) by a conformational change. N-terminal truncation of PrP
Sc
by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in
Escherichia coli
and purified. After refolding rPrP into an α-helical form resembling PrP
C
, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrP
Sc
formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrP
C
into PrP
Sc
, whereas the discontinuous epitope near the C terminus controls this transition.
Coagulation initiated on herpesviruses
Authors: Sutherland M. R. , Raynor C. M. , Leenknegt H. , Wright J. F. , Pryzdial E. L. G.
Published: July 2002

Herpesviruses have been previously correlated to vascular disease and shown to cause thrombogenic and atherogenic changes to host cells. Herein we show that even in the absence of cells, purified cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can initiate thrombin production. Functional assays demonstrated that purified HSV-1 and HSV-2 provide the necessary phospholipid (proPL) for assembling the coagulation factors Xa and Va into prothrombinase, which is responsible for generating thrombin. These observations are consistent with our earlier studies involving CMV. The presence of proPL on all three herpesviruses was confirmed directly by flow cytometry and electron microscopy by using annexin V and factor Va, respectively, as proPL-specific probes. Of equal importance, we found that CMV, HSV-1, and HSV-2 were also able to facilitate factor Xa generation from the inactive precursor factor X, but only when factor VII/VIIa and Ca
2+
were present. Monoclonal antibodies specific for tissue factor (TF), the coagulation initiator, inhibited this factor X activation and, furthermore, enabled identification of TF antigen on each virus type by flow cytometry and electron microscopy. Collectively, these data show that CMV, HSV-1, and HSV-2 can initiate the generation of thrombin by having essential proPL and TF activities on their surface. Unlike the normal cellular source, the viral activity is constitutive and, therefore, not restricted to sites of vascular injury. Thus cell-independent thrombin production may be the earliest event in vascular pathology mediated by herpesviruses.
Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways
Authors: Chow Yu-Hua , O’Brodovich Hugh , Plumb Jonathan , Wen Yanxia , Sohn Kyoung-Jin , Lu Zhan , Zhang Fangyuan , Lukacs Gergely L. , Tanswell A. Keith , Hui Chi-Chung , Buchwald Manuel , Hu Jim
Published: July 2002

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the
Escherichia coli LacZ
gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.
DEK, an autoantigen involved in a chromosomal translocation in acute myelogenous leukemia, binds to the HIV-2 enhancer
Authors: Fu Glenn K. , Grosveld Gerard , Markovitz David M.
Published: July 2002

The product of the
dek
oncogene is the 43-kDa DEK nuclear protein. DEK was first identified in a fusion with the CAN nucleoporin protein in a specific subtype of acute myelogenous leukemia. DEK has also been shown to be an autoantigen in patients with pauciarticular onset juvenile rheumatoid arthritis. Further, the last 65 amino acids of DEK can partially reverse the mutation-prone phenotype of cells from patients with ataxia-telangiectasia. However, in spite of these significant disease associations, the function of DEK has remained unclear. The HIV-2 peri-
ets
(pets) site is a TG-rich element found between the two Elf-1 binding sites in the HIV-2 enhancer. The pets element mediates transcriptional activation whether the enhancer is stimulated by phorbol 12-myristate 13-acetate (PMA) alone, phytohemagluttinin (PHA) alone, PMA plus PHA, soluble antibodies to the T cell receptor, immobilized antibodies to the T cell receptor, or by antigen. Previously, we purified and characterized the pets factor, demonstrating that it is a 43-kDa nuclear protein. We now describe the identification of DEK as this 43-kDa pets factor. Using a modified Southwestern screening procedure, we find that DEK can recognize the pets element. We demonstrate the ability of recombinant DEK to bind specifically to the pets site using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting. “Supershift” EMSA further confirms that DEK is the dominant protein binding to the pets site in T cell extracts. Our findings show that DEK is a site-specific DNA binding protein that is likely involved in transcriptional regulation and signal transduction. This has implications for multiple pathogenic processes, including hematologic malignancies, arthritis, ataxia-telangiectasia, and AIDS caused by HIV-2.
Exploring the basis of peptide–carbohydrate crossreactivity: Evidence for discrimination by peptides between closely related anti-carbohydrate antibodies
Authors: Harris Shannon L. , Craig Lisa , Mehroke Jarnail S. , Rashed Michael , Zwick Michael B. , Kenar Kelly , Toone Eric J. , Greenspan Neil , Auzanneau France-Isabelle , Marino-Albernas José-R. , Pinto B. Mario , Scott Jamie K.
Published: July 2002

