bluuebunny/crossref_metadata_2025 huggingface dataset:
Chichester and its cathedral on the eve of the Reformation
Authors: Thomas Spencer
Published: October 2002

Chichester challenges the orthodoxy of urban decline and a decadent church in late medieval England through the proactive role of the Dean and Chapter of the cathedral as landowner and developer; civic-ecclesiastical harmony; an economic and social environment conducive to wealth creation, entrepreneurial activity, investment and real estate management; productive artisans; and social cohesion. Comparisons with other cathedral cities furthers our embryonic understanding of the urban economy in this genre.
Contribution of F‐Actin to Barrier Properties of the Blood‐Joint Pathway
Authors: POLI A. , COLEMAN P.J. , MASON R.M. , LEVICK J.R.
Published: October 2002

Objectives: Because fibroblast filamentous actin (F‐actin) influences cutaneous interstitial matrix swelling pressure (5), we investigated whether F‐actin in fibroblast‐derived synoviocytes influences the hydraulic permeability of the transsynovial interstitial pathway. The study also tested whether F‐actin in fenestrated synovial endothelium contributes to the blood‐joint barrierin vivo.

Methods: The clearance of Evans blue‐albumin (EVA) from plasma into the knee joint cavity was determined in joint infused with F‐actin disrupting cytochalasin D (1–200 µM), latrunculin B (100 µM) or vehicle in anesthetized rabbits. The hydraulic permeability of the lining was determined as the slope relating net trans‐synovial flowimageto intra‐articular pressurePj. Synovium was examined histologically after i.v. Monastral blue to assess endothelial leakiness.

Results: EVA permeationin vivowas increased up to 25‐fold by cytochalasin (p= 0.0002, ANOVA), with an EC50of 23 µM (95% confidence limits 13–43 µM). Washout quickly reversed the increase. Latrunculin had a similar effect. F‐actin disruption switchedimagefrom drainage (control) to filtration into the cavity at lowPjin vivoand raised the conductanceimageby 2.13 (p= 0.001, ANOVA). Circulatory arrest abolished these effects. Monastral blue revealed numerous endothelial leaks.

Conclusions: F‐actin is crucial to the barrier function of fenestrated endotheliumin situ. No significant effect of synoviocyte F‐actin on matrix permeability was detected.
Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis,
Authors: Fatland Beth L. , Ke Jinshan , Anderson Marc D. , Mentzen Wieslawa I. , Cui Li Wei , Allred C. Christy , Johnston Jerry L. , Nikolau Basil J. , Wurtele Eve Syrkin
Published: October 2002

Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A4B4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by theACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of theACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.
Subthalamic GAD Gene Therapy in a Parkinson's Disease Rat Model
Authors: Luo Jia , Kaplitt Michael G. , Fitzsimons Helen L. , Zuzga David S. , Liu Yuhong , Oshinsky Michael L. , During Matthew J.
Published: October 2002

The motor abnormalities of Parkinson's disease (PD) are caused by alterations in basal ganglia network activity, including disinhibition of the subthalamic nucleus (STN), and excessive activity of the major output nuclei. Using adeno-associated viral vector–mediated somatic cell gene transfer, we expressed glutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of the neurotransmitter GABA, in excitatory glutamatergic neurons of the STN in rats. The transduced neurons, when driven by electrical stimulation, produced mixed inhibitory responses associated with GABA release. This phenotypic shift resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype. This strategy suggests that there is plasticity between excitatory and inhibitory neurotransmission in the mammalian brain that could be exploited for therapeutic benefit.
Towards high affinity carbohydrate-binding proteins: Directed evolution of murine galectin-3
Authors: Lundquist Joseph J , Kiburz Brendan M , Wu Jeffrey K , Gibbs Jr. Kenneth D , Toone Eric J
Published: October 2002

Towards a better understanding of the molecular basis of affinity, a directed evolution of murine galectin-3 (G3) was initiated to produce mutants with improved affinity for lactose and N-acetyllactosamine relative to the wild-type protein. A series of N-terminal truncations were developed to facilitate incorporation of the 35 kDa protein into a phage-display construct. Analysis of the various assemblies revealed that all such deletions produced protein unsuitable for use in directed evolution studies. Following fusion of the full-length galectin to p3 of filamentous phage, three libraries were constructed and biopanned for increased affinity for lactose. The first two libraries, of 1 × 105and 1 × 106members, respectively, were assembled through a combination of error-prone PCR and DNA shuffling. A third library was constructed using a modified staggered extension protocol (StEP), but contained only 10 members. Mutants were also engineered site-specifically to test the role of key residues in or near the binding pocket. Analysis of the mutants by ITC identified one mutation (R158G) that produces a twofold increase in affinity for lactose and another that results in a sixfold increase in affinity for N-acetyllactosamine. Solid-phase binding analysis of phage for nonexpressing proteins indicated that two other mutants demonstrated increased binding to beta-methyllactose relative to the wild-type protein. Together these studies validate the evolutionary approach and set the stage for the development of novel carbohydrate-binding proteins.Key words: phage display, directed evolution, galectin, thermodynamics, carbohydrates.
1H NMR studies on the solution conformation of contulakin-G and analogues
Authors: Kindahl Lill , Sandström Corine , Craig A Grey , Norberg Thomas , Kenne Lennart
Published: October 2002

