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Biomedical subjects

W Tao

Publications and source records attributed to W Tao.

At least 19 recordsLinked to original sources
PubMed2004

SDF-1alpha/CXCL12 enhances retroviral-mediated gene transfer into immature subsets of human and murine hematopoietic progenitor cells.

Genetic modification of hematopoietic stem and progenitor cells has the potential to treat diseases affecting blood cells. Oncoretroviral vectors have been used for gene therapy; however, clinical success has been limited in part by low gene transfer efficiencies. We found that the presence of stromal-derived factor 1 (SDF-1alpha)/CXCL12 during retroviral transduction significantly enhanced, in a dose-dependent fashion, gene transfer into immature subsets of high proliferative human and murine hematopoietic progenitor cells. Murine mononuclear bone marrow cells and purified c-Kit(+)Lin(-) bone marrow cells were prestimulated and transduced with the bicistronic retroviral vector MIEG3 on Retronectin-coated surfaces in the presence and absence of SDF-1. SDF-1 enhanced gene transduction of murine bone marrow and c-Kit(+)Lin(-) cells by 35 and 29%, respectively. Moreover, SDF-1 enhanced transduction of progenitors in these populations by 121 and 107%, respectively. SDF-1 also enhanced transduction of human immature subsets of high proliferative progenitors present in either nonadherent mononuclear or CD34(+) umbilical cord blood cells. Transduction of hematopoietic progenitors was further increased by preloading Retronectin-coated plates with retrovirus using low-speed centrifugation followed by increasing cell-virus interactions through brief centrifugation during the transduction procedure. These results may be of clinical relevance.

Animals↗
PubMed2001

Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1.

We report the isolation and cloning of the Wrch-1 (Wnt-1 responsive Cdc42 homolog) cDNA. Wrch-1 is a novel gene whose mRNA level increases in response to Wnt-1 signaling in Wnt-1 transformed cells, Wnt-1 transgene induced mouse mammary tumors, and Wnt-1 retrovirus infected cells. Wrch-1 encodes a homolog of the Rho family of GTPases. It shares 57% amino acid sequence identity with Cdc42, but possesses a unique N-terminal domain that contains several putative PXXP SH3-binding motifs. Like Cdc42, Wrch-1 can activate PAK-1 and JNK-1, and induce filopodium formation and stress fiber dissolution. Active Wrch-1 stimulates quiescent cells to reenter the cell cycle. Moreover, overexpression of Wrch-1 phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the effects of Wnt-1 signaling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation.

Amino Acid Sequence↗
PubMed2001

Effects of phosphocreatine on apoptosis in a cell-free system.

The characteristic morphological and biochemical changes during caspase-mediated apoptosis can be reproduced to a large extent in a Xenopus laevis egg extract cell-free system by addition of mouse liver nuclei and exogenous cytochrome c. We show that in this system phosphocreatine accelerated the apoptotic morphological changes of the nuclei, but selectively inhibited DNA fragmentation. Western blot showed that the degradation of lamins A and C is accelerated, which is possibly responsible for the nuclear changes during cell apoptosis. However, the degradation of ICAD/DFF45-like protein in the egg extracts is inhibited in a time-dependent manner. Exogenous creatine, ATP, and several organic acids have no effect on DNA fragmentation, excluding the possibility that creatine, ATP, or acidic conditions resulting from phosphocreatine are responsible for inhibiting DNA fragmentation. Lithium chloride, a kinase inhibitor, can overcome the phosphocreatine effects and can restore DNA fragmentation. Our results indicate that phosphocreatine protects ICAD/DFF45-like protein from proteolysis, probably through kinase actions, resulting in its resistance to caspase cleavage and leading to an inhibition of DNA fragmentation.

Adenosine Triphosphate↗
PubMed2001

Distribution and transcription activity of nucleolar DNA in higher plant cells.

By using the NAMA-Ur DNA selective staining method, we have observed in situ the location of nucleolar DNA in onion cells and found it at the boundary between fibrillar centres (FC) and dense fibrillar component (DFC) in transcriptionally active nucleolus. We have also used anti-NOR serum, which is identified as the RNA Polymerase I transcription factor (UBF) antibody, to study its reactivity with higher plant cells and demonstrated this factor associated to the DFC but not present at the interior of FC. Finally, by employing anti-DNA/RNA hybrid antibodies, we labeled the transcriptionally active rRNA genes in active nucleolus and testified that at the boundary between FC and DFC. The results provide the evidence that the boundary between FC and DFC is the genuine transcription site of rRNA genes in nucleolus.

