bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Distinct Jacalin-Related Lectins with a Different Specificity and Subcellular Location Are Major Vegetative Storage Proteins in the Bark of the Black Mulberry Tree
Authors: Van Damme Els J.M. , Hause Bettina , Hu Jialiang , Barre Annick , Rougé Pierre , Proost Paul , Peumans Willy J.
Published: October 2002

Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.
The Abundant Class III Chitinase Homolog in Young Developing Banana Fruits Behaves as a Transient Vegetative Storage Protein and Most Probably Serves as an Important Supply of Amino Acids for the Synthesis of Ripening-Associated Proteins
Authors: Peumans Willy J. , Proost Paul , Swennen Rony L. , Van Damme Els J.M.
Published: October 2002

Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a β-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas.
Photorespiratory NH4 + Production in Leaves of Wild-Type and Glutamine Synthetase 2 Antisense Oilseed Rape
Authors: Husted Søren , Mattsson Marie , Möllers Christian , Wallbraun Michael , Schjoerring Jan K.
Published: October 2002

Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15°C and 25°C increased photorespiratory NH4  + production from 0.7 to 3.5 μmol m−2 s−1. Despite the 5-fold increase in the rate of NH4  + production, the NH4  + concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH4  + production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH4  + concentrations than wild-type plants and no overall correlation between GS activity and tissue NH4  + concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14°C to 27°C or to a trifold increase in the O2 to CO2 ratio did not show any change in steady-state leaf tissue NH4  + concentration or in NH3emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH4  + in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH3 fluxes.
Transcriptional and Posttranscriptional Regulation of ArabidopsisTCH4 Expression by Diverse Stimuli. Roles of cis Regions and Brassinosteroids
Authors: Iliev Emanuil A. , Xu Wei , Polisensky Diana H. , Oh Man-Ho , Torisky Rebecca S. , Clouse Steven D. , Braam Janet
Published: October 2002

The Arabidopsis TCH4 gene is up-regulated in expression by diverse environmental and hormonal stimuli. BecauseTCH4 encodes a xyloglucan endotransglucosylase/hydrolase, this change in expression may reflect a recruitment of cell wall-modifying activity in response to environmental stress and growth. How diverse stimuli lead to the common response of TCH4 expression regulation is not known. Here, we show that induction of expression by the diverse stimuli of touch, darkness, cold, heat, and brassinosteroids (BRs) is conferred to reporter genes by the same 102-bp 5′-untranscribed TCH4region; this result is consistent with the idea that shared regulatory elements are employed by diverse stimuli. Distal regions influence magnitude and kinetics of expression and likely harbor regulatory elements that are redundant with those located more proximal to the transcriptional start site. Substitution of the proximal regulatory region sequences in the context of distal elements does not disrupt inducible expression. TCH4 expression induction is transcriptional, at least in part because 5′-untranscribed sequences are sufficient to confer this regulation. However, 5′-untranslated sequences are necessary and sufficient to confer the marked transience of TCH4 expression, most likely through an effect on mRNA stability. Perception of BR is not necessary forTCH4::GUS induction by environmental stimuli because regulation is intact in the BR-insensitive mutant,bri1-2. The full response to auxin, however, requires the functioning of BRI1. Developmental expression ofTCH4 is unlikely to be meditated by BR becauseTCH4::GUS is expressed in BR perception and biosynthetic mutants bri1-2 and det2-1, respectively.
The synthesis of a carbohydrate-like dihydrooxazine and tetrahydrooxazine as putative inhibitors of glycoside hydrolases: A direct synthesis of isofagomine
Authors: Best Wayne M , Macdonald James M , Skelton Brian W , Stick Robert V , Tilbrook D Matthew G , White Allan H
Published: October 2002

The treatment of benzyl 2,3-O-isopropylidene-β-L-xylopyranoside with N-hydroxyphthalimide under Mitsunobu conditions, followed by protecting-group interchange, gave benzyl 4-O-[(tert-butoxycarbonyl)amino]-2,3- O-isopropylidene-α-D-arabinoside. Mild acid hydrolysis and catalytic hydrogenolysis afforded 4-O-[(tert-butoxycarbonyl)amino]-D-arabinose that, upon heating in water, gave the dihydrooxazine [(4R,5S,6R)-5,6-dihydro-4,5-dihydroxy-6-hydroxymethyl-4H-1,2-oxazine] as a crystalline solid. A single-crystal structure determination of this solid showed it to exist in the 5H6 conformation. Reduction of the dihydrooxazine gave the tetrahydrooxazine [(4R,5S,6R)-4,5-dihydroxy-6-hydroxymethyl-3,4,5,6-tetrahydro-2H-1,2-oxazine]. The dihydrooxazine was an effective inhibitor of two β-glucosidases (Ki = 27 and 35 µM). Benzyl 2,3-O-isopropylidene-β-L-xylopyranoside, via the derived imidazylate, was converted into a nitrile that, upon reduction and protecting-group manipulations, gave benzyl 4-C-aminomethyl-4-deoxy-α-D-arabinoside. Treatment of this amine with hydrogen and palladium-on-carbon gave isofagomine.Key words: dihydrooxazine, tetrahydrooxazine, isofagomine, iminosugars, glycosidase inhibitors.
Potentiometric study of equilibrium acidities of some carbon acids in acetonitrile
Authors: Stanczyk-Dunaj M , Galezowski W , Jarczewski A
Published: October 2002

