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Biomedical subjects

A Chang

Publications and source records attributed to A Chang.

At least 55 records · Page 3Linked to original sources
PubMed2001

Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine.

MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following intramuscular administration. In a tissue distribution study in mice, MuStDO 5 plasmid DNA was detected by PCR initially in highly vascularized tissues, while at later time-points the plasmid DNA was detected primarily at the site(s) of injection. In GLP safety studies in mice and rabbits, repeated intramuscular/intradermal administration of the MuStDO 5 vaccine was found to be safe and well tolerated without any evidence of autoimmune pathology.

Adjuvants, Immunologic↗
PubMed2001

Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast.

Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments. In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface. Disruption of rafts led to mistargeting of Pma1p to the vacuole. Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association. One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7. Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer. We suggest that clustering facilitates partition of Pma1p into rafts and transport to the cell surface.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae.

Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

Alleles↗
PubMed2001

Etk/Bmx activation modulates barrier function in epithelial cells.

Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O(2)) exposure, the TER and equivalent active ion transport rate (I(eq)) were reduced to <5% of the normoxia control in Pa-4 cells, whereas both TER and I(eq) were maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of beta-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of beta-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.

Actins↗
PubMed2001

Early events in naphthalene-induced acute Clara cell toxicity. II. Comparison of glutathione depletion and histopathology by airway location.

One of the presumed roles of intracellular glutathione (GSH) is the protection of cells from injury by reactive intermediates produced by the metabolism of xenobiotics. To establish whether GSH depletion is a critical step in the initiation of events that lead to cytotoxicity by P450-activated cytotoxicants, naphthalene, a well-defined Clara cell cytotoxicant, was administered to mice (200 mg/kg) by intraperitoneal injection. Shortly after injection (1, 2, and 3 h), intracellular GSH content was assessed by high performance liquid chromatography or quantitative epifluorescent imaging microscopy and compared with the degree of cytotoxicity as assessed by high resolution histopathology. In highly susceptible airways (distal bronchioles), GSH decreased by 50% in 1 h. Cytoplasmic vacuolization was not visible until 2 h, when GSH had decreased by an additional 50%. By 3 h, cytoplasmic blebbing was extensive. In minimally susceptible airways (lobar and proximal bronchi), GSH depletion varied widely within the population; a small proportion of the cells lost greater than 50% of their GSH by 2 h and a significant percentage of the cells retained most of their GSH throughout the entire 3 h. Cytoplasmic vacuolization was apparent in some of the cells at 2 h but not visible in any cells at 3 h. We conclude that (1) loss of intracellular GSH is an early event that precedes initial signs of cellular damage in Clara cell cytotoxicity; (2) this pattern of loss in relation to early injury is found both in highly susceptible and minimally susceptible airway sites; (3) there is wide cell-to-cell heterogeneity in the response; (4) the heterogeneity in the response profile varies between populations in highly susceptible and minimally susceptible sites; and (5) once the intracellular GSH concentration within the entire cell population drops below a certain threshold, the initial phase of injury becomes irreversible.

Animals↗
PubMed2001

Superoxide dismutase and catalase do not affect the pulmonary hypertensive response to group B streptococcus in the lamb.

The purpose of this study was to determine whether treatment with conjugated antioxidant enzymes could attenuate or abolish pulmonary hypertension induced by group B streptococcus (GBS). Lambs, 3-7 d old, were anesthetized and ventilated. Intravascular catheters were placed in the left ventricle, descending aorta, right atrium, and pulmonary artery for continuous monitoring of intravascular pressures. Cardiac output was measured with radiolabeled microspheres. Measurements were obtained at baseline and 15 and 60 min into a 60-min GBS infusion, and 60 min after GBS was stopped. Blood gas values were held constant and PaO(2) was maintained >100 mm Hg. The control group received saline vehicle only (n = 6), the GBS group received GBS infusion only (n = 9), the enzymes (ENZ) group received polyethylene glycol-superoxide dismutase (PEG-SOD) and polyethylene glycol-catalase (PEG-CAT) treatment only (n = 6), and the ENZ+GBS group received PEG-SOD and PEG-CAT then GBS (n = 9). Plasma samples were obtained to confirm increased superoxide dismutase and catalase activities in the groups receiving enzymes. Compared with baseline, pulmonary vascular resistance increased by 119% and 101% at 15 min and 87% and 81% at 60 min in the GBS and ENZ+GBS groups, respectively. Sixty minutes after the termination of the GBS infusion, PVR returned to baseline in the GBS group but did not in the ENZ+GBS group. Enzyme infusions resulted in at least a ninefold increase in plasma enzyme activities. As opposed to previously published data from endotoxin models, PEG-CAT and PEG-SOD were ineffective in altering the GBS-induced pulmonary hypertensive response in this model. This suggests that acute administration of antioxidant enzymes may not be effective in ameliorating GBS-induced pulmonary hypertension.

Animals↗
PubMed2001

[Transanal endoscopic microsurgery: a forgotten minimally invasive technique].