The conformation of contulakin-G, a bioactive 16 amino acid O-linked glycopeptide (ZSEEGGSNAT*KKPYIL) with the disaccharide β-D-Gal(1[Formula: see text]3)α-D-GalNAc attached to the threonine residue in position 10, has been investigated by 1H NMR spectroscopy. The 1H NMR data for the non-glycosylated peptide and for two glycopeptide analogues, one with the monosaccharide α-D-GalNAc at Thr10 and one with the disaccharide β-D-Gal(1–>3)α-D-GalNAc at Ser7, all of lower bioactivity than contulakin-G, have also been collected. The chemical shifts, NOEs, temperature coefficients of amide protons, and 3JNH,αH-values suggest that all four compounds exist mainly in random coil conformations. Some transient populations of folded conformations are also present in the glycopeptides and turns, probably induced by the sugars, are present in the peptide chain around the site of glycosylation. In the two peptides O-glycosylated at Thr10, the rotation of α-D-GalNAc around the linkage between the sugar and the peptide is restricted. There is evidence for a hydrogen bond between the amide proton of α-D-GalNAc and the peptide chain that could contribute to this torsional rigidity. An intramolecular hydrogen bond between the carbohydrate and the peptide chain does not exist in the peptide O-glycosylated at the Ser7 residue. Key words: conformation, contulakin-G, NMR, O-linked glycopeptide.
Glycodendrimers based on cellobiosyl-derived monomers
Authors: Kalovidouris Stacey A , Turnbull W Bruce , Stoddart J Fraser
Published: October 2002

Reductive amination of suitably functionalized trisaccharide monomers, based on cellobiosylgalacto residues, has made it possible to construct high-molecular-weight glycodendrons and glycodendrimers for the display of large bioactive oligosaccharides and proteins in a well-defined manner. Key words: cellobiose, glycodendrimers, reductive amination, trisaccharide monomers.
Oligosaccharide recognition by antibodies: Synthesis and evaluation of talose oligosaccharide analogues
Authors: Lowary T L , Eichler E , Bundle D R
Published: October 2002

A series of monosaccharide (46), disaccharide (3,712), and trisaccharide (1315) analogs of the native ligand 2, which fills the binding site of monoclonal antibody Se 155.4, have been synthesized and their bioactivity measured by solid- and solution-phase assays. The syntheses of disaccharide analogs sought to replace galactose by various alkyl groups at the O-2 position of mannose. The activity of one of these O-2 alkyl analogs was 75% of that observed for the trisaccharide and points to only weak net bonding between the solvent exposed galactose residue and the antibody binding site. The synthesis of talose analogs 13 and 14, where the mannose or galactose residues of 2 were replaced by talose produced ligands with activities from one-third to one-half of that seen for the native ligand 2. These activity changes did not exhibit discernable correlations with the ability of talose to disrupt water of solvation.Key words: abequose, 3,6-dideoxy-D-xylo-hexose, talose disaccharide and trisaccharide, antibody oligosaccharide interactions, molecular recognition of carbohydrates, water in antibody complexes, Salmonella LPS, monoclonal antibody Se 155.4, bacterial O-antigen.
The reactions of 3-alkylindoles with cyclopropanes: An unusual rearrangement leading to 2,3-disubstitution
Authors: England Dylan B , Woo Tom K , Kerr Michael A
Published: October 2002

Indoles that bear an alkyl substituent in the 3-position, when treated with cyclopropanediesters, typically undergo a [3 + 2]-annulation reaction in a kinetically controlled process (1[Formula: see text]4). If the reaction is performed at elevated temperatures for a longer period of time, a rearrangement of the putative intermediate 3 occurs in which the alkylating species undergoes a migration to the 2-position followed by loss of a proton to reform the benzopyrrole ring. The yields range from 78 to 10%. If a 3-allylindole is used in combination with a cyclopropanediester, which is further substituted with an alkenyl moiety, the product is an effective ring closing metathesis substrate and can be converted to the 1,3,3a,4,5,6-hexahydro-1H-pyrido[3,2,1-jk]carbazole system. A mechanistic discussion of the rearrangement process is presented.
Anchimeric assistance in hexosaminidases
Authors: Mark Brian L , James Michael NG
Published: October 2002