The pKavalues for 30 C-acids of the type CH·R1·R2·R3 where the R1, R2, and R3substituents are H, alkyl, and aryl, and the most common electron withdrawing groups used in studies of proton-transfer reaction  are given herein. The pKavalues have been measured potentiometrically in acetonitrile, using a glass electrode. These values were determined in a buffer solution consisting of a C-acid and its tetrabutylammonium salt, at constant ionic strength (0.001 M), at 25°C. The measurements were done under nitrogen, to prevent oxidation of the anion. The influence of water within the sample of the tetrabutylammonium hydroxide base was negligible. The lowest pKavalue of 19.1 was found for bis(4-nitrophenyl)cyanomethane, while the largest pKas that could be measured were limited by the basicity of the hydroxide ion, and did not exceed 28. A satisfactory agreement between spectrophotometric and potentiometric acidities was achieved. Key words: C-acids, pKavalues, acetonitrile, potentiometry, proton transfer.
Stratification by ancillary data in multisource forest inventories employing k-nearest-neighbour estimation
Authors: Katila M , Tomppo E
Published: October 2002

The Finnish multisource national forest inventory (MS-NFI) utilizes optical area satellite images and digital maps in addition to field plot data to produce georeferenced information, thematic maps, and small-area statistics. In the early version, forestry land (FRYL) was taken directly from the numerical map data. Such data may be outdated and can contain significant errors, for example, the FRYL area is typically overestimated and the mean volume is underestimated. A statistical calibration method has been introduced to reduce the map errors on multisource forest resource estimates. It is based on large-area estimates of map errors, a confusion matrix among land-use classes of the field sample plots, and corresponding map information. The method has some drawbacks: calculations are more complicated than in the original MS-NFI and some field plots may have negative expansion factors. The paper presents a new stratified MS-NFI method to reduce the effect of inaccurate map data on the forest-resource estimates. In this method, the k-nearest-neighbour (k-NN) estimation is applied by strata. All the field plots within each map stratum, independently of their land-use classification by field crew, are used to estimate the areas of land-use classes and forest variables of that stratum. The method was tested on two large areas containing three Landsat 5 TM scenes and field-inventory data from the ninth NFI. The stratified MS-NFI is essentially a different estimation method compared with the calibrated MS-NFI, which calibrates the MS-NFI estimates more or less systematically in one direction. The stratified MS-NFI was found to be statistically simpler and there were fewer significant errors in the estimates than in the calibrated MS-NFI.
SAKHAROV'S INDUCED GRAVITY: A MODERN PERSPECTIVE
Authors: VISSER MATT
Published: October 2002

Sakharov's 1967 notion of "induced gravity" is currently enjoying a significant resurgence. The basic idea, originally presented in a very brief 3-page paper with a total of four formulas, is that gravity is not "fundamental" in the sense of particle physics. Instead it was argued that gravity (general relativity) emerges from quantum field theory in roughly the same sense that hydrodynamics or continuum elasticity theory emerges from molecular physics. In this article we will translate the key ideas into modern language, and explain the various versions of Sakharov's idea currently on the market.
GRAVITATIONAL FIELD OF A ROTATING GRAVITATIONAL DYON
Authors: DADHICH N. , TURAKULOV Z. YA.
Published: October 2002

We have obtained the general solution of the Einstein vacuum equation for the axially symmetric stationary metric in which both the Hamilton-Jacobi equation for particle motion and the Klein - Gordon equation are separable. It can be interpreted to describe the gravitational field of a rotating dyon, a particle endowed with both gravoelectric (mass) and gravomagnetic (NUT parameter) charges. Further, there also exists a duality relation between the two charges and the radial and the polar angle coordinates which keeps the solution invariant. The solution can however be transformed into the known Kerr - NUT solution indicating its uniqueness under the separability of equations of motion.
THERMODYNAMICS OF HORIZONS: A COMPARISON OF SCHWARZSCHILD, RINDLER AND de SITTER SPACETIMES
Authors: PADMANABHAN T.
Published: October 2002

