bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV-Positive Patients and the Doctor-Patient Relationship: Perspectives From the Margins
Authors: McCoy Liza
Published: June 2005

The doctor-patient relationship and the medical consultation are important resources for the health work of people living with chronic illness. In this article, the author examines physician-based outpatient health care from the standpoint of women and men who live with HIV in conditions of economic and social marginality. She draws on focus group and interview conversations with 79 HIV-positive individuals in southern Ontario. Using the approach of institutional ethnography, she offers a close reading of patients’ descriptions of what they consider good doctoring. Areas of best practice that enhance access to health care examined here include doctors’ interactional styles, ways of providing treatment options and treatment information, and ways of addressing the specific needs and life circumstances of patients living in poverty and social marginality.
Normalization in Families Raising a Child Who Is Medically Fragile/Technology Dependent and Developmentally Delayed
Authors: Rehm Roberta S. , Bradley Joan F.
Published: June 2005

The authors combined and analyzed parent data from two field studies examining family experiences in raising children who were both medically fragile and developmentally delayed or disabled to compare these families’ experiences to published attributes of normalization. Normalization is usually considered a useful conceptual and coping strategy for families of children with chronic conditions, but it has not been examined in families whose children have both complex physical and developmental disabilities. Developmental delays compounded the effects of the children’s physical chronic conditions, severely affecting how families organized and managed their daily lives, with the result that families did not fit currently established attributes of normalization. Instead, parents recognized normal and positive aspects of their lives while acknowledging the profound challenges that their families faced. Parents concluded that it was possible to have a good life that was not necessarily normal by usual standards.
Emotional reactions of parents after the birth of an infant with extremely low birth weight
Authors: Eriksson Birgitta Sandén , Pehrsson Gunnel
Published: June 2005

Emotional reactions of parents after the birth of an extremely low birth weight (ELBW) infant were followed during the early period of hospital care. Ten pairs of parents participated. The process concerning feelings of sadness, anger, fear/anxiety and joy was measured by Single Systems Design (SSD). The infants were born in gestation weeks 23-27. The emotional reactions of the mothers and fathers did not differ significantly. There was emotional confusion during the first weeks of parenthood, then the negative feelings decreased and joy increased. But at the end of a long period of care, the negative feelings reappeared. It is important to be aware that the first weeks after birth can be marked by emotional confusion and that negative feelings can return when the infant’s medical status has been brought under control. The latter might be explained emotionally as a delayed crisis reaction.
The experience of being ill as narrated by hospitalized children aged 7-10 years with short-term illness
Authors: Forsner Maria , Jansson Lilian , Sørlie Venke
Published: June 2005

Children’s illness has been investigated through the eyes of parents and nurses but the child’s own perspective has been largely ignored. The aim of this study is to illuminate the 7-10-year-olds’ experiences of being ill. Three girls and four boys were interviewed and narrated their experience about short-term illness. The data obtained was subjected to a thematic qualitative content analysis. The analysis suggests that the children combined reality and imagination and contrasts seemed to coexist such as being scared/confident, sad/cosy and hurt/having fun. They felt caught and tried to escape. The experience of illness as narrated by children can lead to a richer understanding and influence the way we care for paediatric patients.
Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis
Authors: Potula Raghava , Poluektova Larisa , Knipe Bryan , Chrastil Jesse , Heilman David , Dou Huanyu , Takikawa Osamu , Munn David H. , Gendelman Howard E. , Persidsky Yuri
Published: June 2005

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism. IDO activity is linked with immunosuppression by its ability to inhibit lymphocyte proliferation, and with neurotoxicity through the generation of quinolinic acid and other toxins. IDO is induced in macrophages by HIV-1 infection, and it is up regulated in macrophages in human brain tissue with HIV-1 encephalitis (HIVE). Using a model of HIVE, we investigated whether IDO inhibitor 1-methyl-d-tryptophan (1-MT) could affect the generation of cytotoxic T lymphocytes (CTLs) and clearance of virus-infected macrophages from the brain. Severe combined immunodeficient mice were reconstituted with human peripheral blood lymphocytes, and encephalitis was induced by intracranial injection of autologous HIV-1-infected monocyte-derived macrophages (MDMs). Animals treated with 1-MT demonstrated increased numbers of human CD3+, CD8+, CD8+/interferon-γ+ T cells, and HIV-1gag/pol-specific CTLs in peripheral blood compared with controls. At week 2 after MDM injection in the basal ganglia, mice treated with 1-MT showed a 2-fold increase in CD8+ T lymphocytes in the areas of the brain containing HIV-1-infected MDMs compared with untreated controls. By week 3, 1-MT-treated mice showed 89% reduction in HIV-infected MDMs in brain as compared with controls. Thus, manipulation of immunosuppressive IDO activity in HIVE may enhance the generation of HIV-1-specific CTLs, leading to elimination of HIV-1-infected macrophages in brain.
A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus
Authors: Charbonnier Yvan , Gettler Brian , François Patrice , Bento Manuela , Renzoni Adriana , Vaudaux Pierre , Schlegel Werner , Schrenzel Jacques
Published: June 2005

Background

DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus.