Configuration retaining glycosidases catalyse the hydrolysis of glycosidic bonds via a double displacement mechanism, typically involving two key active site carboxyl groups (Glu or Asp). One of the enzymic carboxyl groups functions as a general acidbase catalyst, the other acts as a nucleophile. Alternatively, configuration-retaining hexosaminidases from the sequence-related glycosidase families 18, 20, and 56 lack a suitably positioned enzymic nucleophile; instead, they use the carbonyl oxygen atom of the neighbouring C2-acetamido group of the substrate. The carbonyl oxygen atom of the 2-acetamido group provides anchimeric assistance to the enzyme catalyzed reaction by acting as an intramolecular nucleophile, attacking the anomeric center and forming a cyclized oxazolinium ion intermediate that is stereochemically equivalent to the glycosylenzyme intermediate formed in the "normal" double displacement mechanism. Although there is little sequence similarity between families 18, 20, and 56 hexosaminidases, X-ray crystallographic studies demonstrate that they have evolved similar catalytic domains and active site architectures that are designed to distort the bound substrate so that the C2-acetamido group can become appropriately positioned to participate in catalysis. The substrate distortion allows for a substrate-assisted catalytic reaction that displays all the general characteristics of the classic double-displacement mechanism including the formation of a covalent intermediate.Key words: glycoside hydrolase, hexosaminidase, glycosidase, substrate-assisted catalysis, anchimeric assistance.
REFINED SIMULATION FOR THE DIFFUSION IN Sivia CHANDRASEKHAR HOPPING
Authors: ROY S. D. D. , RAMACHANDRAN K.
Published: October 2002

The method employed by Chandrasekhar for the astronomical bodies is brought down here to study the diffusion in Silicon. A continuous position probability density for the diffusing particle, ω(r, t) representing the position of the diffusing particle at any time t, is used in the evaluation of the diffusion constant. The results agree reasonably well with the available experimental and theoretically reported values. The existence of "traps" in the semiconducting systems has been clearly brought out by this simulation technique.
COSMIC CENSORSHIP: A CURRENT PERSPECTIVE
Authors: JOSHI PANKAJ S.
Published: October 2002

End state of gravitational collapse and the related cosmic censorship conjecture continue to be amongst the most important open problems in gravitation physics today. My purpose here is to bring out several aspects related to gravitational collapse and censorship, which may help towards a better understanding of the issues involved. Possible physical constraints on gravitational collapse scenarios are considered. It is concluded that the best hope for censorship lies in analyzing the genericity and stability properties of the currently known classes of collapse models which lead to the formation of naked singularities, rather than black holes, as the final state of collapse and which develop from a regular initial data.
THE EQUIVALENCE PRINCIPLE AND QUANTUM MECHANICS: A THEME IN HARMONY
Authors: UNNIKRISHNAN C. S.
Published: October 2002

There have been many speculations that the equivalence principle might be violated in situations involving quantum systems in a classical gravitational field. Also, several authors have stated that experiments involving quantum systems contain quantum signatures, specific to the gravitational interaction, that are fundamentally different from what could be explored in classical experiments. I critically assess these statements and prove the validity of the equivalence principle for general quantum systems in a classical gravitational field. Further, I argue that there should not be any expectation of violation of the equivalence principle in the neutrino sector. I end with some comments on a basic formulation of the equivalence principle in the quantum context.
CREATION OF DIRAC PARTICLES IN THE PRESENCE OF A CONSTANT ELECTRIC FIELD IN AN ANISOTROPIC BIANCHI I UNIVERSE
Authors: VILLALBA VÍCTOR M. , GREINER WALTER
Published: October 2002

In this article we compute the density of Dirac particles created by a cosmological anisotropic Bianchi I universe in the presence of a constant electric field. We show that the particle distribution becomes thermal when one neglects the electric interaction.
Effect of rapamycin on the cyclosporin A–resistant CD28-mediated costimulatory pathway
Authors: Ghosh Paritosh , Buchholz Meredith A. , Yano Shingo , Taub Dennis , Longo Dan L.
Published: October 2002

The consequences of T-cell activation depend exclusively on costimulation during antigen–T-cell receptor interaction. Interaction between the T-cell coreceptor CD28 and its ligand B7 during antigen-antigen receptor engagement results in full activation of T cells, the outcomes of which are proliferation and effector functions. The ability of CD28 to costimulate the production of interleukin-2 (IL-2) explains the importance of this costimulation. The signaling event mediated by CD28 engagement has been proposed to have 2 components: one is sensitive to the immunosuppressive drug cyclosporin A (CsA), and the other one is CsA-resistant. In this report, we demonstrate that the CsA-resistant pathway is sensitive to the immunosuppressive drug rapamycin. Treatment with rapamycin blocked IL-2 production after activation of human peripheral blood T cells with phorbol ester (PMA) and anti-CD28 (CsA-resistant pathway), whereas this drug did not have any effect on PMA plus ionomycin stimulation (CsA-sensitive pathway). The inhibitory effect of rapamycin was on messenger RNA stability and translation, rather than on IL-2 transcription or protein turnover.
Host defense role of platelets: engulfment of HIV andStaphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation
Authors: Youssefian Tayebeh , Drouin Arnaud , Massé Jean-Marc , Guichard Josette , Cramer Elisabeth M.
Published: October 2002