The notions of temperature, entropy and 'evaporation', usually associated with spacetimes with horizons, are analyzed using general approach and the following results, applicable to different spacetimes, are obtained at one go. (i) The concept of temperature associated with the horizon is derived in a unified manner and is shown to arise from purely kinematic considerations. (ii) QFT near any horizon is mapped to a conformal field theory without introducing concepts from string theory. (iii) For spherically symmetric spacetimes (in D = 1 + 3) with a horizon at r = l, the partition function has the generic form Z ∝ exp [S - βE], where S = (1/4)4πl2 and |E| = (l/2). This analysis reproduces the conventional result for the black hole spacetimes and provides a simple and consistent interpretation of entropy and energy E = - (1/2)H-1 for deSitter spacetime. The classical Einstein's equations for this spacetime can be expressed as a thermodynamic identity, TdS - dE = PdV with the same variables. (iv) For the Rindler spacetime the entropy per unit transverse area turns out to be (1/4) while the energy is zero. (v) In the case of a Schwarzschild black hole there exist quantum states (like Unruh vacuum) which are not invariant under time reversal and can describe black hole evaporation. There also exist quantum states (like Hartle-Hawking vacuum) in which temperature is well-defined but there is no flow of radiation to infinity. In the case of deSitter universe or Rindler patch in flat spacetime, one usually uses quantum states analogous to Hartle-Hawking vacuum and obtains a temperature without the corresponding notion of evaporation. It is, however, possible to construct the analogues of Unruh vacuum state in the other cases as well. The implications are briefly discussed.
ODD STATISTICS IN ODD DIMENSIONS FOR ODD COUPLINGS
Authors: SRIRAMKUMAR L.
Published: October 2002

We consider the response of a uniformly accelerated monopole detector that is coupled non-linearly to the nth power of a quantum scalar field in (D + 1)-dimensional flat spacetime. We show that, when (D + 1) is even, the response of the detector in the Minkowski vacuum is characterized by a Bose-Einstein factor for all n. Whereas, when (D + 1) is odd, we find that a Fermi-Dirac factor appears in the detector response when n is odd, but a Bose-Einstein factor arises when n is even. We emphasize the point that, since, along the accelerated trajectory, the Wightman function and, as a result, the (2n)-point function satisfy the Kubo-Martin-Schwinger condition (as required for a scalar field) in all dimensions, the appearance of a Fermi-Dirac factor (instead of the expected Bose-Einstein distribution) for odd(D + 1)andn reflects a peculiar feature of the detector rather than imply a fundamental change in field theory.
BLACK HOLE AREA QUANTIZATION
Authors: DAS SAURYA , RAMADEVI P. , YAJNIK U. A.
Published: October 2002

It has been argued by several authors that the quantum mechanical spectrum of black hole horizon area must be discrete. This has been confirmed in different formalisms, using different approaches. Here we concentrate on two approaches, the one involving quantization on a reduced phase space of collective coordinates of a Black Hole and the algebraic approach of Bekenstein. We show that for non-rotating, neutral black holes in any spacetime dimension, the approaches are equivalent. We introduce a primary set of operators sufficient for expressing the dynamical variables of both, thus mapping the observables in the two formalisms onto each other. The mapping predicts a Planck size remnant for the black hole.
Hoganite and paceite, two new acetate minerals from the Potosi mine, Broken Hill, Australia
Authors: Hibbs D. E. , Kolitsch U. , Leverett P. , Sharpe J. L. , Williams P. A.
Published: October 2002

Hoganite, copper(II) acetate monohydrate, and paceite (pronounced ‘pace-ite’), calcium(II) copper(II) tetraacetate hexahydrate, occur as isolated crystals embedded in ferruginous gossan from the Potosi Pit, Broken Hill, New South Wales, Australia. They are associated with goethite, hematite, quartz, linarite, malachite, azurite, cerussite and cuprian smithsonite. Hoganite is bluish green with a pale blue streak and a Mohs hardness of 1½; it possesses perfect {001} and distinct {110} cleavages and has a conchoidal fracture. Chemical analysis of hoganite gave (wt.%) C 23.85; H 3.95; Cu 31.6; Fe 0.4; O (by difference) 40.2, yielding an empirical formula of C4H7.89O5.07Cu1.00Fe0.01. The simplified formula is C4H8O5Cu or Cu(CH3COO)2.H2O, the mineral being identical to the synthetic compound of the same formula. Single-crystal X-ray data for hoganite are: monoclinic, space group C2/c, a = 13.162(3), b = 8.555(2), c = 13.850(3)Å, β = 117.08(3)°, Z = 8. The density, calculated from single-crystal data, is 1.910 g cm−3. The strongest lines in the X-ray powder pattern are [dobs (Iobs) (hkl)] 6.921 (100) (011); 3.532 (28) (202); 6.176 (14) (200); 3.592 (11) (22); 5.382 (10) (11); 2.278 (10) (204); 5.872 (9) (002). Hoganite (orientation presently unknown) is biaxial positive with α = 1.533(2), β = 1.541(3), γ = 1.554(2), 2V(meas.) = 85(5)°, 2V(calc.) = 76.8°, dispersion is r < v, medium (white light); it is strongly pleochroic with X = blue, Y = pale bluish, Z = pale bluish green and absorption X > Y > Z. The mineral is named after Graham P. Hogan of Broken Hill, New South Wales, Australia, a miner and well-known collector of Broken Hill minerals.