Results

We describe an efficient procedure for selecting 40–60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling.

Conclusion

This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.
Cell membrane array fabrication and assay technology
Authors: Yamazaki Victoria , Sirenko Oksana , Schafer Robert J , Nguyen Luat , Gutsmann Thomas , Brade Lore , Groves Jay T
Published: June 2005

Background

Microarray technology has been used extensively over the past 10 years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. By contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult.

Results

In this article, we describe a novel technology for generating cell membrane microarrays for performing high throughput biology. Our robotically-arrayed supported membranes are physiologically fluid, a critical property which differentiates this technology from other previous membrane systems and makes it useful for studying cellular processes on an industrialized scale. Membrane array elements consist of a solid substrate, above which resides a fluid supported lipid bilayer containing biologically-active molecules of interest. Incorporation of transmembrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as interactions with live intact cells. The fluidity of these molecules in the planar lipid bilayer facilitates dimerization and other higher order interactions necessary for biological signaling events. In order to demonstrate the utility of our fluid membrane array technology to ligand/receptor studies, we investigated the multivalent binding of the cholera toxin B-subunit (CTB) to the membrane ganglioside GM1. We have also displayed a number of bona fide drug targets, including bacterial endotoxin (also referred to as lipopolysaccharide (LPS)) and membrane proteins important in T cell activation.

Conclusion

We have demonstrated the applicability of our fluid cell membrane array technology to both academic research applications and industrial drug discovery. Our technology facilitates the study of ligand/receptor interactions and cell-cell signaling, providing rich qualitative and quantitative information.
Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV
Authors: Lu Kristina T , Dryer Rebecca L , Song Charles , Covey Lori R
Published: June 2005

Our previous investigation of a patient (pt1) with non-X-linked hyper-immunoglobulin M syndrome revealed a CD40-mediated defect in B cell activation that resulted in low CD23 expression and absence of germ-line transcription and class-switch recombination. These deficiencies were complemented in vitro by a high threshold of sustained sinaling through CD40. To further analyze the signaling defect in pt1 B cells, two types of Epstein-Barr virus lymphoblastoid cell lines (LCLs) were generated that either constitutively expressed the viral transforming protein latent membrane protein-1 (LMP1; pt1-LCL) or expressed it under the control of a tet-inducible promoter (pt1-LCLtet). Because LMP1 signals through the CD40 pathway, the pt1-LCL and pt1-LCLtet lines allow comparison of downstream functions in response to either constitutive LMP1 signals or regulated LMP1 and CD40 signals. Immortalized pt1-LCLs were initially CD23lo/CD38hi and reverted to a CD23hi/CD38lo phenotype upon extended growth in culture, suggesting that the CD40 defect was reversed by selection and/or constitutive expression of LMP1. In contrast, pt1-LCLtet cells retained the CD23lo/CD38hi phenotype after extended periods of culture and failed to up-regulate CD23 in response to CD40 signals. Analysis of pt1-LCLtet cells in response to the CD40 signals in the presence or absence of LMP1 revealed that mitogenic activation resulted only from LMP1 and not CD40, indicating a difference in the response of pt1 B cells to these two distinct signals. Together, these data demonstrate that the pt1-LCLtet cells maintain the CD40-related defect and provide a unique approach to study the independent effects of LMP1- and CD40-directed signals.
Effect of plasmid backbone modification by different human CpG motifs on the immunogenicity of DNA vaccine vectors
Authors: Coban Cevayir , Ishii Ken J , Gursel Mayda , Klinman Dennis M , Kumar Nirbhay
Published: June 2005

DNA vaccines, in general, have been found to be poorly immunogenic in nonhuman primates and humans as compared with mie. As the immunogenicity of DNA plasmids relies, to a large extent, on the presence of CpG motifs as built in adjuvants, we addressed the issue of poor immunogenicity by inserting recently identified CpG oligonucleotides (ODN) optimal for human (K-type or D-type CpG ODN) into the backbone of plasmid VR1020. We found that plasmid DNA containing K-type CpG motifs or D-type CpG motifs significantly enhanced the up-regulation of surface molecules and production of interleukin-6 from human peripheral blood mononuclear cells (PBMC) and stimulated monocytes to develop into functionally mature dendritic cells (DC) compared with unmodified plasmid. Monocyte maturation into DC was through plasmacytoid DC present in the culture. It is interesting that the K-type CpG motif-modified plasmid stimulated significant levels of interferon (IFN)-γ and IFN-α from human PBMC. Immunization of mice with D-type CpG motif-modified plasmid, encoding Plasmodium falciparum surface protein 25, yielded enhanced antigen-specific antibodies. Taken together, these results suggest that insertion of immunomodulatory human CpG motifs into plasmid DNA can improve immunogenicity of DNA vaccines.
Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy
Authors: Feili-Hariri Maryam , Falkner Dewayne H , Morel Penelope A
Published: June 2005