To investigate the molecular basis of antigenic mimicry by peptides, we studied a panel of closely related mAbs directed against the cell-wall polysaccharide of group AStreptococcus. These antibodies have restrictedV-gene usage, indicating a shared mechanism of binding to a single epitope. Epitope mapping studies using synthetic fragments of the cell-wall polysaccharide supported this conclusion. All of the mAbs isolated crossreactive peptides from a panel of phage-displayed libraries, and competition studies indicated that many of the peptides bind at or near the carbohydrate binding site. Surprisingly, the peptides isolated by each mAb fell into distinct consensus-sequence groups that discriminated between the mAbs, and in general, the peptides bound only to the mAbs used for their isolation. Similar results were obtained with polyclonal antibodies directed against synthetic oligosaccharide fragments of the streptococcal cell-wall polysaccharide. Thus, the peptides appear to be specific for their isolating antibodies and are not recognized by the same mechanism as their carbohydrate counterparts.
Buried asparagines determine the dimerization specificities of leucine zipper mutants
Authors: Zeng Xiangang , Herndon Aaron M. , Hu James C.
Published: July 2002

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo- and heterodimers through a short α-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at thea,d,e, andgpositions of the (abcdefg)nheptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at fourapositions in the dimer interface, using a genetic test for the specificity of dimer formation by λ repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C/EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at theapositions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.
Direct antidiabetic effect of leptin through triglyceride depletion of tissues
Authors: Shimabukuro Michio , Koyama Kazunori , Chen Guoxun , Wang May-Yun , Trieu Falguni , Lee Young , Newgard Christopher B. , Unger Roger H.
Published: July 2002

Leptin is currently believed to control body composition largely, if not entirely, via hypothalamic receptors that regulate food intake and thermogenesis. Here we demonstrate direct extraneural effects of leptin to deplete fat content of both adipocytes and nonadipocytes to levels far below those of pairfed controls. In cultured pancreatic islets, leptin lowered triglyceride (TG) content by preventing TG formation from free fatty acids (FFA) and by increasing FFA oxidation.
In vivo
hyperleptinemia, induced in normal rats by adenovirus gene transfer, depleted TG content in liver, skeletal muscle, and pancreas without increasing plasma FFA or ketones, suggesting intracellular oxidation. In islets of obese Zucker Diabetic Fatty rats with leptin receptor mutations, leptin had no effect
in vivo
or
in vitro
. The TG content was ≈20 times normal, and esterification capacity was increased 3- to 4-fold. Thus, in rats with normal leptin receptors but not in Zucker Diabetic Fatty rats, nonadipocytes and adipocytes esterify FFA, store them as TG, and later oxidize them intracellularly via an “indirect pathway” of intracellular fatty acid metabolism controlled by leptin. By maintaining insulin sensitivity and preventing islet lipotoxicity, this activity of leptin may prevent adipogenic diabetes.
t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses
MLL
with hCDC
rel
, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes
Authors: Megonigal Maureen D. , Rappaport Eric F. , Jones Douglas H. , Williams Terence M. , Lovett Brian D. , Kelly Kara M. , Lerou Paul H. , Moulton Thomas , Budarf Marcia L. , Felix Carolyn A.
Published: July 2002

We examined the
MLL
genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical
MLL
gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence
in situ
hybridization with an
MLL
-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting
MLL
. Known 5′ sequence from
MLL
but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′
MLL
genomic sequence to the 5′ ends of
Bam
HI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The
MLL
genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDC
rel
(human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both
MLL
and hCDC
rel
contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of
MLL
exon 7 to hCDC
rel
exon 3, indicating that an
MLL
-hCDC
rel
chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.
Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity
Authors: Wang Hua , Diamond Rochelle A. , Rothenberg Ellen V.
Published: July 2002

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8–cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.
eskimo1mutants ofArabidopsisare constitutively freezing-tolerant
Authors: Xin Zhanguo , Browse John
Published: July 2002

Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants ofArabidopsisthat are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-typeArabidopsiswas increased from −5.5°C to −12.6°C by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from −6.8°C to −10.6°C in the absence of acclimation. Plants with mutations at theeskimo1(esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation inesk1plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization ofesk1mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.
T cell-independent type I antibody response against B cell epitopes expressed repetitively on recombinant virus particles
Authors: Fehr Thomas , Skrastina Dace , Pumpens Paul , Zinkernagel Rolf M.
Published: July 2002