Paceite is dark blue with a pale blue streak and a Mohs hardness of 1½; it possesses perfect {100} and {110} cleavages and has an uneven fracture. Chemical analysis of paceite gave (wt.%) C 21.25; H 5.3; Ca 9.0; Cu 14.1; O (by difference) 50.35, yielding an empirical formula of C8H23.77O14.23Ca1.02-Cu1.00. The simplified formula is C8H24O14CaCu or CaCu(CH3COO)4.6H2O, the mineral being identical to the synthetic compound of the same formula. Unit-cell data (refined from X-ray powder diffraction data) for paceite are: tetragonal, space group I4/m, a = 11.155(4), c = 16.236(17)Å, Z = 4. The density, calculated from refined cell data, is 1.472 g cm−3. The strongest lines in the X-ray powder pattern are [dobs (Iobs) (hkl)] 7.896 (100) (110); 3.530 (20) (310); 5.586 (15) (200); 8.132 (8) (002); 9.297 (6) (101); 2.497 (4) (420); 3.042 (3) (321). Paceite is uniaxial positive with ω = 1.439(2) and ɛ = 1.482(3) (white light); pleochroism is bluish with a greenish tint (O), pale bluish with a greyish tint (E), and absorption O ⩾ E. The mineral is named after Frank L. Pace of Broken Hill, New South Wales, Australia, an ex-miner and well-known collector of Broken Hill minerals.
Intrinsic pathway of blood coagulation contributes to thrombogenicity of atherosclerotic plaque
Authors: Ananyeva Natalya M. , Kouiavskaia Diana V. , Shima Midori , Saenko Evgueni L.
Published: October 2002

Thrombosis is the major mechanism underlying acute complications of atherosclerosis. Although thrombogenicity of atherosclerotic plaques has been ascribed to activation of the extrinsic pathway of blood coagulation, in the present study we investigated contribution of the intrinsic factor VIII (fVIII)–dependent pathway. We found that in vitro exposure of human macrophages and smooth muscle cells (SMCs) to atherogenic oxidized low-density lipoprotein (oxLDL) enhances their ability to support activity of 2 major complexes of the intrinsic pathway, Xase and prothrombinase, leading to a 20- and 10-fold increase in thrombin formation, respectively. In contrast, human aortic endothelial cells were less responsive to oxLDL. The increase in the intrinsic procoagulant activity was related to formation of additional fVIII binding sites due to enhanced translocation of phosphatidylserine to the outer surface of oxLDL-treated cells and a 5-fold higher affinity of interaction between components of the Xase complex, activated factors VIII and IX. Processes occurring at early apoptotic stages, including changes in the cell membrane induced by free radicals, may be related to activation of the intrinsic pathway as suggested by effects of inhibitors of early apoptosis on thrombin formation. Immunohistochemical studies on human atherectomy specimens revealed the presence of fVIII in the vicinity of macrophages and SMCs in atheromatous regions with massive deposits of oxLDL, supporting the possible involvement of the intrinsic pathway in thrombus formation in vivo. Our data predict that the intrinsic pathway significantly enhances thrombogenicity of atherosclerotic lesions after removal of the endothelial layer and exposure of SMCs and macrophages to blood flow.
Experience with gemtuzumab ozogamycin (“mylotarg”) and all-trans retinoic acid in untreated acute promyelocytic leukemia
Authors: Estey Elihu H. , Giles Francis J. , Beran Miloslav , O'Brien Susan , Pierce Sherry A. , Faderl Stefan H. , Cortes Jorge E. , Kantarjian Hagop M.
Published: October 2002

We administered gemtuzumab ozogamycin (“mylotarg”; 9 mg/m2 day 1 or 5) and all-trans retinoic acid (ATRA) to 19 patients with untreated acute promyelocytic leukemia (APL). There were 3 patients who also received idarubicin because of a white blood cell (WBC) count of more than 30 000/μL. In complete remission (CR), patients were to receive 8 courses of mylotarg (9 mg/m2 every 4 to 5 weeks) and ATRA; idarubicin was added only for persistent or recurrent polymerase chain reaction (PCR) positivity. The CR rate was 16/19 (84%). All 12 patients tested to date were PCR-negative 2 to 4 months from CR date; none of the 7 patients evaluated subsequently have reverted to PCR positivity (median follow-up in CR was 5 months, up to 14 months). Mylotarg was well tolerated. A median of 5 post-CR courses have been given to date with 3 patients having currently received 8 post-CR courses, and 4 patients receiving 7 post-CR courses. Mylotarg appears active in APL, and repeated administration is feasible.
Single-cell analysis of CD30+ cells in lymphomatoid papulosis demonstrates a common clonal T-cell origin
Authors: Steinhoff Matthias , Hummel Michael , Anagnostopoulos Ioannis , Kaudewitz Peter , Seitz Volkhard , Assaf Chalid , Sander Christian , Stein Harald
Published: October 2002