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced ow levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-γ, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-γ. In contrast, the GM + IL-15 DCs induced higher IFN-γ production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.
Growth and Differentiation of Astrocytes and Neural Progenitor Cells on Micropatterned Polymer Films
Authors: RECKNOR JENNIFER B. , SAKAGUCHI D S. , MALLAPRAGADA S.K.
Published: June 2005

Abstract: This paper investigates the influence of micropatterned polymers and chemical modification on neural progenitor cell growth and differentiation in co‐culture systems with astrocytes. We sought to develop stategies to facilitate nerve regeneration using a synergistic combination of guidance cues, investigating the cellular mechanisms of nerve repair using adult rat hippocampal progenitor cells (AHPCs). Our studies have shown that this synergistic combination of physical, chemical, and biological cues can lead to oriented growth of astrocytes and progenitor cells, can control and accelerate neurite outgrowth and alignment in vitro, and may influence differentiation of progenitor cells.
Infusion of Human Umbilical Cord Blood Ameliorates Neurologic Deficits in Rats with Hemorrhagic Brain Injury
Authors: NAN ZHENHONG , GRANDE ANDREW , SANBERG CYNDY D. , SANBERG PAUL R. , LOW WALTER C.
Published: June 2005

Abstract: Umbilical cord blood is a rich source of hematopoietic stem cells. It is routinely used for transplantation to repopulate cells of the immune system. Recent studies, however, have demonstrated that intravenous infusions of umbilical cord blood can ameliorate neurologic deficits associated with ischemic brain injury in rodents. Moreover, the infused cells penetrate into the parenchyma of the brain and adopt phenotypic characteristics typical of neural cells. In the present study we tested the hypothesis that the administration of umbilical cord blood can also diminish neurologic deficits caused by intracerebral hemorrhage (ICH). Intracerebral hemorrhage is a major cause of morbidity and mortality, and at the present time there are no adequate therapies that can minimize the consequences of this cerebrovascular event. ICH was induced in rats by intrastriatal injections of collagenase to cause bleeding in the striatum. Twenty‐four hours after the induction of ICH rats received intravenous saphenous vein infusions of human umbilical cord blood (2.4 × 106 to 3.2 to 106 cells). Animals were evaluated using a battery of tests at day 1 after ICH, but before the administration of umbilical cord blood, and at days 7, and 14 after ICH (days 6 and 13, respectively, after cord blood administration). These tests included a neurological severity test, a stepping test, and an elevated body‐swing test. Animals with umbilical cord blood infusions exhibited significant improvements in (1) the neurologic severity test at 6 and 13 days after cord blood infusion in comparison to saline‐treated animals (P < 0.05); (2) the stepping test at day 6 (P < 0.05); and (3) the elevated body‐swing test at day 13 (P< 0.05). These results demonstrate that the administration of human umbilical cord blood cells can ameliorate neurologic deficits associated with intracerebral hemorrhage.
How Wnt Signaling Affects Bone Repair by Mesenchymal Stem Cells from the Bone Marrow
Authors: GREGORY CARL A. , GUNN WILLIAM G. , REYES EMIGDIO , SMOLARZ ANGELA J. , MUNOZ JAMES , SPEES JEFFREY L. , PROCKOP DARWIN J.
Published: June 2005

Abstract: Human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo, and under appropriate conditions they differentiate into osteoblasts ex vivo. The cells provide a convenient cell culture model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose hMSCs to cell cycle entry and prevent osteogenesis. Our investigation of the role of Wnt signaling in osteogenesis by hMSCs ex vivo has demonstrated that osteogenesis proceeds in response to bone morphogenic protein 2 stimulation and is sustained by Wnt signaling. In the presence of Dkk‐1, an inhibitor of Wnt signaling, the cascade is disrupted, resulting in inhibition of osteogenesis. Peptide mapping studies have provided peptide Dkk‐1 agonists and the opportunity for the production of blocking antibodies. Anti‐Dkk‐1 strategies are clinically relevant since high serum levels of Dkk‐1 are thought to contribute to osteolytic lesion formation in multiple myeloma and possibly some forms of osteosarcoma. Specific inhibitors of glycogen synthetase kinase 3β (GSK3β), which mimic Wnt signaling, may also have a therapeutic benefit by enhancing in vitro osteogenesis despite the presence of Dkk‐1. Antibodies that block Dkk‐1 and GSK3β inhibitors may provide novel opportunities for the enhancement of bone repair in a variety of human diseases such as multiple myeloma and osteosarcoma.
Graft/Host Relationships in the Developing and Regenerating CNS of Mammals
Authors: OUREDNIK VÁCLAV , OUREDNIK JITKA
Published: June 2005

