bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of binding‐induced changes in dynamics suggests a model for sequence‐nonspecific binding of ssDNA by replication protein A
Authors: Bhattacharya Shibani , Botuyan Maria‐Victoria , Hsu Fred , Shan Xi , Arunkumar A.I. , Arrowsmith Cheryl H. , Edwards Aled M. , Chazin Walter J.
Published: September 2002

Single‐stranded‐DNA‐binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence‐nonspecific binding of ssDNA, NMR chemical shift and 15N relaxation experiments were performed on an isolated ssDNA‐binding domain (RPA70A) from the human SSB replication protein A. The backbone 13C, 15N, and 1H resonances of RPA70A were assigned for the free protein and the d‐CTTCA complex. The binding‐induced changes in backbone chemical shifts were used to map out the ssDNA‐binding site. Comparison to results obtained for the complex with d‐C5 showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R1 and R2) and 1H–15N NOE values were measured for RPA70A in the absence and presence of d‐CTTCA. Analysis of the data using the Model‐Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA‐binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence‐nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.
Crystal structure of the ADP‐dependent glucokinase from Pyrococcus horikoshii at 2.0‐Å resolution: A large conformational change in ADP‐dependent glucokinase
Authors: Tsuge Hideaki , Sakuraba Haruhiko , Kobe Toru , Kujime Akira , Katunuma Nobuhiko , Ohshima Toshihisa
Published: September 2002

Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP‐dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three‐dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large α/β‐domains and small domains. However, a marked adjustment of the two domains, which is a 10‐Å translation and a 20° rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo‐phGK and ADP‐tlGK structures. The ADP‐binding loop (430–439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction.
Naturally Occurring Substitutions in the P/V Gene Convert the Noncytopathic Paramyxovirus Simian Virus 5 into a Virus That Induces Alpha/Beta Interferon Synthesis and Cell Death
Authors: Wansley Elizabeth K. , Parks Griffith D.
Published: September 2002

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-α/β) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI−) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-α/β signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI− had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI− were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI− mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI− but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-β promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI− but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI− mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI− growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI− by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI− induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-α/β synthesis, and virus-induced apoptosis.
Molecular Genetic Evidence that the Hydrophobic Anchors of Glycoproteins E2 and E1 Interact during Assembly of Alphaviruses
Authors: Strauss Ellen G. , Lenches Edith M. , Strauss James H.
Published: September 2002

Chimeric alphaviruses in which the 6K and glycoprotein E1 moieties of Sindbis virus are replaced with those of Ross River virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. We have sequenced the entire domain encoding the E2, 6K, and E1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. In three independent passage series, amino acid 380 of E2, which is in the transmembrane domain, was mutated from the original isoleucine to serine in two instances and to valine once. We have now changed this residue to seven others by site-directed mutagenesis and tested the effects of these mutations on the growth of both the chimera [SIN(RRE1)] and of parental Sindbis. These results indicate that the transmembrane domains of glycoproteins E2 and E1 of alphaviruses interact in a sequence-dependent manner and that this interaction is required for efficient budding and assembly of infectious virions.
Promoter Sequences Required for Reactivation of Epstein-Barr Virus from Latency
Authors: Binné Ulrich K. , Amon Wolfgang , Farrell Paul J.
Published: September 2002

A luciferase reporter system with stably transfected
oriP
plasmids in Akata Burkitt's lymphoma cells provides a quantitative assay for the BZLF1 Zp promoter in response to B-cell receptor (BCR) activation by cross-linking with anti-immunoglobulin. In this system, detailed kinetic studies of promoter activity are possible. Previously reported promoter elements upstream of −221 from the transcription start and the ZIIR sequence had little effect on the Zp promoter, but the ZI and ZIIIA elements were essential for early activation. The ZIIIB element mediates autoactivation. Mutation of the ZV repressor sequence greatly increased the induction of the promoter but did not make it constitutively active. Zp transcription in response to BCR cross-linking declined after a few hours; this decline was reduced and delayed by acyclovir or phosphonoacetic acid, indicating that viral DNA replication or a late viral gene can play a role in the switch off of the Zp promoter. Late expression of the LMP1 protein may account for this.
Functional Dissection of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Involved in Latent DNA Replication and Transcription of Terminal Repeats of the Viral Genome
Authors: Lim Chunghun , Sohn Hekwang , Lee Daeyoup , Gwack Yousang , Choe Joonho
Published: September 2002