Lymphomatoid papulosis (LyP) represents an intriguing cutaneous T-cell lymphoproliferative disorder with a histologic appearance resembling malignant lymphoma. This finding strongly contrasts with the benign clinical course of the disease. However, in 10% to 20% of cases, LyP can precede, coexist with, or follow malignant lymphoma. In these cases, the same T-cell population has been shown to be present in the LyP as well as in the associated lymphoma. In most LyP cases, there is—despite the sometimes extremely long course of the disease—no evolution of a secondary lymphoma. The investigation of these uncomplicated LyP cases for the presence of clonal T-cell receptor rearrangements has produced heterogeneous results. This might be explained by biologic or technical reasons arising from analyzing whole tissue DNA extracts. To definitively clarify whether the large atypical CD30+ cells in LyP without associated lymphoma all belong to the same clone or represent individually rearranged T cells, we analyzed the T-cell receptor–γ rearrangements of single CD30+ as well as of single CD30− cells isolated from 14 LyP lesions of 11 patients. By using this approach we could demonstrate that the CD30+ cells represent members of a single T-cell clone in all LyP cases. Moreover, in 3 patients the same CD30+ cell clone was found in anatomically and temporally separate lesions. In contrast, with only a few exceptions, the CD30− cells were polyclonal in all instances and unrelated to the CD30+ cell clone. Our results demonstrate that LyP unequivocally represents a monoclonal T-cell disorder of CD30+ cells in all instances.
Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1
Authors: Zhou Quansheng , Zhao Ji , Wiedmer Therese , Sims Peter J.
Published: October 2002

Phospholipid scramblase 1 (PLSCR1) is an endofacial plasma membrane protein proposed to participate in transbilayer movement of phosphatidylserine and other phospholipids. In addition to its putative role in the reorganization of plasma membrane phospholipids, PLSCR1 is a substrate of intracellular kinases that imply its possible participation in diverse signaling pathways underlying proliferation, differentiation, or apoptosis. Because PLSCR1 is prominently expressed in a variety of blood cells, we evaluated PLSCR activity in platelets and erythrocytes, and cytokine-dependent growth of hematopoietic precursor cells, of PLSCR1 knock-out mice. Adult PLSCR1−/− mice showed no obvious hematologic or hemostatic abnormality, and blood cells from these animals normally mobilized phosphatidylserine to the cell surface upon stimulation. Whereas blood cell counts in adult PLSCR1−/− mice were normal, in both fetus and newborn animals neutrophil counts were significantly depressed relative to age-matched wild type (WT). Furthermore, when compared with WT, hematopoietic precursor cells from PLSCR1−/− mice showed defective colony formation and impaired differentiation to mature granulocytes as stimulated by stem cell factor and granulocyte colony-stimulating factor (G-CSF). By contrast, PLSCR1−/− cells showed normal colony formation stimulated by interleukin-3 or granulocyte-macrophage CSF, and expansion of megakaryocytic and erythroid progenitors by thrombopoietin or erythropoietin was unaffected. Stem cell factor and G-CSF were also found to induce marked increases in PLSCR1 levels in WT cells. Consistent with in vitro assays, PLSCR1−/− mice treated with G-CSF showed less than 50% of the granulocytosis observed in identically treated WT mice. These data provide direct evidence that PLSCR1 functionally contributes to cytokine-regulated cell proliferation and differentiation and suggest it is required for normal myelopoiesis.
Role of GITR in activation response of T lymphocytes
Authors: Ronchetti Simona , Nocentini Giuseppe , Riccardi Carlo , Pandolfi Pier Paolo
Published: October 2002

In this study, we describe the generation and characterization of mice in which GITR gene (TNFRSF18 [tumor necrosis factor receptor superfamily 18]), a member of the TNFRSF expressed mainly on T lymphocytes, has been ablated (GITR−/− mice). Results indicate that GITR inactivation does not impair the normal development of the lymphoid organs but modulates T-cell activation. In fact, whenGITR−/− T lymphocytes are activated by treatment with an anti-CD3 monoclonal antibody they proliferate more than wild-type cells. Moreover, activatedGITR−/− T lymphocytes express higher levels of interleukin-2 receptor, produce larger amounts of interleukin-2, and are more sensitive to activation-induced cell death than controls. These results suggest that GITR is involved in the regulation of T-cell receptor/CD3–driven T-cell activation and programmed cell death.
An exon 10 deletion in the mouse ferrochelatase gene has a dominant-negative effect and causes mild protoporphyria
Authors: Magness Scott T. , Maeda Nobuyo , Brenner David A.
Published: October 2002