Abstract: A new light was shed on the utility of neural grafts when it was recognized that donor tissues and cells offer more than a source of immature progenitors potentially capable of cell replacement: First, they have the inherent capacity to produce multiple trophic and tropic factors promoting cell survival and tissue plasticity often characteristic of the immature central nervous system (CNS). Second, by their interaction with the host microenvironment via cell/cell and cell/ECM interactions, these grafts are capable of re‐establishing homeostasis, which can be, for example, reflected in rescue and protection of host elements from harmful influences. This second capacity of donor cells relies, in part, also on a “dormant” but still present regenerative capacity of mature or even aged CNS and on the possibility of its mobilization in the damaged nervous system by neural grafts. For this to occur efficiently after transplantation, a bi‐directional dialogue between donor and host cells must gradually be established, in which both “partners” transmit signals /cell/cell contact, molecular messengers), “listen to” and “understand” each other and are able to react by modifying their own plasticity‐ and development‐related programs. Thus, for the best possible recovery of functionality in the injured adult and aged nervous system, neurotransplantation must always try to find optimal conditions for all three of the mentioned qualities of neural grafts, especially for the protection and/or reactivation of neural circuitry embedded in non‐neurogenic CNS areas. Once fully understood, this newly recognized aspect of neurotransplantation (and topic of this review) might, someday, even allow the recovery of systems that would otherwise be doomed, such as cognition‐ and experience‐related circuitry.
Hearing Loss in Mitochondrial Disorders
Authors: HSU CHANG‐HUNG , KWON HAEYOUNG , PERNG CHERNG‐LIH , BAI REN‐KUI , DAI PU , WONG LEE‐JUN C.
Published: June 2005

Abstract: Hearing loss is a common clinical feature in mitochondria‐syndrome disorders. The underlining molecular etiology of hearing loss has not been fully investigated. In this study, 83 patients with mitochondrial syndromic hearing loss were evaluated clinically and their blood and tissue samples were examined molecularly. Using modified Walker's criteria, 31, 31, 14, and 7 patients had been classified as having definite, probable, possible, and unlikely diagnosis of mitochondrial disease, respectively. Deleterious mtDNA point mutations and/or abnormal mtDNA content or multiple deletions were identified in 20 patients with definite diagnosis and 2 patients with probable diagnosis. In addition to known, undisputed pathogenic mutations, several novel mutations believed to be clinically significant were found. Furthermore, abnormal mtDNA content and mtDNA deletions were found in some of the cases. Evaluation of clinical and diagnostic features associated with hearing loss revealed that cardiomyopathy, lactic acidosis, deficient respiratory chain enzyme complex activities, histochemical and ultrastructural abnormalities in mitochondria, and abnormal brain imaging results occurred significantly more frequently in patients with mtDNA alterations than in those without. This study revealed that the majority of the mtDNA defects in patients with mitochondrial syndromic hearing loss affect the overall mitochondrial gene expression.
Analysis of Proteome Bound to D‐Loop Region of Mitochondrial DNA by DNA‐Linked Affinity Chromatography and Reverse‐Phase Liquid Chromatography/Tandem Mass Spectrometry
Authors: CHOI YON‐SIK , RYU BO‐KYUNG , MIN HYE‐KI , LEE SANG‐WON , PAK YOUNGMI KIM
Published: June 2005

Abstract: Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D‐loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D‐loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D‐loop DNA of mtDNA. We amplified D‐loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr‐activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep‐gradient ultracentrifugation. The D‐loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS‐PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse‐phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D‐loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single‐stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D‐loop binding proteins such as histone family proteins and high‐mobility group proteins.
Analysis of Heteroplasmy in Hypervariable Region II of Mitochondrial DNA in Maternally Related Individuals
Authors: LO MEI‐CHEN , LEE HORNG‐MO , LIN MING‐WEI , TZEN CHIN‐YUAN
Published: June 2005