OBJECTIVES: The aim of this study was to evaluate transanal endoscopic microsurgery in patients with benign and malignant rectal tumours with special reference to feasibility, morbidity, and recurrence rate. METHODS: Forty-three patients underwent transanal endoscopic microsurgical excision of rectal tumours between 1996 and 2000. The histological diagnosis was benign adenoma in 30 and invasive carcinoma in 13. The mean height of the tumour above the anal verge was 11.2 +/- 3 cm and the mean diameter of the lesion was 3.4 +/- 1.5 cm. RESULTS: The mean operative time was 85 +/- 26 min and in one case (2%), it was necessary to convert to an anterior resection. The morbidity rate was 18%. Mean hospital stay was 3.9 +/- 2.4 days. Complete excision of the tumour with histological confirmation was achieved in 42 cases (98%). With a mean follow-up of 26 months, benign tumour recurrence was observed in one patient (3%). Of the 13 patients with carcinoma, two had immediate further radical resection. For the remaining 11 patients, with a mean follow-up of 19 months, the recurrence rate was 75% for T2 tumours and nil for T1 tumours. CONCLUSIONS: Transanal endoscopic microsurgery is safe and feasible technique which should have a useful place in the management of sessile adenomas of the mid and upper rectum. Its role in the management of rectal cancer is limited, although it may be appropriate for carefully selected cases.

Adenoma↗
PubMed2000

Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1.

The small evolutionarily conserved protein Dim1p/hDim1/Dib1p/DML-1 was initially defined as a factor essential for progression through the G2/M transition, and shown to be required to maintain the steady state level of a component of the fission yeast anaphase promoting complex/cyclosome. More recently, Dib1p has been defined as a component of the U4/U6.U5 tri-snRNP, required for pre-mRNA splicing. To investigate the mechanism(s) of Dim1 function, reiterative two-hybrid screening was performed to identify interacting proteins. Proteins thus identified were solely those involved in pre-mRNA splicing or related functions, and one partner induced a striking synthetic phenotype when co-expressed with hDim1 in mammalian cells. Saturating alanine scanning mutagenesis of Dim1 allowed delineation of amino acids essential for its ability to interact with its defined partners: mapping these residues on the structural coordinates of hDim1 defined an interactive sector of the protein. Finally, depletion studies have recently shown that Dim1 function is essential for pre-mRNA splicing in yeast. We find that elimination of DML-1 expression in C. elegans by RNA interference leads to embryonal lethality during gastrulation, marked by a failure to correctly express early zygotic transcripts. These results parallel the arrest phenotypes associated with global disruption of zygotic gene expression, suggesting that Dim1 proteins maintain an essential function in gene expression in higher eukaryotes.

Animals↗
PubMed2000

Effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts.

CONTEXT: A growing proportion of young children in the United States participate in day care, and these children are considered to be at high risk for influenza infection. Whether vaccinating day care children reduces household transmission of influenza is not known. OBJECTIVE: To evaluate the effect of vaccinating day care children on reducing influenza-related morbidity among their household contacts. DESIGN: Single-blind, randomized controlled trial conducted during the 1996-1997 influenza season. SETTING: Ten day care centers for children of US Navy personnel in San Diego, Calif. PARTICIPANTS: A total of 149 day care attendees (aged 24-60 months) and their families were randomized; 127 children and their 328 household contacts received 2 vaccine doses and were included in the analysis. INTERVENTIONS: Inactivated influenza vaccine was administered to 60 children with 162 household contacts, and hepatitis A vaccine as a control was administered to 67 age-matched children with 166 household contacts. MAIN OUTCOME MEASURES: Information regarding febrile respiratory illnesses and related morbidity for household contacts of influenza-vaccinated vs control children (subgrouped by influenza-vaccinated and unvaccinated contacts), obtained by telephone interviews with parents every 2 weeks from November 1996 through April 1997. RESULTS: Influenza-unvaccinated household contacts (n = 120) of influenza-vaccinated day care children had 42% fewer febrile respiratory illnesses (P =.04) compared with unvaccinated household contacts of control children. Among school-aged household contacts (aged 5-17 years), there was an 80% reduction among contacts of vaccinated children (n = 28) vs contacts of unvaccinated children (n = 31) in febrile respiratory illnesses (P =.01), as well as reductions of more than 70% in school days missed (P =.02), reported earaches (P =.02), physician visits (P =.007), physician-prescribed antibiotics (P =.02), and adults who missed work to take care of ill children (P =.04). CONCLUSIONS: These results indicate that vaccinating day care children against influenza helps reduce influenza-related morbidity among their household contacts, particularly among school-aged contacts. Future studies should be conducted in civilian populations to assess the full effect of vaccinating day care children against influenza. JAMA. 2000;284:1677-1682.

Adolescent↗
PubMed2000

Treatment of solid tumours in children with tumour-lysate-pulsed dendritic cells.