Protoporphyria is generally inherited as an autosomal dominant disorder. The enzymatic defect of protoporphyria is a deficiency in ferrochelatase, which chelates iron and protoporphyrin IX to form heme. Patients with protoporphyria have decreased ferrochelatase activities that range from 5% to 30% of normal caused by heterogeneous mutations in the ferrochelatase gene. The molecular mechanism by which the ferrochelatase activity is decreased to less than an expected 50% is unresolved. In this study, we assessed the effect of a ferrochelatase exon 10 deletion, a common mutation in human protoporphyria, introduced into the mouse by gene targeting. F1 crosses produced (+/+), (+/−), and (−/−) mice at a ratio of 1:2:0; (−/−) embryos were detected at 3.5 days postcoitus, consistent with embryonic lethality for the homozygous mutant genotype. Heterozygotes demonstrated equivalent levels of wild-type and mutant ferrochelatase messenger RNAs and 2 immunoreactive proteins that corresponded to the full-length and an exon 10–deleted ferrochelatase protein. Ferrochelatase activities in the heterozygotes were an average of 37% of normal, and protoporphyrin levels were elevated in erythrocytes and bile. Heterozygous mice exhibited skin photosensitivity but no liver disease. These results lend support for a dominant-negative effect of a mutant allele on ferrochelatase activity in patients with protoporphyria.
Bismuth 213–labeled anti-CD45 radioimmunoconjugate to condition dogs for nonmyeloablative allogeneic marrow grafts
Authors: Sandmaier Brenda M. , Bethge Wolfgang A. , Wilbur D. Scott , Hamlin Donald K. , Santos Erlinda B. , Brechbiel Martin W. , Fisher Darrell R. , Storb Rainer
Published: October 2002

To lower treatment-related mortality and toxicity of conventional marrow transplantation, a nonmyeloablative regimen using 200 cGy total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for postgrafting immunosuppression was developed. To circumvent possible toxic effects of external-beam γ irradiation, strategies for targeted radiation therapy were investigated. We tested whether the short-lived (half-life, 46 minutes) α-emitter bismuth 213 (213Bi) conjugated to an anti-CD45 monoclonal antibody (mAb) could replace 200 cGy TBI and selectively target hematopoietic tissues in a canine model of nonmyeloablative DLA-identical marrow transplantation. Biodistribution studies using iodine 123–labeled anti-CD45 mAb showed uptake in blood, marrow, lymph nodes, spleen, and liver. In a dose-escalation study, 7 dogs treated with the 213Bi–anti-CD45 conjugate (213Bi dose, 0.1-5.9 mCi/kg [3.7-218 MBq/kg]) without marrow grafts had no toxic effects other than a mild, reversible suppression of blood counts. On the basis of these studies, 3 dogs were treated with 0.5 mg/kg 213Bi-labeled anti-CD45 mAb (213Bi doses, 3.6, 4.6, and 8.8 mCi/kg [133, 170, and 326 MBq/kg]) given in 6 injections 3 and 2 days before grafting of marrow from DLA-identical littermates. The dogs also received MMF (10 mg/kg subcutaneously twice daily the day of transplantation until day 27 afterward) and CSP (15 mg/kg orally twice daily the day before transplantation until 35 days afterward). The therapy was well tolerated except for transient elevations in levels of transaminases in 3 dogs, followed by, in one dog, ascites. All dogs had prompt engraftment and achieved stable mixed hematopoietic chimerism, with donor contributions ranging from 30% to 70% after more than 27 weeks of follow-up. These results will form the basis for additional studies in animals and later the design of clinical trials using213Bi as a nonmyeloablative conditioning regimen with minimal toxicity.
Nondisjunction of chromosomes leading to hyperdiploid childhood B-cell precursor acute lymphoblastic leukemia is an early event during leukemogenesis
Authors: Panzer-Grümayer E. Renate , Fasching Karin , Panzer Simon , Hettinger Klaudia , Schmitt Klaus , Stöckler-Ipsiroglu Sylvia , Haas Oskar A.
Published: October 2002

A hyperdiploid karyotype is found in 30% of B-cell precursor acute lymphoblastic leukemias in childhood. The time of nondisjunction of chromosomes leading to hyperdiploidy during leukemogenesis is unknown. We used the 3 clonotypic immunoglobulin heavy chain (IgH) gene rearrangements as molecular markers for each of the 3 chromosomes 14 in a case with hyperdiploid acute lymphoblastic leukemia to define the order of events—namely, somatic recombination and nondisjunction of chromosomes—during leukemia development. A partial sequence homology of the incomplete DJH rearrangement with 1 of the 2 nonfunctional VDJH rearrangements suggests that the doubling of chromosomes had occurred after this DJHrearrangement and thus during early B-cell differentiation. The occurrence of the nondisjunction of chromosomes as well as ongoing rearrangement processes in utero were confirmed by the presence of all 3 IgH rearrangements in neonatal blood spots, providing the first evidence that hyperdiploidy formation is an early event in leukemogenesis in these leukemias.
A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice
Authors: Ziegler Sandra , Bürki Kurt , Skoda Radek C.
Published: October 2002

Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5′-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis.
The DAU allele cluster of the RHDgene
Authors: Wagner Franz F. , Ladewig Birgit , Angert Katharina S. , Heymann Guido A. , Eicher Nicole I. , Flegel Willy A.
Published: October 2002

Variant D occurs frequently in Africans. However, considerably lessRHD alleles have been described in this population compared with Europeans. We characterized 5 new RHD alleles, dubbedDAU-0 to DAU-4, that shared a T379M substitution and occurred in a cDe haplotype.DAU-1 to DAU-4 were detected in Africans with partial D phenotypes. They harbored one and 2 additional missense mutations, respectively, dispersed throughout the RhD protein. An anti-D immunization was found in DAU-3. DAU-0carrying T379M only was detected by screening European blood donors and expressed a normal D phenotype. Within the phylogeny of theRHD alleles, DAU formed an independent allele cluster, separate from the DIVa, weak D type 4, and Eurasian D clusters. The characterization of the RH phylogeny provided a framework for future studies on RH alleles. The identification of theDAU alleles increased the number of known partial D alleles in Africans considerably. DAU alleles may be a major cause of antigen D variability and anti-D immunization in patients of African descent.
Functional involvement of Akt signaling downstream of Jak1 in v-Abl–induced activation of hematopoietic cells
Authors: Oki Shinji , Limnander Andre , Danial Nika N. , Rothman Paul B.
Published: October 2002

Activation of intracellular signaling pathways is important for cellular transformation and tumorigenesis. The nonreceptor tyrosine kinases Jak1 and Jak3, which bind to the v-Abl oncoprotein, are constitutively activated in cells transformed with the Abelson murine leukemia virus. A mutant of p160 v-Abl lacking the Jak1-binding region (v-Abl Δ858-1080) has a significant defect in Jak/STAT (signal transducers and activators of transcription) activation, cytokine-independent cell growth/survival, and tumorigenesis. To identify the pathways downstream of Jak kinases in v-Abl–mediated signaling, we examined the activation of several signaling molecules by p160 v-Abl or the v-Abl Δ858-1080 mutant. We demonstrate that, in addition to the decreased Ras activation, signaling through phosphatidylinositol-3 kinase and Akt are impaired in cells expressing mutant v-Abl. The proliferative defect of v-Abl Δ858-1080 was rescued by activated v-Akt and was also moderately rescued by activated v-H-Ras. However, constitutive active phosphatidylinositol-3 kinase (p110CAAX) did not complement this effect. Cells expressing v-Abl Δ858-1080 demonstrated reduced tumor formation in nude mice. In contrast, cells coexpressing v-Akt with v-Abl Δ858-1080 demonstrated reduced latency and increased frequency of tumor formation in nude nice compared with cells expressing v-Abl Δ858-1080 alone, whereas v-H-Ras or p110CAAX had minimum effects on tumor formation. These results suggest that Jak1–dependent Akt activation is important in v-Abl–mediated transformation.
High hepatitis B virus (HBV) DNA viral load as the most important risk factor for HBV reactivation in patients positive for HBV surface antigen undergoing autologous hematopoietic cell transplantation
Authors: Lau George K. K. , Leung Yu-hung , Fong Daniel Y. T. , Au Wing-yan , Kwong Yok-lam , Lie Albert , Hou Ji-lin , Wen Yu-mei , Nanj Amin , Liang Raymond
Published: October 2002

The risk factors for hepatitis due to hepatitis B virus (HBV) reactivation in patients positive for hepatitis B surface antigen (HBsAg) treated with autologous hematopoietic cell transplantation (HCT) are unknown. We evaluated 137 consecutive patients (23 positive for HBsAg, 37 positive for hepatitis B surface antibody, and 77 negative for HBV) who underwent HCT. Serial serum ALT were measured before transplant and after transplant at 1 to 4 weekly intervals for the first year and then at 2 to 12 weekly intervals thereafter. Before HCT, basic core promoter (T1762/A1764) and precore (A1896) HBV variants were determined in HBsAg-positive and HBV DNA–positive (by polymerase chain reaction assay) patients by direct sequencing and serum HBV DNA quantitation using the Digene Hybrid Capture II assay. Cox proportional hazards analysis was used to assess the association between pretransplantation HBV virologic and host factors and occurrence of hepatitis due to HBV reactivation. After HCT, hepatitis due to HBV reactivation was more common in HBsAg-positive patients than in HBsAg-negative patients (hazard ratio, 33.3; 95% confidence interval [CI], 7.35-142.86;P < .0001). HBsAg-positive patients with detectable serum HBV DNA before HCT (on Digene assay) had a significantly higher risk of hepatitis due to HBV reactivation than HBsAg-positive patients with no detectable serum HBV DNA (adjusted hazard ratio, 9.35; 95% CI, 1.65-52.6; P = .012). Thus, we found that hepatitis due to HBV reactivation is common in HBsAg-positive patients undergoing autologous HCT. A high HBV DNA level (>105 copies/mL) was the most important risk factor for HBV reactivation, and its lowering by administration of nucleoside analogues before transplantation should be considered.
Macrophage colony stimulating factor modulates the development of hematopoiesis by stimulating the differentiation of endothelial cells in the AGM region
Authors: Minehata Ken-ichi , Mukouyama Yoh-suke , Sekiguchi Takashi , Hara Takahiko , Miyajima Atsushi
Published: October 2002