Recombinant viral or virus-like particles offer new tools for vaccine development. This study investigated hepatitis B core antigen (HBcAg) capsids and RNA phage Qβ coats as carriers of a foreign epitope to induce antibody responses in mice. HBcAg capsids were shown to induce T cell-independent (TI) antibodies. We found that these particles behave as antigen-specific TI type 1 (TI-1) Ag comparable to other rigidly structured viruses. When a 5-aa long epitope of the pre-S1 domain of hepatitis B surface antigen (HBsAg) was introduced into the optimal position of the HBc molecule, it also behaved as a TI-1 Ag. Best efficiency of the antibody response to the foreign epitope was achieved by a compensatory deletion after the epitope to retain the regular structure of the HBcAg capsid with a highly repetitive superficial exposition of the foreign epitope. For recombinant Qβ phage coats, a much more efficient antibody response to the foreign epitope was achieved when the foreign epitope was expressed repetitively on a particulate derivate of Qβ phage coats. Thus, recombinant virus particles are suitable vaccine carriers for the introduction of foreign B cell epitopes, if precise structural requirements are fulfilled.
Expression of mouse telomerase catalytic subunit in embryos and adult tissues
Authors: Martín-Rivera Luis , Herrera Eloísa , Albar Juan P. , Blasco María A.
Published: July 2002

Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing the stable maintenance of chromosomes during multiple cell divisions. Here, we describe the isolation and characterization of the catalytic subunit of mouse telomerase, mTERT (
m
ouse
te
lomerase
r
everse
t
ranscriptase), an essential protein component of the telomerase complex. During embryonic development, mTERT mRNA is abundantly expressed in the whole embryo, especially in regions of intense proliferation. We found that the mTERT mRNA expression in both embryonic and adult tissues is independent of the essential RNA component of telomerase, mTR, and therefore, of the formation of active telomerase complexes. mTERT protein is present exclusively in tissues with telomerase activity, such as testis, spleen, and thymus. mTERT protein is barely detectable in the thymus of mTR
−/−
mice, suggesting that mTERT protein stability in this tissue may depend on the actual assembly of active telomerase complexes. Finally, we found that mouse and human telomerase catalytic subunit is located in the cell nucleus, and its localization is not regulated during cell cycle progression.
Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin
Authors: Sheng Shijie , Truong Binh , Fredrickson Derek , Wu Ruilian , Pardee Arthur B. , Sager Ruth
Published: July 2002

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion
in vitro
and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-
d
-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (<0.5 μM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA
in vitro
suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.
The structure and organization within the membrane of the helices composing the pore-forming domain of
Bacillus thuringiensis
δ-endotoxin are consistent with an “umbrella-like” structure of the pore
Authors: Gazit Ehud , Rocca Paolo La , Sansom Mark S. P. , Shai Yechiel
Published: July 2002

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by
Bacillus thuringiensis
δ-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices α4 and α5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the “umbrella model”). Our results also support the suggestion that α7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.
Electrostatic interaction between helical macromolecules in dense aggregates: An impetus for DNA poly- and meso-morphism
Authors: Kornyshev Alexei A. , Leikin Sergey
Published: July 2002

DNA exhibits a surprising multiplicity of structures when it is packed into dense aggregates. It undergoes various polymorphous transitions (e.g., from the
B
to
A
form) and mesomorphous transformations (from hexagonal to orthorhombic or monoclinic packing, changes in the mutual alignment of nearest neighbors, etc). In this report we show that such phenomena may have their origin in the specific helical symmetry of the charge distribution on DNA surface. Electrostatic interaction between neighboring DNA molecules exhibits strong dependence on the patterns of molecular surface groups and adsorbed counter-ions. As a result, it is affected by such structural parameters as the helical pitch, groove width, the number of base pairs per helical turn, etc. We derive expressions which relate the energy of electrostatic interaction with these parameters and with the packing variables characterizing the axial and azimuthal alignment between neighboring macromolecules. We show, in particular, that the structural changes upon the
B
-to-
A
transition reduce the electrostatic energy by ≈kcal/mol per base pair, at a random adsorption of counter ions. Ion binding into the narrow groove weakens or inverts this effect, stabilizing
B
-DNA, as it is presumably the case in Li
+
-DNA assemblies. The packing symmetry and molecular alignment in DNA aggregates are shown to be affected by the patterns of ion binding.
The role of mismatch repair in the prevention of base pair mutations in
Saccharomyces cerevisiae
Authors: Earley Marie C. , Crouse Gray F.
Published: July 2002