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the maintenance of the viral genome during latent infection. LANA1 colocalizes with KSHV episomes on the host chromosome and mediates their maintenance by attaching these viral structures to host chromosomes. Data from long-term selection of drug resistance in cells conferred by plasmids containing the terminal repeat (TR) sequence of KSHV revealed that KSHV TRs and LANA1 act as
cis
and
trans
elements of viral latent replication, respectively. In this study, we further characterized the
cis
- and
trans
-acting elements of KSHV latent replication by using a transient replication assay with a methylation-sensitive restriction enzyme,
Dpn
I. Transient reporter and replication assays disclosed that the orientation and basal transcriptional activity of TR constructs did not significantly affect the efficiency of replication. However, at least two TR units were necessary for efficient replication. The N-terminal 90 amino acids comprising the chromosome-binding domain of LANA1 were required for the mediation of LANA1 C-terminal DNA-binding and dimerization domains to support the transient replication of KSHV TRs. LANA1 interacted with components of the origin recognition complexes (ORCs), similar to Epstein-Barr virus nuclear antigen 1. Our data suggest that LANA1 recruits ORCs to KSHV TRs for latent replication of the viral genome.
Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Nuclear Localization Mediates Early Viral mRNA Expression
Authors: Zhang Jielin , Crumpacker Clyde S.
Published: September 2002

An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 × 10
9
cells/day and 10.3 × 10
9
virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.
Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration
Authors: Johnson-Léger Caroline A. , Aurrand-Lions Michel , Beltraminelli Nicola , Fasel Nicolas , Imhof Beat A.
Published: September 2002

The molecular mechanisms underlying lymphocyte extravasation remain poorly characterized. We have recently identified junctional adhesion molecule-2 (JAM-2), and have shown that antibodies to JAM-2 stain high endothelial venules (HEVs) within lymph nodes and Peyer patches of adult mice. Here we show that mouse lymphocytes migrate in greater numbers across monolayers of endothelioma cells transfected with JAM-2. The significance of these findings to an understanding of both normal and pathologic lymphocyte extravasation prompted us to clone the human homologue of JAM-2. We herein demonstrate that an anti–JAM-2 antibody, or a soluble JAM-2 molecule, blocks the transmigration of primary human peripheral blood leukocytes across human umbilical vein endothelial cells expressing endogenous JAM-2. Furthermore, we show that JAM-2 is expressed on HEVs in human tonsil and on a subset of human leukocytes, suggesting that JAM-2 plays a central role in the regulation of transendothelial migration.
Characterization of hyaluronan synthase expression and hyaluronan synthesis in bone marrow mesenchymal progenitor cells: predominant expression of HAS1 mRNA and up-regulated hyaluronan synthesis in bone marrow cells derived from multiple myeloma patients
Authors: Calabro Anthony , Oken Martin M. , Hascall Vincent C. , Masellis Anna M.
Published: September 2002

Hyaluronan (HA) is suggested to play a role in the pathophysiology of multiple myeloma. To further investigate the role of HA in this disease, we examined hyaluronan synthase (Has) gene expression and HA production in bone marrow mesenchymal progenitor cells (bmMPCs) derived from multiple myeloma patients. The relative abundance of mRNA for each HAS gene was determined using competitive reverse transcription–polymerase chain reaction (cRT-PCR), whereas HA production was detected by fluorophore-assisted carbohydrate electrophoresis (FACE). We determined the basal expression of Has isoforms in myeloma bmMPCs and then compared this expression with expression in healthy donor bmMPCs. Of the 3 Has isoforms, Has1 mRNA was expressed predominantly in myeloma bmMPCs, with expression 7.6-fold greater than Has2. Compared with normal bmMPCs, Has1 mRNA expression was 20-fold greater in myeloma bmMPCs. Normal bmMPCs predominantly expressed Has2 mRNA (8.2-fold greater than myeloma bmMPCs). Upon coculture of myeloma bmMPCs with plasma cells, Has1 transcript was strongly attenuated. FACE results show that myeloma bmMPCs synthesize 5.7-fold more HA than those from healthy donors. These data suggest that myeloma bmMPCs could be an important component of the myeloma pathophysiology in vivo by their increased expression of extracellular matrix (ECM) components relevant to plasma cell growth and survival.
Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality
Authors: Suzuki Norio , Ohneda Osamu , Takahashi Satoru , Higuchi Masato , Mukai Harumi Y. , Nakahata Tatsutoshi , Imagawa Shigehiko , Yamamoto Masayuki
Published: September 2002