Platelets can bind and phagocytose infectious microorganisms and so enable their transport for a prolonged time. To investigate the subcellular events of these interactions, platelets were incubated either with Staphylococcus aureus or with HIV and analyzed by electron microscopy (EM) and immuno-EM. HIV and bacteria internalization occurred exclusively within platelets showing morphological evidence of activation. Platelet activation enhanced the degree of bacterial internalization. Immunolabeling revealed that the engulfing vacuoles and the open canalicular system (OCS) were composed of distinct antigens. The engulfing vacuoles eventually became the site of prominent α-granule release. In platelets incubated with HIV, characteristic endocytic vacuoles were identified close to the plasma membrane, tightly surrounding 1 or 2 HIV particles. Virus particles were also located within the OCS. Immunogold labeling for the viral core protein p24 confirmed the presence of HIV within platelets. Finally, examination of platelets from a patient with acquired immunodeficiency syndrome and high viremia suggested that HIV endocytosis may also occur in vivo.
Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL)
Authors: Lundin Jeanette , Kimby Eva , Björkholm Magnus , Broliden Per-Anders , Celsing Fredrik , Hjalmar Viktoria , Möllgård Lars , Rebello Peppy , Hale Geoff , Waldmann Herman , Mellstedt Håkan , Österborg Anders
Published: October 2002

This phase II study determined the efficacy and safety of alemtuzumab, a humanized anti-CD52 monoclonal antibody, delivered subcutaneously as first-line therapy, over a prolonged treatment period of 18 weeks in 41 patients with symptomatic B-cell chronic lymphocytic leukemia (B-CLL). Injections were administered subcutaneously 3 times per week, from week 2 to 3 onward. An overall response rate (OR) of 87% (95% CI, 76%-98%; complete remission [CR], 19%; partial remission [PR], 68%) was achieved in 38 evaluable patients (81% of intent-to-treat population). CLL cells were cleared from blood in 95% patients in a median time of 21 days. CR or nodular PR in the bone marrow was achieved in 66% of the patients and most patients achieved this after 18 weeks of treatment. An 87% OR (29% CR) was achieved in the lymph nodes. The median time to treatment failure has not yet been reached (18+ months; range, 8-44+ months). Transient injection site skin reactions were seen in 90% of patients. Rigor, rash, nausea, dyspnea, and hypotension were rare or absent. Transient grade IV neutropenia developed in 21% of the patients. Infections were rare, but 10% patients developed cytomegalovirus (CMV) reactivation. These patients rapidly responded to intravenous ganciclovir. One patient, allergic to cotrimoxazole prophylaxis, developedPneumocystis carinii pneumonia. Alemtuzumab is highly effective as first-line treatment in patients with B-CLL. Prolonged treatment is important for maximal bone marrow response. Subcutaneous administration induced very few “first-dose” flulike symptoms and may reduce health care costs in comparison with the intravenous infusions.
Transcobalamin II 775G>C polymorphism and indices of vitamin B12 status in healthy older adults
Authors: Miller Joshua W. , Ramos Marisa I. , Garrod Marjorie G. , Flynn Margaret A. , Green Ralph
Published: October 2002

A common polymorphism (775G>C) in the vitamin B12 transport protein, transcobalamin II (TCII), has been identified in which proline replaces arginine at codon 259. We determined the influence of TCII genotype on indices of B12 status, including total serum B12, the amount of B12 bound to TCII (holoTCII), methylmalonic acid, and homocysteine, in 128 healthy older adults (ages 40-88 years). Mean total B12 and homocysteine concentrations were not significantly different among the 3 genotypes. Mean holoTCII concentration was significantly higher in those subjects homozygous for the proline form of TCII (PP) compared with those homozygous for the arginine form (RR) and heterozygotes (PR) (P ≤ .006). In addition, mean methylmalonic acid concentrations were significantly lower in the PP and PR groups compared with the RR group (P ≤ .02). The PP genotype may be more efficient in delivering B12 to tissues, resulting in enhanced B12 functional status. TCII genotype may thus influence susceptibility to B12 deficiency.
Prospective study of hepatitis C viral infection as a risk factor for subsequent B-cell neoplasia
Authors: Rabkin Charles S. , Tess Beatriz H. , Christianson Roberta E. , Wright William E. , Waters David J. , Alter Harvey J. , van den Berg Bea J.
Published: October 2002

Several case-control studies have found increased prevalence of hepatitis C virus (HCV) in patients with non-Hodgkin lymphoma (NHL) and other B-cell lymphoproliferative disorders. We examined whether HCV infection preceded the development of these neoplasms in a prospective cohort study of 48 420 individuals in northern California. Stored sera from 95 subjects with NHL (n = 57), multiple myeloma (n = 24), or Hodgkin disease (n = 14) diagnosed a mean of 21 years after phlebotomy were screened for antibodies to HCV as well as viral RNA, based on previous reports of antibody-negative viremia. Sera from 4 cases and one of 95 age-, sex-, and race-matched controls were repeatedly reactive by enzyme immunoassay, but none were confirmed by recombinant immunoblot assay; none of the case sera had HCV RNA by reverse transcription– polymerase chain reaction. Although acquisition in later life cannot be ruled out, these prospective data do not support a substantial role of chronic HCV infection in the etiology of B-cell neoplasia.
Functional characterization of recombinant FV Hong Kong and FV Cambridge
Authors: Norstrøm Eva , Thorelli Elisabeth , Dahlbäck Björn
Published: October 2002