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In
Saccharomyces cerevisiae
, the products of three genes homologous to
Escherichia coli
mutS
—
MSH2
,
MSH3
, and
MSH6
—function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the
CYC1
gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of
MSH2
or
MSH6
, but not
MSH3
, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.
Rapidly induced auditory plasticity: The ventriloquism aftereffect
Authors: Recanzone Gregg H.
Published: July 2002

Cortical representational plasticity has been well documented after
peripheral and central injuries or improvements in perceptual and motor
abilities. This has led to inferences that the changes in cortical
representations parallel and account for the improvement in performance
during the period of skill acquisition. There have also been several
examples of rapidly induced changes in cortical neuronal response
properties, for example, by intracortical microstimulation or by
classical conditioning paradigms. This report describes similar rapidly
induced changes in a cortically mediated perception in human subjects,
the ventriloquism aftereffect, which presumably reflects a
corresponding change in the cortical representation of acoustic space.
The ventriloquism aftereffect describes an enduring shift in the
perception of the spatial location of acoustic stimuli after a period
of exposure of spatially disparate and simultaneously presented
acoustic and visual stimuli. Exposure of a mismatch of 8° for 20–30
min is sufficient to shift the perception of acoustic space by
approximately the same amount across subjects and acoustic frequencies.
Given that the cerebral cortex is necessary for the perception of
acoustic space, it is likely that the ventriloquism aftereffect
reflects a change in the cortical representation of acoustic space.
Comparisons between the responses of single cortical neurons in the
behaving macaque monkey and the stimulus parameters that give rise to
the ventriloquism aftereffect suggest that the changes in the cortical
representation of acoustic space may begin as early as the primary
auditory cortex.
Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Authors: Körkkö Jarmo , Annunen Susanna , Pihlajamaa Tero , Prockop Darwin J. , Ala-Kokko Leena
Published: July 2002

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.
Cytokine-induced survival of activated T cellsin vitroandin vivo
Authors: Vella Anthony T. , Dow Steven , Potter Terry A. , Kappler John , Marrack Philippa
Published: July 2002

Many antigen-specific T cells die after exposure to antigen in animals. These cells also die if they are isolated from animals shortly after activation and cultured. Various cytokines were tested for their ability to interfere with thisin vitrodeath. Surprisingly, tumor necrosis factor α and other inflammatory cytokines did not prevent thein vitrodeath of activated T cells, even though these cytokines do prevent activated T cell death in animals. Therefore, the inflammatory cytokines probably act on T cellsin vivovia an intermediary factor. Four cytokines, interleukin (IL)-2, IL-4, IL-7, and IL-15, did prevent activated T cell deathin vitro, with IL-4 and IL-15 more effective than IL-2 or IL-7. These cytokines share a component of their receptors, the common γ chain, γc. Therefore, their collective ability to protect activated T cells from death may be mediated by signals involving γc. To assess their activityin vivo, two of the cytokines, IL-2 and IL-4, were expressed in animals at local sites of superantigen responses. Both cytokines increased the numbers of T cells found at the local sites 14 days later. Interleukin 4 was more effective than IL-2, even though IL-2 stimulates T cell proliferation better than IL-4. This result suggested that IL-4 and related cytokines can promote T cell survivalin vivoas well asin vitro. The ability of these cytokines to prevent the death of activated T cells may be important at certain stages of immune responses in animals.
Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects
Authors: Ghivizzani Steven C. , Lechman Eric R. , Kang Richard , Tio Caroline , Kolls Jay , Evans Christopher H. , Robbins Paul D.
Published: July 2002

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.
Novel phenomena in dilute electron systems in two dimensions
Authors: Sarachik M. P. , Kravchenko S. V.
Published: July 2002

For the past two decades, all
two-dimensional systems of electrons were believed to be insulating in
the limit of zero temperature. Recent experiments provide evidence for
an unexpected transition to a conducting phase at very low electron
densities. The nature of this phase is not understood and is currently
the focus of intense theoretical and experimental attention.
Global and local implications of biotechnology and climate change for future food supplies
Authors: Evenson Robert E.
Published: July 2002