Erythropoietin (Epo) and its receptor (EpoR) are indispensable to erythropoiesis. Although roles besides angiogenesis, such as neuroprotection and heart development, have been reported for the Epo-EpoR system, the precise contribution of Epo-EpoR to these nonhematopoietic tissues requires clarification. Exploiting aGATA-1 minigene cassette with hematopoietic regulatory domains, we established 2 lines of transgene-rescued EpoR-null mutant mice expressing EpoR exclusively in the hematopoietic lineage. Surprisingly, despite the lack of EpoR expression in nonhematopoietic tissues, these mice develop normally and are fertile. As such, we could exploit them for analyzing the roles of the Epo-EpoR system in adult hematopoiesis and in nonhematopoietic tissues. These rescued lines showed a differential level of EpoR expression in erythroid cells; one expressed approximately 40%, and the other expressed 120% of the wild-type EpoR level. A colony formation assay showed that erythroid progenitors in the 2 mutant lines exhibit distinct sensitivity to Epo. The circulating Epo level was much higher in the transgenic line with a lower EpoR expression. In response to induced anemia, the plasma Epo concentrations increased in both lines. Notably, the timing of the peak of plasma Epo concentration was delayed in both lines of rescued mice compared with wild type, suggesting that, in wild-type mice, nonhematopoietic EpoR contributes to the regulation of plasma Epo concentration. We thus conclude that nonhematopoietic expression of EpoR is dispensable to normal mouse development and that the expression level of EpoR regulates erythropoiesis by controlling the sensitivity of erythroid progenitors to Epo.
Successful treatment of posttransplantation lymphoproliferative disorder (PTLD) following renal allografting is associated with sustained CD8+ T-cell restoration
Authors: Porcu Pierluigi , Eisenbeis Charles F. , Pelletier Ronald P. , Davies Elizabeth A. , Baiocchi Robert A. , Roychowdhury Sameek , Vourganti Srinivas , Nuovo Gerard J. , Marsh William L. , Ferketich Amy K. , Henry Mitchell L. , Ferguson Ronald M. , Caligiuri Michael A.
Published: September 2002

Posttransplantation lymphoproliferative disorder (PTLD) is a life-threatening Epstein-Barr virus (EBV)–associated B-cell malignancy occurring in 1% to 2% of renal transplantation patients. Host- and PTLD-related factors determining the likelihood of tumor response following reduction of immune suppression (IS) and antiviral therapy remain largely unknown. Standard therapy for PTLD is not well established. Eleven consecutive renal transplantation patients who developed EBV-positive PTLD 8 to 94 months after allografting were uniformly treated with acyclovir and IS reduction. All PTLDs were EBV-positive diffuse large B-cell lymphomas. Ten patients (91%) obtained a durable complete response (CR), and 9 (82%) have remained in continuous CR with a median follow-up of 29 months. Five patients (45%) lost their allograft. Of these, 4 patients had PTLD affecting the transplanted kidney. Peripheral blood CD8+ T cells increased significantly (P = .0078) from baseline in 8 responders available for analysis. One of 2 patients whose absolute CD8+ T-cell count subsequently dropped to baseline after IS reduction relapsed. The expanded CD8+ T cells from 2 responders specifically recognized an immunodominant peptide from the EBV lytic gene BZLF-1. Another lytic EBV gene, thymidine kinase, was expressed in all 8 PTLDs tested. IS reduction and antiviral therapy for PTLD after renal transplantation is a highly successful therapeutic combination, but the risk of graft rejection is significant, particularly in patients with PTLD involving the renal allograft. A sustained expansion of CD8+ T cells and a cellular immune response to EBV lytic antigens may be important for PTLD clearance in renal transplantation patients.
Hemin induces neuroglobin expression in neural cells
Authors: Zhu Yonghua , Sun Yunjuan , Jin Kunlin , Greenberg David A.
Published: September 2002