Abstract: Mitochondrial DNA sequences have been widely employed for identity investigation. However, the presence of a heteroplasmic site may complicate sequence analysis for forensic purposes when two samples are compared. To study this potential problem, we analyzed the hypervariable region of the displacement loop in five maternally related individuals, that is, grandmother, mother, one son, and two daughters. The results showed that three of them had a heteroplasmic site at nucleotide position (np) 204, located in the hypervariable region II. By using Bayesian inference to assess the significance of the mother‐offspring pairs, a likelihood ratio of 1.78 × 105 was obtained. Therefore, Bayesian inference does not place the prior odds into the context of the two different likelihood ratios derived from the DNA evidence. On the other hand, the chromatogram of the denaturing high‐performance liquid chromatography system proved that the single peak in a sequencing chromatogram at np 204 was, in fact, heteroplasmic in nature. This study demonstrated that heteroplasmy is a common occurrence in tissue from normal individuals and should be taken into account in forensic investigation when samples appear to differ at a single nucleotide position by direct sequencing.
Enhanced Generation of Mitochondrial Reactive Oxygen Species in Cybrids Containing 4977‐bp Mitochondrial DNA Deletion
Authors: JOU MEI‐JIE , PENG TSUNG‐I , WU HONG‐YUEH , WEI YAU‐HUEI
Published: June 2005

Abstract: The poor bioenergetic state in mitochondria containing mtDNA with the 4977‐bp deletion has been well documented. However, information on mitochondrial reactive oxygen species (ROS) generation at rest or under intense oxidative stress in mitochondria lacking the 4977‐bp mtDNA fragment inside intact living cells was insufficient. We used cybrids containing truncated mtDNA lacking the 4977‐bp fragment and measured ROS levels inside cybrids by fluorescence probe, 2′,7′‐dichlorodihydrofluorescein (DCF), and confocal microscopy. Mitochondrial ROS at resting state was slightly higher in cybrids containing 4977‐bp deletion mtDNA as compared to cybrids without mtDNA defects. For intense oxidative stress treatment, cybrids were treated with 5 mM H2O2 for 10 min. Consecutive DCF images were acquired after H2O2 had been washed away. Progressive increase of DCF signals, especially in the mitochondrial area, was observed in cybrids containing 4977‐bp deletion mtDNA, even long after the brief, intense H2O2 treatment. This result suggests that a feed‐forward, self‐accelerating vicious cycle of mitochondrial ROS production could be initiated in cybrids containing 4977‐bp deletion fragment mitochondria after brief, intense H2O2 treatment. This mechanism may play an important role in the pathophysiology of the disease process caused by mitochondria containing mtDNA with the 4977‐bp deletion.
Oxidative Stress‐Induced Depolymerization of Microtubules and Alteration of Mitochondrial Mass in Human Cells
Authors: LEE CHENG‐FENG , LIU CHUN‐YI , HSIEH RONG‐HONG , WEI YAU‐HUEI
Published: June 2005

Abstract: Mitochondrial biogenesis is a biological process that has been intensively studied over the past few years. However, the detailed molecular mechanism underlying this increase in mitochondria remains unclear. To investigate the mechanism of such a mitochondrial proliferation, we examined alterations in mitochondria of human osteosarcoma 143B cells that had been treated with 100 to 500 μM hydrogen peroxide (H2O2) for 48 h. The results showed that mitochondrial mass of the cell was increased with the increase of the concentration of H2O2. On the other hand, by using real‐time PCR techniques, we observed the changes of mitochondrial DNA (mtDNA) content in the cells exposed to oxidative stress. The copy number of mtDNA was increased by treatment with a low dose of H2O2 but was drastically decreased after treatment with H2O2 higher than 300 μM. Transmission electron microscopic images revealed that mitochondria were abnormally proliferated in cells exposed to oxidative stress. Moreover, we found that the percentage of 143B cells arrested at the G2/M phase increased upon treatment with H2O2. Immunostaining and microtubule fractionation assay revealed that microtubules were depolymerized in the cells that had been treated with H2O2. To understand the effect of microtubules depolymerization on the mitochondrial mass, we treated the cells with several kinds of microtubule‐active drugs, which arrest cultured cells at the G2/M phase. The results showed that mitochondrial mass and mtDNA copy number all were increased after such treatments. Taking these findings together, we suggest that oxidative stress‐induced microtubule derangement is one of the molecular events involved in the increase of mitochondrial mass upon treatment of human cells with H2O2.
Antisense RNA to Inducible Nitric Oxide Synthase Reduces Cytokine‐Mediated Brain Endothelial Cell Death
Authors: YANG DING‐I , CHEN SHAWEI , EZEKIEL UTHAYASHANKER R. , XU JAN , WU YINGJI , HSU CHUNG Y.
Published: June 2005