The development of improved technology for agricultural production and its diffusion to farmers is a process requiring investment and time. A large number of studies of this process have been undertaken. The findings of these studies have been incorporated into a quantitative policy model projecting supplies of commodities (in terms of area and crop yields), equilibrium prices, and international trade volumes to the year 2020. These projections show that a “global food crisis,” as would be manifested in high commodity prices, is unlikely to occur. The same projections show, however, that in many countries, “local food crisis,” as manifested in low agricultural incomes and associated low food consumption in the presence of low food prices, will occur. Simulations show that delays in the diffusion of modern biotechnology research capabilities to developing countries will exacerbate local food crises. Similarly, global climate change will also exacerbate these crises, accentuating the importance of bringing strengthened research capabilities to developing countries.
Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues
Authors: Mann Michael J. , Gibbons Gary H. , Hutchinson Howard , Poston Robert S. , Hoyt E. Grant , Robbins Robert C. , Dzau Victor J.
Published: July 2002

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but theirin vivoapplication is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability ofex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.
Substrate binding and catalysis by ribonuclease P from cyanobacteria and
Escherichia coli
are affected differently by the 3′ terminal CCA in tRNA precursors
Authors: Pascual Alberto , Vioque Agustín
Published: July 2002

We have studied the effect of the 3′ terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium
Synechocystis
sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3′ terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in
Escherichia coli
RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330–332 in the J15/16 loop of
Synechocystis
RNase P RNA from GGU to CCA has a modest effect on
k
cat
for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that
Synechocystis
RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3′ terminal CCA is not involved.
JE-2147: A dipeptide protease inhibitor (PI) that potently inhibits multi-PI-resistant HIV-1
Authors: Yoshimura Kazuhisa , Kato Ryohei , Yusa Keisuke , Kavlick Mark F. , Maroun Victor , Nguyen Aline , Mimoto Tsutomu , Ueno Takamasa , Shintani Makoto , Falloon Judith , Masur Henry , Hayashi Hideya , Erickson John , Mitsuya Hiroaki
Published: July 2002

We designed, synthesized, and identified JE-2147, an allophenylnorstatine-containing dipeptide HIV protease inhibitor (PI), which is potent against a wide spectrum of HIV-1, HIV-2, simian immunodeficiency virus, and various clinical HIV-1 strains
in vitro
. Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9–11 different anti-HIV therapeutics after 32–83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all of the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains, with IC
50
values ranging from 13–41 nM (<2-fold changes in IC
50
compared with that of wild-type HIV-1). The emergence of JE-2147-resistant HIV-1 variants
in vitro
was substantially delayed compared with that of HIV-1 resistant to another allophenylnorstatine-containing compound, KNI-272, and other related PIs. Structural analysis revealed that the presence of a flexible P2′ moiety is important for the potency of JE-2147 toward wild-type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. Further development of JE-2147 for treating patients harboring multi-PI-resistant HIV-1 is warranted.
Complexity of terminal airspace geometry assessed by lung computed tomography in normal subjects and patients with chronic obstructive pulmonary disease
Authors: Mishima Michiaki , Hirai Toyohiro , Itoh Harumi , Nakano Yasutaka , Sakai Hiroaki , Muro Shigeo , Nishimura Koichi , Oku Yoshitaka , Chin Kazuo , Ohi Motoharu , Nakamura Takashi , Bates Jason H. T. , Alencar Adriano M. , Suki Bèla
Published: July 2002

Increases in the low attenuation areas (LAA) of chest x-ray computed tomography images in patients with chronic obstructive pulmonary disease (COPD) have been reported to reflect the development of pathological emphysema. We examined the statistical properties of LAA clusters in COPD patients and in healthy subjects. In COPD patients, the percentage of the lung field occupied by LAAs (LAA%) ranged from 2.6 to 67.6. In contrast, LAA% was always <30% in healthy subjects. The cumulative size distribution of the LAA clusters followed a power law characterized by an exponent
D
. We show that
D
is a measure of the complexity of the terminal airspace geometry. The COPD patients with normal LAA% had significantly smaller
D
values than the healthy subjects, and the
D
values did not correlate with pulmonary function tests except for the diffusing capacity of the lung. We interpret these results by using a large elastic spring network model and find that the neighboring smaller LAA clusters tend to coalesce and form larger clusters as the weak elastic fibers separating them break under tension. This process leaves LAA% unchanged whereas it decreases the number of small clusters and increases the number of large clusters, which results in a reduction in
D
similar to that observed in early emphysema patients. These findings suggest that
D
is a sensitive and powerful parameter for the detection of the terminal airspace enlargement that occurs in early emphysema.