Neuroglobin is a newly identified vertebrate globin that binds O2 and is expressed in cerebral neurons. We found recently that neuronal expression of neuroglobin is stimulated by hypoxia and ischemia and protects neurons from hypoxic injury. Here we report that, like hemoglobin and myoglobin, neuroglobin expression can also be induced by hemin. Induction was concentration dependent and time dependent, with maximal (about 4-fold) increases in neuroglobin mRNA and protein levels occurring with 50 μM hemin and at 8 to 24 hours. The inductive effect of hemin was attenuated by the protein kinase G inhibitor KT5823 and the soluble guanylate cyclase inhibitor LY83583, was mimicked by treatment with 8-bromo–cyclic guanosine 3′,5′-monophosphate, and was accompanied by a greater than 10-fold increase in cGMP levels, suggesting that it is mediated through protein kinase G and soluble guanylate cyclase. In contrast, hypoxic induction of neuroglobin was blocked by the mitogen-activated protein kinase/extracellular signal–regulated kinase kinase inhibitor PD98059, indicating that hemin and hypoxia regulate neuroglobin expression by different mechanisms. These results provide evidence for regulation of neuroglobin expression by at least 2 signal transduction pathways.
Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors
Authors: Kottaridis Panagiotis D. , Gale Rosemary E. , Langabeer Stephen E. , Frew Marion E. , Bowen David T. , Linch David C.
Published: September 2002

FLT3 mutations, either internal tandem duplications (ITDs) or aspartate residue 835 (D835) point mutations, are present in approximately one third of patients with acute myeloid leukemia (AML) and have been associated with an increased relapse rate. We have studied FLT3 mutations in paired presentation and relapse samples to ascertain the biology of these mutations and to evaluate whether they can be used as markers of minimal residual disease. At diagnosis, 24 patients were wild-type FLT3, and 4 acquired a FLT3 mutation at relapse (2 D835+, 2 ITD+), with a further patient acquiring an ITD at second relapse. Of 20 patients positive at diagnosis (18 ITD+, 2 D835+), 5 who were all originally ITD+ had no detectable mutation at relapse, as determined by a sensitive radioactive polymerase chain reaction. One of these patients had acquired an N-Ras mutation not detectable at presentation. Furthermore, another patient had a completely different ITD at relapse, which could not be detected in the presentation sample. These results indicate that FLT3 mutations are secondary events in leukemogenesis, are unstable, and thus should be used cautiously for the detection of minimal residual disease.
Frequency and load of hepatitis B virus DNA in first-time blood donors with antibodies to hepatitis B core antigen
Authors: Hennig Holger , Puchta Ines , Luhm Jürgen , Schlenke Peter , Goerg Siegfried , Kirchner Holger
Published: September 2002

The objective of this study was to determine the frequency and load of hepatitis B virus (HBV) DNA in anti-HBc–positive first-time blood donors; it was designed to contribute to determining whether anti-HBc screening of blood donations might reduce the residual risk of posttransfusion HBV infection. A total of 14 251 first-time blood donors were tested for anti-HBc using a microparticle enzyme immunoassay; positive results were confirmed by a second enzyme-linked immunosorbent assay (ELISA). For the detection of HBV DNA from plasma samples, we developed a novel and highly sensitive real-time polymerase chain reaction (PCR) assay. The 95% detection limit of the method amounted to 27.8 IU/mL, consistent with the World Health Organization (WHO) international standard for HBV DNA. A total of 216 blood donors (1.52%) tested anti-HBc–positive in both tests, and 205 of them (16 HBsAg+, 189 HBsAg−) were tested for HBV DNA. In 14 (87.5%) of the HBsAg-positive blood donors, HBV DNA was repeatedly detected, and in 3 (1.59%) of the HBsAg-negative donors, HBV DNA was also found repeatedly. In the 3 HBV DNA–positive, HBsAg-negative cases, anti-HBe and anti-HBs (> 100 IU/L) were also detectable. HBV DNA in HBsAg-negative as well as HBsAg-positive samples was seen at a low level. Thus, HBV DNA is sometimes found in HBsAg-negative, anti-HBc–positive, and anti-HBs–positive donors. Retrospective studies on regular blood donors and recipients are necessary to determine the infection rate due to those donations. Routine anti-HBc screening of blood donations could probably prevent some transfusion-transmitted HBV infections.
Inherited predisposition to CLL is detectable as subclinical monoclonal B-lymphocyte expansion
Authors: Rawstron Andy C. , Yuille Martin R. , Fuller Julie , Cullen Matthew , Kennedy Ben , Richards Stephen J. , Jack Andrew S. , Matutes Estella , Catovsky Daniel , Hillmen Peter , Houlston Richard S.
Published: September 2002