Definitive hematopoietic stem cells arise in the aorta–gonad–mesonephros (AGM) region from hemangioblasts, common precursors for hematopoietic and endothelial cells. Previously, we showed that multipotential hematopoietic progenitors and endothelial cells were massively produced in primary culture of the AGM region in the presence of oncostatin M. Here we describe a role for macrophage–colony-stimulating factor (M-CSF) in the development of hematopoietic and endothelial cells in AGM culture. The number of hematopoietic progenitors including multipotential cells was significantly increased in the AGM culture of op/opembryos. The addition of M-CSF to op/op AGM culture decreased colony-forming unit (CFU)-GEMM, granulocyte macrophage–CFU, and erythroid–CFU, but it increased CFU-M. On the other hand, the number of cells expressing endothelial markers, vascular endothelial-cadherin, intercellular adhesion molecule 2, and Flk-1 was reduced in op/op AGM culture. The M-CSF receptor was expressed in PCLP1+CD45− cells, the precursors of endothelial cells, and M-CSF up-regulated the expression of more mature endothelial cell markers—VCAM-1, PECAM-1, and E-selectin—in PCLP1+CD45− cells. These results suggest that M-CSF modulates the development of hematopoiesis by stimulating the differentiation of PCLP-1+CD45− cells to endothelial cells in the AGM region.
Ca++-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin
Authors: Salzer Ulrich , Hinterdorfer Peter , Hunger Ursula , Borken Cordula , Prohaska Rainer
Published: October 2002

Cytosolic Ca++ induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glycosyl phosphatidylinositol–linked proteins, free of cytoskeletal components, and depleted of the major transmembrane proteins. Both types of vesicles contain 2 as-yet-unrecognized red cell proteins, synexin and sorcin, which translocate from the cytosol to the membrane upon Ca++ binding. In nanovesicles, synexin and sorcin are the most abundant proteins after hemoglobin. In contrast, the microvesicles are highly enriched in stomatin. The membranes of both microvesicles and nanovesicles contain lipid rafts. Stomatin is the major protein of the microvesicular lipid rafts, whereas synexin and sorcin represent the major proteins of the nanovesicular rafts in the presence of Ca++. Interestingly, the raft proteins flotillin-1 and flotillin-2 are not found in the vesicles but remain in the red cell membrane. These data indicate the presence of different types of lipid rafts in the erythrocyte membrane with distinct fates after Ca++ entry. Synexin, which is known to be vital to the process of membrane fusion, is suggested to be a key component in the process of vesicle release from erythrocytes.
Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy
Authors: Mount Jane D. , Herzog Roland W. , Tillson D. Michael , Goodman Susan A. , Robinson Nancy , McCleland Mark L. , Bellinger Dwight , Nichols Timothy C. , Arruda Valder R. , Lothrop Clinton D. , High Katherine A.
Published: October 2002

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)–mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 × 1012 vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.
Extensive in vivo self-renewal, long-term reconstitution capacity, and hematopoietic multipotency of Pax5-deficient precursor B-cell clones
Authors: Schaniel Christoph , Gottar Marie , Roosnek Eddy , Melchers Fritz , Rolink Antonius G.
Published: October 2002

Self-renewal, pluripotency, and long-term reconstitution are defining characteristics of single hematopoietic stem cells.Pax5−/− precursor B cells apparently possess similar characteristics. Here, using serial transplantations, with in vitro recloning and growth of the bone marrow–homed donor cells occurring after all transplantations, we analyzed the extent of self-renewal and hematopoietic multipotency ofPax5−/− precursor B-cell clones. Moreover, telomere length and telomerase activity in these clones was analyzed at various time points. Thus far, 5 successive transplantations have been performed. Clones transplanted for the fifth time, which have proliferated for more than 150 cell divisions in vitro, still repopulate the bone marrow with precursor B cells and reconstitute these recipients with lymphoid and myeloid cells. During this extensive proliferation, Pax5−/− precursor B cells shorten their telomeres at 70 to 90 base pairs per division. Their telomerase activity remains at 3% of that of HEK293 cancer cells during all serial in vivo transplantations/in vitro expansions. Together, these data show thatPax5−/− precursor B-cell clones possess extensive in vivo self-renewal capacity, long-term reconstitution capacity, and hematopoietic multipotency, with their telomeres shortening at the normal rate.