Abstract: We test whether inhibition of inducible nitric oxide synthase (iNOS) can exert a cytoprotective effect on cerebral endothelial cells upon stimulation by pro‐inflammatory cytokines. Mouse brain endothelial cells were stably transfected to express an antisense RNA against iNOS driven by an endothelium‐specific von Willebrand factor (vWF) promoter. Upon stimulation with tumor necrosis factor‐α (TNF‐α) plus interferon‐γ (IFN‐γ), antisense transfectants showed less iNOS enzymatic activity with less nitric oxide (NO) when compared to the sense control cells. Correspondingly, the antisense cells showed a reduced LDH release and less cytosolic content of oligonucleosomes. These findings establish a cell‐specific antisense strategy and confirm the cytotoxic role of iNOS expression in cultured cerebral endothelial cells.
Enhancement of Cisplatin‐Induced Apoptosis and Caspase 3 Activation by Depletion of Mitochondrial DNA in a Human Osteosarcoma Cell Line
Authors: YEN HSIU‐CHUAN , TANG YI‐CHIA , CHEN FAN‐YI , CHEN SHIH‐WEI , MAJIMA HIDEYUKI J.
Published: June 2005

Abstract: Cisplatin is an anticancer drug that can induce apoptosis. In this study, we investigated the effect of mitochondrial DNA (mtDNA) depletion on cisplatin‐induced cell death using a human osteosarcoma cell line (143B) and mtDNA‐depleted 143B cells (143B‐ρ0). Results showed that cisplatin decreased cell survival in 143B‐ρ0 cells. Moreover, cisplatin induced a greater extent of apoptosis‐associated DNA fragmentation and caspase 3 activation in 143B‐ρ0 cells. The release of mitochondrial cytochrome c into cytosol by cisplatin was enhanced more obviously in 143B cells than in 143B‐ρ0 cells; however, in the control group of 143B‐ρ0 cells, it was already dramatically greater. Depletion of mtDNA may increase sensitivity of cells to cisplatin‐induced apoptosis by enhancing caspase 3 activation via both cytochrome c‐dependent and ‐independent pathways.
Oxidative Toxicity in BV‐2 Microglia Cells: Sesamolin Neuroprotection of H2O2 Injury Involving Activation of p38 Mitogen‐Activated Protein Kinase
Authors: HOU ROLIS CHIEN‐WEI , WU CHIA‐CHUAN , HUANG JING‐RONG , CHEN YUH‐SHUEN , JENG KEE‐CHING G.
Published: June 2005

Abstract: Reactive oxygen species (ROS) has been proposed to play a pathogenic role in neuronal injury. Sesame antioxidants that inhibit lipid peroxidation and regulate cytokine production may suppress ROS generation. In this study, we focused on the effect of sesamolin on H2O2‐induced neurotoxicity and ROS production in the murine microglial cell line BV‐2. Results indicate that the H2O2 elicited BV‐2 cell death in a concentration‐ and time‐dependent manner. ROS generation in BV‐2 cells was time‐dependently increased by the H2O2 treatment. Sesamolin reduced ROS generation in BV‐2 cells. p38 mitogen‐activated protein kinase (MAPK) and caspase‐3 were also activated in BV‐2 cells under H2O2 stress. Sesamolin was able to inhibit H2O2‐induced p38 MAPK and caspase‐3 activation and cell death. In addition, sesamolin preserved superoxide dismutase and catalase activities in BV‐2 cells under H2O2 stress. In conclusion, sesamolin protects microglia against H2O2‐induced cell injury and this protective effect was accompanied by its inhibition of p38 MAPK and caspase‐3 activation and ROS production.
Identification of Three Mutations in the Cu,Zn‐Superoxide Dismutase (Cu,Zn‐SOD) Gene with Familial Amyotrophic Lateral Sclerosis: Transduction of Human Cu,Zn‐SOD into PC12 Cells by HIV‐1 TAT Protein Basic Domain
Authors: CHOU CHIH‐MING , HUANG CHANG‐JEN , SHIH CHWEN‐MING , CHEN YI‐PING , LIU TSANG‐PAI , CHEN CHIEN‐TSU
Published: June 2005

Abstract: The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu,Zn‐SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. Wild‐type SOD1 was constructed into a transcription‐translation expression vector to examine the SOD1 production in vitro. Wild‐type SOD1 was highly expressed in Escherichia coli. Active SOD1 was expressed in a metal‐dependent manner. To investigate the possible roles of genetic causes of ALS, a human Cu,Zn‐SOD gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of human immunodeficiency virus type 1 in a bacterial expression vector to produce a genetic in‐frame Tat‐SOD1 fusion protein. The expressed and purified Tat‐SOD1 fusion proteins in E. coli can enter PC12 neural cells to observe the cellular consequences. Denatured Tat‐SOD1 was successfully transduced into PC12 cells and retained its activity via protein refolding. Three point mutations, E21K, D90V, and D101G, were cloned by site‐directed mutagenesis and showed lower SOD1 activity. In undifferentiated PC12 cells, wild‐type Tat‐SOD1 could prevent DNA fragmentation due to superoxide anion attacks generated by 35 mM paraquat, whereas mutant Tat‐D101G enhanced cell death. Our results demonstrate that exogenous human Cu,Zn‐SOD fused with Tat protein can be directly transduced into cells, and the delivered enzymatically active Tat‐SOD exhibits a cellular protective function against oxidative stress.
Curcumin Inhibits ROS Formation and Apoptosis in Methylglyoxal‐Treated Human Hepatoma G2 Cells
Authors: CHAN WEN‐HSIUNG , WU HSIN‐JUNG , HSUUW YAN‐DER
Published: June 2005