Monoclonal chronic lymphocytic leukemia (CLL)–phenotype cells are detectable in 3.5% of otherwise healthy persons using flow cytometric analysis of CD5/CD20/CD79b expression on CD19-gated B cells. To determine whether detection of such CLL-phenotype cells is indicative of an inherited predisposition, we examined 59 healthy, first-degree relatives of patients from 21 families with CLL. CLL-phenotype cells were detected in 8 of 59 (13.5%) relatives, representing a highly significant increase in risk (P = .00002). CLL-phenotype cell levels were stable with time and had the characteristics of indolent CLL. Indolent and aggressive clinical forms were found in family members, suggesting that initiation and proliferation involves distinct factors. The detection of CLL-phenotype cells provides a surrogate marker of carrier status, potentially facilitating gene identification through mapping in families and direct analysis of isolated CLL-phenotype cells.
Role of Cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb-MYH11
Authors: Kundu Mondira , Chen Amy , Anderson Stacie , Kirby Martha , Xu LiPing , Castilla Lucio H. , Bodine David , Liu Pu Paul
Published: September 2002

Core-binding factor β (CBFβ) and CBFα2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFβ or CBFα2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFβ and CBFα2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion ofCBFB with MYH11, the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for aCbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb−/−embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfβ in hematopoietic cells. In midgestation embryos, Cbfβ was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and thatCbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfβ was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfβ was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.
Biphasic transmittance waveform in the APTT coagulation assay is due to the formation of a Ca++-dependent complex of C-reactive protein with very-low–density lipoprotein and is a novel marker of impending disseminated intravascular coagulation
Authors: Toh Cheng Hock , Samis John , Downey Colin , Walker John , Becker Lev , Brufatto Nicole , Tejidor Liliana , Jones Greg , Houdijk Wim , Giles Alan , Koschinsky Marlys , Ticknor Larry O. , Paton Ray , Wenstone Richard , Nesheim Michael
Published: September 2002

A decrease in light transmittance before clot formation, manifesting as a biphasic waveform (BPW) pattern in coagulation assays, was previously correlated with the onset of disseminated intravascular coagulation (DIC). In this study of 1187 consecutive admissions to the intensive care unit, the degree of this change on admission predicts DIC better than D-dimer measurements. Additionally, the BPW preceded the time of DIC diagnosis by 18 hours, on average, in 56% (203 of 362) of DIC patients. The BPW is due to the rapid formation of a precipitate and coincident turbidity change on recalcification of plasma. The isolated precipitate contains very-low–density lipoprotein (VLDL) and C-reactive protein (CRP). The addition of CRP and Ca++ to normal plasma also causes the precipitation of VLDL and IDL, but not LDL or HDL. The Kd of the CRP/VLDL interaction is 340 nM, and the IC50 for Ca++ is 5.0 mM. In 15 plasmas with the BPW, CRP was highly elevated (77-398 μg/mL), and the concentration of isolated VLDL ranged from 0.082 to 1.32 mM (cholesterol). The turbidity change on recalcification correlates well with the calculated level of the CRP–VLDL complex. Clinically, the BPW better predicts for DIC than either CRP or triglyceride alone. The complex may have pathophysiological implications because CRP can be detected in the VLDL fraction from sera of patients with the BPW, and the VLDL fraction has enhanced prothrombinase surface activity. The complex has been designated lipoprotein complexed C-reactive protein.
Mammary Gland Development in Transgenic Male Mice Expressing Human P450 Aromatase
Authors: Li Xiangdong , Wärri Anni , Mäkelä Sari , Ahonen Tommi , Streng Tomi , Santti Risto , Poutanen Matti
Published: September 2002