In factor V (FV) Cambridge (Arg306Thr) and Hong Kong (Arg306Gly), a cleavage site for anticoagulant activated protein C (APC), which is crucial for the inactivation of FVa, is lost. Although patients carrying FV Hong Kong have a normal APC response, those with FV Cambridge were reported to be APC resistant. To elucidate the molecular characteristics of the 2 FV mutants, we recreated them in a recombinant system and evaluated their functional properties. The 2 FV variants yielded identical APC resistance patterns, with APC responses being intermediate to those of wild-type FV and FV Leiden (Arg506Gln), which is known to be associated with the APC resistance phenotype. In the absence of protein S, APC mediated FVa inactivation curves obtained with the 2 variants were identical, resulting in partial FVa inactivation. In the presence of protein S, both FVa variants were almost completely inactivated because of protein S stimulation of the cleavage at Arg679. In a FVIIIa degradation system, both FV variants demonstrated slightly impaired APC cofactor activity. The ability of APC to cleave at Arg506 and at Arg679 in FVa Cambridge and Hong Kong and the slight decrease in APC cofactor activity of the 2 FV variants may explain the low thrombotic risk associated with these Arg306 mutations. In conclusion, we demonstrate that recombinant FV Cambridge and Hong Kong behave identically in in vitro assays and provide a mechanism for the low thrombotic risk associated with these FV mutations.
Rapamycin inhibits macropinocytosis and mannose receptor–mediated endocytosis by bone marrow–derived dendritic cells
Authors: Hackstein Holger , Taner Timucin , Logar Alison J. , Thomson Angus W.
Published: October 2002

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that use 2 major pathways for antigen uptake: constitutive macropinocytosis and mannose receptor–mediated endocytosis. Efficient endocytosis is critical for DCs to fulfill their sentinel function in immunity. We investigated the influence of the immunosuppressive macrolide rapamycin on macropinocytosis of fluorescein isothiocyanate (FITC)–albumin and mannose receptor–mediated endocytosis of FITC-dextran by murine bone marrow–derived DCs by flow cytometry. The data show that (1) at a low, physiologically relevant concentration (1 ng/mL), rapamycin impairs macropinocytosis and mannose receptor–mediated endocytosis; (2) the effects are independent of DC maturation and can be demonstrated specifically in immature CD11c+ major histocompatibility complex (MHC) class IIlo DCs by 3-color flow cytometry; (3) inhibition of endocytosis is not related to apoptotic cell death; and (4) molar excess of the structurally related molecule FK506 inhibits the actions of rapamycin. The inhibitory effects of rapamycin on DC endocytosis were confirmed in vivo. To our knowledge, this is the first report that a clinically relevant immunosuppressant inhibits DC endocytosis.
Stomatin is a major lipid-raft component of platelet α granules
Authors: Mairhofer Mario , Steiner Marianne , Mosgoeller Wilhelm , Prohaska Rainer , Salzer Ulrich
Published: October 2002

Lipid rafts are detergent-resistant, cholesterol- and sphingolipid-rich membrane domains that are involved in important cellular processes such as signal transduction and intracellular trafficking. Stomatin, a major lipid-raft component of erythrocytes and epithelial cells, is also an abundant platelet protein. Microscopical methods and subcellular fractionation showed that stomatin is located mainly at the α-granular membrane. The lipid-raft marker proteins flotillin-1 and flotillin-2 were also present in platelets but excluded from α granules. Stomatin and the flotillins were associated with Triton X-100–insoluble lipid rafts. Whereas stomatin was partly soluble in Triton X-100, it was insoluble in the detergents Lubrol and 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (CHAPS). Flotation experiments after CHAPS lysis of platelets revealed a distinct set of lipid-raft–associated proteins, which were identified by matrix-assisted laser desorption/ionization mass spectrometry as stomatin, flotillin-1, flotillin-2, CD36, CD9, integrin αIIbβ3, and the glucose transporter GLUT-3. Stomatin, the flotillins, and CD36 were exclusively present in this lipid-raft fraction. Activation of platelets by calcium ionophore A23187 or thrombin led to translocation of stomatin to the plasma membrane, cleavage by calpain, and specific sorting into released microvesicles. In conclusion, this study demonstrated the existence of α-granular lipid rafts and suggests an important role for stomatin in the organization and function of α granules.
5′ CpG island hypermethylation is associated with transcriptional silencing of the p21CIP1/WAF1/SDI1 gene and confers poor prognosis in acute lymphoblastic leukemia
Authors: Roman-Gomez Jose , Castillejo Juan Antonio , Jimenez Antonio , Gonzalez Maria Gracia , Moreno Fernanda , Rodriguez Maria del Carmen , Barrios Manuel , Maldonado Juan , Torres Antonio
Published: October 2002