Abstract: Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti‐inflammatory properties. In the present study, we investigated the effect of curcumin on MG‐induced apoptotic events in human hepatoma G2 cells. We report that curcumin prevented MG‐induced cell death and apoptotic biochemical changes such as mitochondrial release of cytochrome c, caspase‐3 activation, and cleavage of PARP (poly [ADP‐ribose] polymerase). Using the cell permeable dye 2′,7′‐dichlorofluorescein diacetate (DCF‐DA) as an indicator of reactive oxygen species (ROS) generation, we found that curcumin abolished MG‐stimulated intracellular oxidative stress. The results demonstrate that curcumin significantly attenuates MG‐induced ROS formation, and suggest that ROS triggers cytochrome c release, caspase activation, and subsequent apoptotic biochemical changes.
Prevention of Cellular Oxidative Damage by an Aqueous Extract of Anoectochilus formosanus
Authors: WANG LENG‐FANG , LIN CHUN‐MAO , SHIH CHWEN‐MING , CHEN HUI‐JU , SU BORCHERNG , TSENG CHENG‐CHUANG , GAU BAO‐BIH , CHENG KUR‐TA
Published: June 2005

Abstract: Anoectochilus formosanus (AF) is a popular folk medicine in Taiwan whose pharmacological effects have been characterized. In this work we investigated the antioxidant properties of an aqueous extract prepared from AF. The AF extract was capable of scavenging H2O2 in a dose‐dependent manner. We induced oxidative stress in HL‐60 cells, either by the addition of hydrogen peroxide (H2O2) or by the xanthine/xanthine oxidase reaction. Apoptosis caused by oxidative damage was displayed by DNA fragmentation on gel electrophoresis, and the apoptotic fraction was quantified with flow cytometry. The cell damage induced by oxidative stress was prevented by the plant extract in a concentration‐dependent manner. Furthermore, the proteolytic cleavage of poly(ADP‐ribose) polymerase during the apoptotic process was also inhibited by AF extract. Our results provide the basis for determining an AF extract to be an antioxidant.
Effects of Gonadotrophin‐Releasing Hormone Agonists on Apoptosis of Granulosa Cells
Authors: TSAI NU‐MAN , HSIEH RONG‐HONG , AU HENG‐KIEN , SHIEH MING‐JER , HUANG SHIH‐YI , TZENG CHII‐RUEY
Published: June 2005

Abstract: Granulosa cells are known to contribute to maturation of oocytes, and most of the growth factors exert their action via granulosa cells. It has been established that granulosa cell death during follicular atresia and luteolysis results from apoptosis. However, the precise mechanistic pathways of granulosa cell apoptosis have not yet been defined. In this study, we determined the proportions of apoptosis in granulosa cells treated with two kinds of gonadotrophin‐releasing hormone agonists (GnRHa): buserelin and leuprorelin depot. The incidences of DNA fragmentation of human granulosa cells treated with buserelin and leuprorelin were 54.33% and 39.02%, respectively. The proportions of apoptotic bodies were 6.04% and 4.29%, respectively. There was a significant difference in the proportions of DNA fragmentation between the two kinds of GnRHa‐treated granulosa cells. The apoptosis pathway and associated protein expression in granulosa cells treated with GnRHa were also determined. The Bax molecule, a pro‐apoptosis protein, was expressed in granulosa cells undergoing apoptosis. In contrast, Bcl‐2, an anti‐apoptosis protein, could not be detected in the same group of granulosa cells. The distribution of cytochrome c determined via immunostaining showed a diffuse pattern, which most likely indicated that cytochrome c was translocated from mitochondria into the cytoplasm. Western blotting showed the expressions of caspase‐9 and caspase‐3 in patients' granulosa cells. The GnRHa effects on granulosa cells indicated a higher incidence of DNA fragmentation and apoptotic bodies in the buserelin‐treated than in the leuprorelin depot‐treated group. The granulosa cells go through the mitochondria‐dependent apoptosis pathway; the indicated pro‐apoptosis protein Bax was expressed and induced cytochrome c release from mitochondria, which then activated caspase‐9 and caspase‐3 until cell death occurred.
Tunicate Gonadotropin-Releasing Hormone (GnRH) Peptides Selectively Activate Ciona intestinalis GnRH Receptors and the Green Monkey Type II GnRH Receptor
Authors: Tello Javier A. , Rivier Jean E. , Sherwood Nancy M.
Published: June 2005