We recently generated a transgenic mouse strain that expresses the human aromatase gene under the ubiquitin C promoter (AROM+). We have previously shown that in these mice the serum estradiol concentration is highly elevated, whereas the testosterone concentration is decreased. In the present study we examined mammary gland development in AROM+ male mice at different ages and found that the mammary glands of AROM+ males undergo ductal and alveolar development morphologically resembling that of terminally differentiated female mammary glands, expressing mRNA for a milk protein gene (β-casein). The male mammary glands also express multiple hormone receptors typical for female mammary gland: estrogen receptor α and β, progesterone receptor, and PRL receptor. Furthermore, data showed activation of the Stat5 (signal transducer and activator of transcription 5) signaling pathway in the AROM+ male mammary gland. Interestingly, the phenotype observed is in part reversible. Treatment with finrozole, a specific aromatase inhibitor, caused an involution of the differentiated phenotype of the mammary gland, marked by the disappearance of alveolar structures and the majority of the tertiary side branches of the ducts. The present animal model is a valuable tool for better understanding the cellular and molecular mechanisms involved in the development of gynecomastia.
Identification and Characterization of a Selective, Nonpeptide Follicle-Stimulating Hormone Receptor Antagonist
Authors: Arey Brian J. , Deecher Darlene C. , Shen Emily S. , Stevis Panayiotis E. , Meade Edwin H. , Wrobel Jay , Frail Donald E. , López Francisco J.
Published: September 2002

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-{4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene}-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [125I]hFSH with an IC50 value of 5.4 ± 2.3 μm. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [125I]hFSH binding to BV-tFSHR with an IC50 value of 10 ± 2.8 μm. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC50 value of 3 ± 0.6 μm. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.
Showing you can do it: Homework in therapy for children and adolescents with anxiety disorders
Authors: Hudson Jennifer L. , Kendall Philip C.
Published: September 2002

This article discusses the application of homework tasks in an empirically supported treatment for children (aged 8–13 years) with anxiety disorders. Within this program, homework tasks—through practice and rehearsal in the child's natural environment—are an important ingredient to enable the child to increase his or her mastery of the information/skills covered in each session. The homework tasks also provide an opportunity for the therapist to check the child's grasp of the session content through unassisted application of the material. This article discusses issues that arise in the application of homework tasks with a child population (e.g., compliance). Homework in both child‐ and family‐focused treatment is discussed. The issues addressed regarding homework, although raised in the context of anxious children in a cognitive behavioral treatment program, may be broadly applied to children in psychotherapy. © 2002 Wiley Periodicals, Inc. J Clin Psychol/In Session 58: 525–534, 2002.
Potential predictability of Eurasian snow cover
Authors: Schlosser C. Adam , Dirmeyer Paul A.
Published: September 2002

Potential predictability and skill of simulated Eurasian snow cover are explored using a suite of seasonal ensemble hindcasts (i.e. retrospective forecasts), an ensemble climate simulation (spanning the years 1982–1998) and observations. Using remotely sensed observations of snow cover, we find significant point‐wise correlation over the North Atlantic and North Pacific between winter and spring averaged sea‐surface temperatures and Eurasian snow cover area. The observed correlation shows no discernible pattern related to the El Niño‐Southern Oscillation (ENSO). The hindcasts show correlation patterns similar to the observations. However, the climate simulation shows an exaggerated ENSO pattern. The results underscore the importance of initialization in seasonal climate forecasts, and that the observed potential predictability of Eurasian snowcover cannot be solely attributed to ENSO. Copyright © 2003 Royal Meteorological Society.
Electron‐dense material in the cell sall/plasma membrane interface of specialized cortical cells of peanut nodules.
Authors: Bal Arya K.
Published: September 2002

The nitrogen‐fixing peanut root nodules have 2‐3 layers of specialized cortical cells surrounding the infected zone. These cells are morphologically distinct from the other cortical cells due to the presence of prominent amyloplasts, lipid bodies (oleosomes), large microbodies and localized pockets of electron‐dense material at the plasma membrane‐cell wall interface. The material is resistant to solubilization by hexane and chloroform. Electron density is enhanced by p‐phenylenediamine staining and heavy metal ions such as iron, uranium and osmium. The silver proteinate staining method for suberin has shown positive results. Peroxidative activity can be demonstrated by diaminobenzidine reaction. The material appears to be proteinaceous, but could be complexed with suberin or phenolics. Although the exact nature of this material remains to be clarified, its unique position in the cells of specialized tissue of the cortex relates it to the regulation of bidirectional transport of gases and solutes during symbiosis in the root nodules.
Relationship between cell adherence and proteoglycan synthesis in cultures of human peripheral blood mononuclear cells: effects of concanavalin A.
Authors: Anastassiades T. P. , Chopra R. K. , Ford P. M. , Wood A.
Published: September 2002