The p21 is a downstream effector of p53/p73 and belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs). It is, therefore, a potential tumor suppressor gene and probably plays an important role in tumor development. Moreover, reduced expression of p21 has been reported to have prognostic value in several human malignancies. In contrast with other CDKIs, mutational inactivation of p21 is infrequent, but gene inactivation by an alternative mechanism seems to be the general pathway. In this study, we analyzed the methylation status of the p21 promoter region using semiquantitative polymerase chain reaction in 124 patients with acute lymphoblastic leukemia (ALL). We observed p21 hypermethylation in bone marrow cells from 41% (51 of 124) of ALL patients. Hypermethylation within promoter strongly correlated with decreased p21 messenger RNA expression in tumoral cells. Clinical, molecular, and laboratory features and complete remission rate did not differ significantly between hypermethylated and normally methylated patients. Estimated disease-free survival (DFS) and overall survival at 7 and 9 years, respectively, were 59% and 65% for healthy patients and 6% and 8% for hypermethylated patients (P = .00001 andP = .006). Multivariate analysis of potential prognostic factors demonstrated that p21 methylation status was an independent prognostic factor in predicting DFS (P = .0001). Our results indicate that the p21 gene is subject to methylation regulation at the transcription level in ALL and seems to be an important factor in predicting the clinical outcome of these patients.
Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss
Authors: Thornley Ian , Sutherland Robert , Wynn Robert , Nayar Rakash , Sung Lillian , Corpus George , Kiss Thomas , Lipton Jeff , Doyle John , Saunders Fred , Kamel-Reid Suzanne , Freedman Melvin , Messner Hans
Published: October 2002

Our inability to purify hematopoietic stem cells (HSCs) precludes direct study of many aspects of their behavior in the clinical hematopoietic stem cell transplantation (HSCT) setting. We indirectly assessed stem/progenitor cell behavior in the first year after HSCT by examining changes in neutrophil telomere length, X-inactivation ratios, and cycling of marrow progenitors in 25 fully engrafted allogeneic HSCT recipients. Donors were sampled once and recipients at engraftment and 2 to 6 months and 12 months after HSCT. Telomere length was measured by an in-gel hybridization technique, X-inactivation ratios were measured by the human androgen receptor assay, and cell cycle status was determined by flow cytometric analysis of pyronin Y- and Hoechst 33342–stained CD34+CD90+ and CD34+CD90− marrow cells. Compared with their donors, recipients' telomeres were shortened at engraftment (−424 base pairs [bp]; P < .0001), 6 months (−495 bp; P = .0001) after HSCT, and 12 months after HSCT (−565 bp; P < .0001). There was no consistent pattern of change in telomere length from 1 to 12 months after HSCT; marked, seemingly random, fluctuations were common. In 11 of 11 informative recipients, donor X-inactivation ratios were faithfully reproduced and maintained. The proportion of CD34+CD90+ progenitors in S/G2/M was 4.3% in donors, 15.7% at 2 to 6 months (P < .0001) after HSCT, and 11.5% at 12 months after HSCT (P < .0001, versus donors; P = .04, versus 2-6 months). Cycling of CD34+ CD90−progenitors was largely unchanged. We infer that (1) HSCT-induced accelerated telomere loss is temporary and unlikely to promote graft failure or clonal hematopoietic disorders and (2) the striking fluctuations in telomere length and variation in pattern of telomere loss reflect stochastic determination of HSC fate after HSCT.
Human single chain antibodies against heparin: selection, characterization, and effect on coagulation
Authors: van de Westerlo Els M. A. , Smetsers Toon F. C. M. , Dennissen Michel A. B. A. , Linhardt Robert J. , Veerkamp Jacques H. , van Muijen Goos N. P. , van Kuppevelt Toin H.
Published: October 2002

Heparin, located in mast cells and basophilic granulocytes, is widely used as an anticoagulant. It belongs to a class of linear polysaccharides called glycosaminoglycans (GAGs). Using phage display technology, we have selected 19 unique human antiheparin antibodies. Some antibodies react almost exclusively with heparin, others also react with the structurally related heparan sulfate, and some with chondroitin sulfate. In all cases, sulfate groups are essential for binding. For activity of some antibodies, O-sulfation is more important than N-sulfation. Antibodies are reactive with heparin in mast cells. Each antibody showed a defined staining pattern on cryosections of rat kidney, pancreas, and testis. Enzymatic digestion with glycosidases on tissue sections further indicated that the antibodies are specific for GAGs. All antibodies recognize a unique epitope. The effect of the antibodies on heparin as an anticoagulant was also studied. There were 3 antibodies that were very effective inhibitors of heparin action in the activated partial thromboplastin time (APTT) clotting assay, and their effect was related to the amount of heparin bound. Some antibodies reacted strongly with the pentasaccharide, which interacts with antithrombin III. The human antibodies selected represent unique tools to study the structure, location, and function of heparin and related GAGs, and some may be used as blocking agents.
A hereditary bleeding disorder of dogs caused by a lack of platelet procoagulant activity
Authors: Brooks Marjory B. , Catalfamo James L. , Brown H. Alex , Ivanova Pavlina , Lovaglio Jamie
Published: October 2002