In vertebrates, GnRH binds to its receptor and stimulates predominantly Gq/11-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.
Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy
Authors: Ho Thai H. , Savkur Rajesh S. , Poulos Michael G. , Mancini Michael A. , Swanson Maurice S. , Cooper Thomas A.
Published: June 2005

Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.
Formin-induced actin cables are required for polarized recruitment of the Ste5 scaffold and high level activation of MAPK Fus3
Authors: Qi Maosong , Elion Elaine A.
Published: June 2005

Little is known about how a mitogen-activated protein kinase (MAPK) cascade is targeted to specific sites at the plasma membrane during receptor stimulation. In budding yeast, the Ste5 scaffold is recruited to a receptor-coupled G protein during mating pheromone stimulation, allowing the tethered MAPK cascade to be activated by Ste20, a Cdc42-anchored kinase. Here we show that stable recruitment of Ste5 at cortical sites requires the formin Bni1, Bni1-induced actin cables, Rho1 and Myo2. Rho1 directs recruitment of Bni1 via the Rho-binding domain, and Bni1 mediates localization of Ste5 through actin cables and Myo2, which co-immunoprecipitates with Ste5 during receptor stimulation. Bni1 is also required for polarized recruitment and full activation of MAPK Fus3, which must bind Ste5 to be activated, and polarized recruitment of Cdc24, the guanine exchange factor that binds Ste5 and promotes its recruitment to the G protein. In contrast, Bni1 is not important for activation of MAPK Kss1, which can be activated while not bound to Ste5 and does not accumulate at cortical sites. These findings reveal that Bni1 mediates the formation of a Ste5 scaffold/Fus3 MAPK signaling complex at polarized sites, and suggests that a pool of Ste5 may translocate along formin-induced actin cables to the cell cortex.
Thermodynamic methods for performance analysis of chillers
Authors: Ng K. C. , Wang X. L.
Published: June 2005

This paper presents a simple thermodynamic model that captures accurately the behaviour of all types of real chillers or cooling devices. In addition to its predictive capability, it also provides a diagnostic means for the in situ monitoring of chiller performance. To enhance the understanding of the chiller cycles, a novel way of formulating the classic T-s diagrams is provided for all chiller types, ranging from the mechanical, heat-driven and solid-state chillers.
Modular regenerative adsorption cycles with fixed beds, applied to trigeneration
Authors: Critoph R. E.
Published: June 2005

A regenerative adsorption cycle based on the use of low-cost tubular modules arranged in a multirow bed is described. It is a variety of ‘thermal wave’ cycle, capable of a very high coefficient of performance (COP). Detailed simulations of system performance have been carried out using measured carbon heat transfer and porosity data. Depending upon a suitable choice of heating fluid flowrate and time between flow reversals, it is possible to achieve air conditioning COPs in excess of 1.1, albeit at a very low specific coding power (SCP) of 10W/kg. The same machine can be operated to achieve a range of COP and SCP combinations, depending on the control strategy.
R134a flow patterns in small-diameter tubes
Authors: Chen L , Tian Y. S. , Karayiannis T. G.
Published: June 2005

R134a vapour-liquid two-phase flow patterns were studied in vertical small-diameter tubes. The observed flow patterns include bubbly, dispersed bubble, slug, churn, annular, and mist flow. Six integrated flow pattern maps, derived for two internal diameters (2.01 and 4.26 mm) and three different pressures (6.0, 10.0, 14.0 bar), are presented. Some transition boundaries, such as slug-churn and churn-annular, were found to be very sensitive to diameter and pressure. In contrast the boundaries of dispersed bubble-churn and bubbly-slug are less affected. The transition boundaries are compared with the existing models for normal size tubes showing significant differences.
Geometrical optimization of bare tube heat exchangers for process industries
Authors: Ma T. Q. , Ooi K. T. , Wong T. N.
Published: June 2005

This paper presents simulation results on the geometrical optimization design of bare tube heat exchangers. By linking a mathematical model with an optimization alogorithm, it is possible to predict which combination of five geometrical variables would produce a given coil capacity of a heat exchanger, the minimum core volume size operating at the minimum pressure drop. A constrained multivariable direct search technique is used in which the five geometrical variables and a mixture of five explicit and implicit constraints are accommodated. Using this design method, three typical sizes of bare tube optimization cases have been studied. The simulation results predict significant performance improvements for heat exchanger design. The range of tube outer diameter in this optimization study is from 4.9 to 9.0 mm.