Dendritic cells are potent stimulators of antigen-specific immune responses, including antitumour responses. We explored the use of tumour-lysate-pulsed dendritic cells in children with relapsed solid tumours. Dendritic cell treatment in children was feasible and apparently not toxic. The treatment was able to produce significant tumour regression in a child with metastatic fibrosarcoma.

Adolescent↗
PubMed2000

Genomic organization of the human kallikrein gene family on chromosome 19q13.3-q13.4.

Kallikreins are a subgroup of serine proteases with diverse physiological functions. Recently, growing evidence indicates that many kallikrein genes are involved in malignancy. In rodents, kallikreins are encoded by a large multigene family, but in humans only three kallikreins were thought to exist. Based on the homology between the human and rodent kallikrein loci, we studied a 300 kb region of genomic sequences around the putative KLK1 gene locus on chromosome 19q13.3-q13.4. By using linear sequence information, restriction analysis, end sequencing, PCR and blotting techniques, as well as bioinformatic approaches, we were able to construct the first detailed map of the human kallikrein gene family. Comparative analysis of genes located in this area, provides strong evidence that the human kallikrein gene family locus on chromosome 19 is considerably larger than previously thought, containing at least fifteen genes. We have established, for the first time, the common structural features that apply to all members of the expanded kallikrein multigene family. Our map specifies the distance between genes to one base pair accuracy, the relative location, and the direction of transcription of all 15 genes. Determination of the true size of the kallikrein family in humans is important for our understanding of the contribution of the kallikreins to human biology and pathophysiology.

Chromosome Mapping↗
PubMed2000

Studies of the 1996-1997 inactivated influenza vaccine among children attending day care: immunologic response, protection against infection, and clinical effectiveness.

A randomized, blinded, pilot study of influenza vaccine administered to children attending day care centers was conducted during the 1996-1997 winter. Vaccine efficacy in preventing serologically proven influenza virus infection was 0.45 (95% confidence limit [CL]: -0.02, 0.69) for influenza B and 0.31 (95% CL: -0.95, 0.73) for influenza A(H3N2). For both influenza A(H3N2) and B, children without preexisting hemagglutination inhibition (HI) antibody to these antigens had lower antibody responses to vaccine, were less likely to develop a serological response, and were more likely to develop serological evidence of influenza infection. Although there were no reductions in respiratory or febrile respiratory illnesses among all vaccinated children, there was a trend for reductions in such illnesses among vaccinated children with preexisting HI antibodies to influenza A(H3N2) and B. Therefore, immunologic priming in young children may be important for vaccine response and for protection against infection. Larger studies are needed in other influenza seasons to assess vaccine efficacy and clinical effectiveness.

Antibodies, Viral↗
PubMed2000

NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions.

Multiple sclerosis (MS) is characterized by multifocal loss of myelin, oligodendrocytes, and axons. Potential MS therapies include enhancement of remyelination by transplantation or manipulation of endogenous oligodendrocyte progenitor cells. Characteristics of endogenous oligodendrocyte progenitors in normal human brain and in MS lesions have not been studied extensively. This report describes the distribution of cells in sections from normal adult human brain and MS lesions by using antibodies directed against NG2, an integral membrane chondroitin sulfate proteoglycan expressed by oligodendrocyte progenitor cells. Stellate-shaped NG2-positive cells were detected in the white and gray matter of normal adult human brain and appeared as abundant as, but distinct from, astrocytes, oligodendrocytes, and microglia. Stellate-shaped or elongated NG2-positive cells also were detected in chronic MS lesions. A subpopulation of the elongated NG2-positive cells expressed the putative apoptotic signaling molecule p75(NTR). TUNEL-positive cells in three active, nine chronic active, and four chronic inactive lesions, however, were p75(NTR)-negative. These studies identify cells with phenotypic markers of endogenous oligodendrocyte progenitors in the mature human CNS and suggest that functional subpopulations of NG2-positive cells exist in MS lesions. Endogenous oligodendrocyte progenitor cells may represent a viable target for future therapies intended to enhance remyelination in MS patients.

Adult↗
PubMed2000

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.

Combinatorial Chemistry Techniques↗
PubMed2000

Identification and characterization of KLK-L4, a new kallikrein-like gene that appears to be down-regulated in breast cancer tissues.

Kallikreins are a subgroup of serine proteases and these proteolytic enzymes have diverse physiological functions in many tissues. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins constitute a large multigene family, but in humans, only three genes were identified. By using the positional candidate gene approach, we were able to identify a new kallikrein-like gene, tentatively named KLK-L4 (for kallikrein-like gene 4). This new gene maps to chromosome 19q13. 3-q13.4, is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK-L4 is expressed in a variety of tissues including prostate, salivary gland, breast, and testis. Our preliminary results show that KLK-L4 is down-regulated, at the mRNA level, in breast cancer tissues and breast cancer cell lines. Its expression is regulated by steroid hormones in the breast cancer cell line BT-474. This gene may be involved in the pathogenesis and/or progression of breast cancer and may find applicability as a novel cancer biomarker.

Amino Acid Sequence↗