Human, blood‐derived mononuclear cells (MC), stimulated with Concanavalin A (Con A), synthesized a chondroitin sulfate (CS) proteoglycan (PG), which was elaborated largely by T‐cells. Following Con A stimulation, the majority of MC adhered to the culture dish by day 2; but as incubation progressed to day 10 the proportion of non‐adherent (NAd) MC increased in a fashion which approximately paralleled the accumulation of PG in the medium. Cell kinetic studies suggest that, following Con A stimulation, there was an inverse relationship between the amount of newly synthesized cellular PG and adherence, which appears to be related to a reciprocal effect on PG synthesis of the declining adherent (Ad) cell density with time of culture. In the stimulated cultures, NAd cells contained much more newly synthesized CS/cell than Ad cells up to day 6 of incubation. Cell type analysis, using monoclonal antibodies against specific cell surface markers, suggested that the higher PG synthesis in the NAd population may, at least in part, be due to a greater proportion of T‐helper cells.
Bovine embryo co‐culture.
Authors: Catt James W.
Published: September 2002

The three methods of culturing bovine zygotes; surrogate oviducts, co‐culture and defined media are compared and contrasted. Surrogate oviducts provide the best environment and so provide a benchmark to compare in vitro culture systems. At present, co‐culture systems provide a convenient method of reliably producing embryos suitable for transfer. This is important for commercial embryo production. The use of defined media is improving and can be as efficacious as co‐culture. The advantage of defined culture systems is the ability to find the requirements of an early embryo and to tailor media to meet them. It is suggested that maturation, not zygote culture, may be the constraint in embryonic development.
The control of Ca2+ homeostasis: role of intracellular rapidly exchanging Ca2+ stores.
Authors: Villa Antonello , Meldolesi Jacopo
Published: September 2002

The role of rapidly exchanging intracellular Ca2+ stores in the control of Ca2+ homeostasis is reviewed. The following issues are discussed: the reasons why such stores exist in eukaryotic cells; the differences between the terminal cisternae of the skeletal muscle sarcoplasmic reticulum, which have direct, physical connection with the T tubules of the plasmalemma, and the Ca2+ stores located in the depth of the cytoplasm, which are stimulated by second messengers; the cytological nature (subcompartments of the ER) of the rapidly exchanging Ca2+ stores and their functional significance. The conclusions introduce recent developments in which intracellular Ca2+ stores have been investigated also by molecular biology techniques.
THE FUNCTION OF HEAT‐SHOCK PROTEINS IN STRESS TOLERANCE
Authors: Venetianer Aniko , Pirity Melinda , Hever‐Szabo Anna
Published: September 2002

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid‐resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid‐sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pretreatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat‐resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat‐inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat‐resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P‐glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P‐gp may participate in protection against environmental stress such as heat.
Interaction of filarial proteins on growth regulation of normal lung epithelial cells in vitro
Authors: Maya A. , Usha S. , Jayaraman K. , Baba Krishnan , Sukumaran M. , Balakrishnan A.
Published: September 2002

An in vitro model to examine the effects of filarial proteins on lung epithelial cells has been developed. Several of these proteins appear in circulation of infected individuals. A close association between tropical pulmonary eosinophilia (TPE) and filariasis has been reported by several workers. [3H]‐thymidine studies do indicate that when optimum concentration of these filarial proteins were added to lung cultures in proliferating and basal/maintenance media a further increase in growth stimulation was observed early in culture. However, on longer exposures and at higher concentrations an inhibitory effect with distinct morphological changes were noted. The dual role of these proteins on lung epithelial cells in vitro may highlight the possibility of a direct interaction of these proteins with lung cells during disease also contributing to tissue damage.
Effect of Extracellular Matrix Proteins on Vascular Smooth Muscle Cell Phenotype
Authors: Hayward I. P. , Bridle K. R. , Campbell G. R. , Underwood P. A. , Campbell J. H.
Published: September 2002

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (Vvmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high Vvmyo (Vvmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest Vvmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high Vvmyo phenotype.
AN ICAM‐1 LIKE CELL ADHESION MOLECULE IS RESPONSIBLE FOR CD34 POSITIVE HAEMOPOIETIC STEM CELLS ADHESION TO BONE‐MARROW STROMA
Authors: RAO S. G. A. , CHITNIS V. S. , DEORA A. , TANAVDE V. , DESAI S. S.
Published: September 2002