We have discovered a novel canine hereditary bleeding disorder with the characteristic features of Scott syndrome, a rare defect of platelet procoagulant activity. Affected dogs were from a single, inbred colony and experienced clinical signs of epistaxis, hyphema, intramuscular hematoma, and prolonged bleeding with cutaneous bruising after surgery. The hemostatic abnormalities identified were restricted to tests of platelet procoagulant activity, whereas platelet count, platelet morphology under light microscopy, bleeding time, clot retraction, and platelet aggregation and secretion in response to thrombin, collagen, and adenosine diphosphate stimulation were all within normal limits. Washed platelets from the affected dogs demonstrated approximately twice normal clotting times in a platelet factor 3 availability assay and, in a prothrombinase assay, generated only background levels of thrombin in response to calcium ionophore, thrombin, or combined thrombin plus collagen stimulation. While platelet phospholipid content was normal, flow cytometric analyses revealed diminished phosphatidylserine exposure and a failure of microvesiculation in response to calcium ionophore, thrombin, and collagen stimulation. Pedigree studies indicate a likely homozygous recessive inheritance pattern of the defect. These findings confirm the importance of platelet procoagulant activity for in vivo hemostasis and provide a large animal model for studying agonist-induced signal transduction, calcium mobilization, and effector pathways involved in the late platelet response of transmembrane phospholipid movement and membrane vesiculation.
Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia
Authors: Crans-Vargas Heather N. , Landaw Elliot M. , Bhatia Smita , Sandusky George , Moore Theodore B. , Sakamoto Kathleen M.
Published: October 2002

Cyclic adenosine monophosphate response-element binding protein (CREB) is a nuclear protein that regulates expression of genes that control cell proliferation, differentiation, and survival. To analyze CREB expression in leukemia cells, we conducted Western blot analysis of bone marrow cells obtained from patients with acute lymphoblastic leukemia, patients with acute myeloid leukemia, and patients without active leukemia. CREB was expressed at a higher frequency in bone marrow cells from patients with acute lymphoid or myeloid leukemia than in patients with leukemia remission or without leukemia. Our results indicate that CREB expression could be a useful marker for leukemia in patients with acute disease and suggest a role for CREB in leukemogenesis.
CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor
Authors: Wang Qian-fei , Friedman Alan D.
Published: October 2002

Potential redundancy among members of the CCAAT/enhancer-binding protein (C/EBP) family in myeloid cells is indicated by the ability of C/EBPβ to replace C/EBPα in vivo, by the expression of granulocyte colony-stimulating factor receptor (G-CSFR) on C/EBPα−/− cell lines, and by our finding that as with C/EBPα–estrogen receptor (C/EBPα-ER), either C/EBPβ-ER or C/EBPδ-ER can induce terminal granulopoiesis in 32D cl3 cells. To assess the consequences of globally inhibiting C/EBPs, we employed KαER, containing a Kruppel-associated box (KRAB) transrepression domain, the C/EBPα DNA-binding domain, and an ER ligand-binding domain. C/EBPs have a common DNA-binding consensus, and activation of KαER repressed transactivation by endogenous C/EBPs 50-fold and reduced endogenous G-CSFR expression. In 32D cl3 cells coexpressing exogenous G-CSFR, activation of KαER prevented and even reversed myeloperoxidase, lysozyme, lactoferrin, and C/EBPε RNA induction by G-CSF. In contrast, induction of PU.1 and CD11b, a gene regulated by PU.1 but not by C/EBPs, was unaffected. A KαER variant incapable of binding DNA owing to an altered leucine zipper did not affect 32D cl3 differentiation. Transduction of KαER into murine hematopoietic progenitor cells suppressed the formation of granulocyte colony-forming units, even in cytokines that enable C/EBPα−/−progenitors to differentiate into neutrophils. The formation of macrophage and of granulocyte-macrophage colony-forming units were also inhibited, but erythroid burst-forming units grew normally. Thus, in 32D cl3 cells and perhaps normal progenitors, C/EBPs are required for granulopoiesis beyond their ability to induce receptors for G-CSF and other cytokines. One requisite activity may be activation of the C/EBPε gene by C/EBPα, as either C/EBPα-ER or C/EBPβ-ER rapidly elevated C/EBPε RNA in 32D cl3 cells in the presence of cycloheximide but not actinomycin D.
Human mast cell progenitors use α4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions
Authors: Boyce Joshua A. , Mellor Elizabeth A. , Perkins Brandy , Lim Yaw-Chyn , Luscinskas Francis W.
Published: October 2002