The microenvironment in the haematopoietic organs plays an important role in regulating and sustaining differentiation and self‐renewal of haematopoietic stem cells. Although crucial for stem cell maintenance and homing, the stromal cell—stem cell interactions are poorly understood. Here we show that an ICAM‐like molecule is responsible for stem cell adhesion to stromal cellsin vitro. The molecule was characterized by a monoclonal antibody 3E10. Immunoblotting results indicated that the molecule had an electrophoretic mobility equal to that of intercellular cell adhesion molecule‐1 (ICAM‐1). Binding inhibition assays, however, showed that inhibition of binding of enriched CD34 cells by 3E10was more prominent in comparison with that of ICAM‐1.
PRIMORDIAL GERM CELL‐DERIVED MOUSE EMBRYONIC GERM (EG) CELLSIN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION
Authors: ROHWEDEL JÜRGEN , SEHLMEYER ULRICH , SHAN JIN , MEISTER ARMIN , WOBUS ANNA M.
Published: September 2002

Embryonic germ (EG) cells of line EG‐1 derived from mouse primordial germ cells were investigated for theirin vitrodifferentiation capacity. By cultivation as embryo‐like aggregates EG‐1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue‐specific genes and proteins as shown by RT‐PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI‐7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI‐7 cells showed a longer G1‐phase and shorter S‐ and G2/M‐phases. Together, our results demonstrate that the differentiation state and capacity of EG cellsin vitroresemble that of totipotent ES cells.
INFLUENCE OF SUCCESSIVE AND COMBINED ULTRAVIOLET A AND B IRRADIATIONS ON MATRIX METALLOELASTASES PRODUCED BY HUMAN DERMAL FIBROBLASTS IN CULTURE
Authors: KUT C. , HORNEBECK W. , GROULT N. , REDZINIACK G. , GODEAU G. , PELLAT B.
Published: September 2002

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three‐fold, at 24h intervals, with UVA (3×5J/cm2), UVB (3×8mJ/cm2), UVA plus UVB (3×5J/cm2and 3×8mJ/cm2) and the levels of 92kDa gelatinase (pro‐MMP9), 72kDa gelatinase (pro‐MMP2) and plasma‐membrane elastase type protease were determined, following subsequent 24‐h culture in 10% serum‐containing medium. UV irradiations had only minor influence (1.4‐fold increase for UVB) on secreted levels of pro‐MMP2and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66kDa activated MMP2production: 2.5‐ and 1.7‐fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro‐MMP9secretion by dermal fibroblast enhancing enzyme levels by 2.5‐, 6.5‐ and 5‐fold for UVA, UVB and UVA+UVB, respectively.
ALPHA‐FETOPROTEIN‐MEDIATED TARGETING—A NEW STRATEGY TO OVERCOME MULTIDRUG RESISTANCE OF TUMOUR CELLS IN VITRO
Authors: MOSKALEVA ELIZAVETA YU. , POSYPANOVA GALINA A. , SHMYREV IGOR I. , RODINA ALLA V. , MUIZHNEK EKATERINA L. , SEVERIN EUGENE S. , KATUKOV VALERY YU. , LUZHKOV YURY M. , SEVERIN SERGEI E.
Published: September 2002

The possibility of overcoming the multidrug resistance of human malignant cells by using doxorubicin conjugated to alpha‐fetoprotein (AFP) was studied. It was shown that this type of antitumour drugs, penetrating the cell by receptor‐mediated endocytosis with AFP as a vehicle, raises the sensitivity of the tumour cells that are resistant due to the expression of the multidrug resistance genemdr1. The sensitivity of antibiotic‐resistant cell lines SKVLB (a human ovarian carcinoma) and MCF‐7 AdrR (a human breast carcinoma) increased by 10‐ and 4‐fold, respectively, when AFP‐conjugated doxorubicin was used. The rationale of using human AFP‐antitumour drug conjugates for the development of new chemotherapeutic approaches to cancer treatment is discussed.
EFFECTS OF ADENOSINE AND EPIDERMAL GROWTH FACTOR ON THE GROWTH OF MOUSE MAMMARY EPITHELIAL CELLS
Authors: YUH I. S. , JANG B. B. , HONG B. J.
Published: September 2002

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24h, EGF (0–100ng/ml) and/or adenosine (0–100μm) was added. Adenosine at concentrations of 1, 10 or 100μmincreased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10ng/ml) into 1 or 10μmadenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100μmadenosine added to various concentrations of EGF (0–100ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100μm), EGF (100ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.