Mast cells (MCs) are central to asthma and other allergic diseases, and for responses to infection and tissue injuries. MCs arise from committed progenitors (PrMCs) that migrate from the circulation to tissues by incompletely characterized mechanisms, and differentiate in situ in perivascular connective tissues of multiple organs. PrMCs derived in vitro from human cord blood were examined for adhesion molecule expression and their ability to adhere to human umbilical vein endothelial cells (HUVECs) under conditions that mimic physiologic shear flow. The PrMCs expressed α4β1, low levels of β7, and the β2-integrins αLβ2 and αMβ2. The PrMCs also expressed PSGL-1, but not L-selectin. At low (0.5 dynes/cm2-1.0 dynes/cm2) shear stress, PrMCs attached and rolled on recombinant E-selectin and P-selectin and VCAM-1. An anti–PSGL-1 monoclonal antibody (mAb) blocked essentially all adhesion to P-selectin but reduced adhesion to E-selectin by only 40%, suggesting PrMCs express other ligands for E-selectin. PrMCs adhered strongly to tumor necrosis factor-α (TNF-α)–activated HUVECs, whereas adhesion to interleukin 4 (IL-4)–activated HUVECs was lower. PrMC adhesion to IL-4–activated HUVECs was totally α4-integrin– and VCAM-1–dependent. Adhesion to TNF-α–activated HUVECs was blocked by 50% by mAbs against α4-integrin, vascular cell adhesion molecule–1 (VCAM-1), E-selectin, or PSGL-1, whereas combinations of mAbs to α4-integrin plus PSGL-1, or VCAM-1 plus E-selectin, blocked adhesion by greater than 70%. Thus, PrMCs derived in vitro predominantly use α4-integrin, VCAM-1, PSGL-1, and other ligands that bind E-selectin for adhesion to cytokine-activated HUVEC monolayers. These observations may explain the abundance of MCs at sites of mucosal inflammation, where VCAM-1 and E-selectin are important inducible receptors.
The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia–like disease by bcr/abl
Authors: He Yiping , Wertheim Jason A. , Xu Lanwei , Miller Juli P. , Karnell Fredrick G. , Choi John K. , Ren Ruibao , Pear Warren S.
Published: October 2002

The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Δ(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Δ(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.
A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis
Authors: Miyashita Hiroki , Yamazaki Tohru , Akada Tetsuya , Niizeki Osamu , Ogawa Minetaro , Nishikawa Shin-ichi , Sato Yasufumi
Published: October 2002

Using polymerase chain reaction–coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.
Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application
Authors: von Bergwelt-Baildon Michael S. , Vonderheide Robert H. , Maecker Britta , Hirano Naoto , Anderson Karen S. , Butler Marcus O. , Xia Zhinan , Zeng Wan Y. , Wucherpfennig Kai W. , Nadler Lee M. , Schultze Joachim L.
Published: October 2002

CD40 engagement is the major signal that induces B cells to efficiently present antigen to T cells. We previously demonstrated that human peripheral blood–derived CD40-activated B cells (CD40–B cells) function as antigen-presenting cells (APCs). Here, we have established a culture system to generate these APCs under clinically applicable conditions using guanylic acid–grade soluble trimeric CD40 ligand. To monitor APC function and antigen loading for these cells, simple and efficient quality control assays have been developed. Using this approach, we demonstrate that CD40–B cells from healthy donors and cancer patients are fully functional and equally expanded in long-term cultures. These B cells boost robust memory T-cell responses, but more importantly, they also prime naive T-cell responses against neoantigens ex vivo. CD40–B cells overcome current obstacles, such as the difficulty of isolation, generation, and long-term expansion observed with other APCs. Therefore, they are an excellent source of professional APCs for immune assessment, antigen discovery, and antigen-specific immunotherapy.
Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia
Authors: Milella Michele , Estrov Zeev , Kornblau Steven M. , Carter Bing Z. , Konopleva Marina , Tari Ana , Schober Wendy D. , Harris David , Leysath Clinton E. , Lopez-Berestein Gabriel , Huang Ziwei , Andreeff Michael
Published: October 2002

Recent studies suggest that the Bcl-2 and mitogen-activated protein kinase (MAPK) pathways together confer an aggressive, apoptosis-resistant phenotype on acute myelogenous leukemia (AML) cells. In this study, we analyzed the effects of simultaneous inhibition of these 2 pathways. In AML cell lines with constitutively activated MAPK, MAPK kinase (MEK) blockade by PD184352 strikingly potentiated the apoptosis induced by the small-molecule Bcl-2 inhibitor HA14-1 or by Bcl-2 antisense oligonucleotides. Isobologram analysis confirmed the synergistic nature of this interaction. Moreover, MEK blockade overcame Bcl-2 overexpression-mediated resistance to the proapoptotic effects of HA14-1. Most importantly, simultaneous exposure to PD184352 significantly (P = .01) potentiated HA14-1–mediated inhibition of clonogenic growth in all primary AML samples tested. These findings show that the Bcl-2 and MAPK pathways are relevant molecular targets in AML and that their concurrent inhibition could be developed into a new therapeutic strategy for this disease.
SIGNIFICANCE OF SOME FUNDAMENTAL PROPERTIES OF EXPLOSIVES, WITH SPECIAL REFERENCE TO GEOPHYSICAL PROSPECTING
Authors: Nash H. E. , Martin J. M.
Published: October 2002

This discussion is concerned with certain factors influencing the results from explosives in geophysical prospecting. The study is intended to acquaint the geophysicist with the pressures developed in the drill hole when the explosive charge is detonated, the temperature of the explosive gases, the amount of available energy released, and the proportion of this energy which is converted into useful seismic energy.