Cell Nucleolus↗
PubMed2001

A new method for protein coexpression in Escherichia coli using two incompatible plasmids.

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.

Apoptosis Regulatory Proteins↗
PubMed2001

Safety and efficacy of a heparin removal device: a prospective randomized preclinical outcomes study.

BACKGROUND: Systemic protamine sulfate for heparin reversal after cardiopulmonary bypass (CPB) is associated with uncommon, but life-threatening adverse reactions. METHODS: In a prospective randomized 3-day outcomes study, a heparin removal device (HRD) group (n = 12; 60-, 80-, 100-kg subgroups) was compared with a matched systemic Protamine group (Protamine; n = 6) for safety and efficacy using an adult swine model of CPB (60 minutes, 28 degrees C). RESULTS: HRD run time was 25 to 38 minutes depending on weight without complications. After HRD, heparin concentration decreased from 4.77 +/- 0.17 to 0.45 +/- 0.06 U/mL (activated clotting time [ACT] 776 +/- 83 to 180 +/- 12 seconds), and in Protamine, 3.94 +/- 0.63 to 0.13 +/- 0.02 U/mL (ACT 694 +/- 132 to 101 +/- 5 seconds) (p = 0.01 between groups, but no significant differences 60 minutes later). No significant difference between HRD and Protamine to 72 hours was seen in plasma-free hemoglobin C3a, heparin concentration, thromboelastogram index, platelet count, activated partial thromboplastin time, anti-thrombin III, fibrinogen, ACT, and tissue histology. CONCLUSIONS: In a prospective randomized outcomes study, HRD achieved predictable reversal of systemic heparinization after CPB with no difference in safety or outcomes compared with protamine.

Adsorption↗
PubMed2001

Structural components of the nuclear body in nuclei of Allium cepa cells.

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Cell Nucleolus↗
PubMed2001

Percutaneous extracorporeal arteriovenous carbon dioxide removal improves survival in respiratory distress syndrome: a prospective randomized outcomes study in adult sheep.

OBJECTIVE: Arteriovenous carbon dioxide removal (AVCO(2)R) uses a simple arteriovenous shunt for CO(2) removal to minimize barotrauma/volutrauma from mechanical ventilation. We performed a prospective randomized outcomes study of AVCO(2)R in our new, clinically relevant model of respiratory distress syndrome. METHODS: Adult sheep (n = 18) received an LD(50) severe smoke inhalation and 40% third-degree burn. When respiratory distress syndrome developed (PaO (2)/FIO (2) < 200 at 40 to 48 hours), animals were randomized to the AVCO(2)R (n = 9) or sham group (n = 9) for 7 days. Ventilator management protocols mandated reductions in minute ventilation, first tidal volume to peak inspiratory pressure less than 30 cm H(2)O, then respiratory rate when PaCO (2) was less than 40 mm Hg. PaO (2) was kept above 60 mm Hg by adjusting FIO (2). When FIO (2) was 0.21, animals were weaned. RESULTS: The study required 2946 animal-hours of critical care with 696 AVCO(2)R hours. One died in each group during model development. AVCO(2)R flow from 820 mL/min to 970 mL/min (11% to 14% cardiac output) removed CO(2) at a rate of 92 to 116 mL/min (mean 103 mL/min; 93%-97% of CO(2) production). Heart rate, mean arterial pressure, cardiac output, and pulmonary arterial wedge pressure remained relatively constant. Within 48 hours, AVCO(2)R allowed significant ventilator reductions versus baseline in the following measurements: tidal volume (420 to 270 mL), peak inspiratory pressure (25 to 14 cm H(2)O), minute ventilation (13 to 5 L/min), respiratory rate (26 to 16 breaths/min), and FIO (2) (0.88 to 0.35). Ventilator-free days with AVCO(2)R were 3.9 versus 0.2 (P <.01) for sham animals, and ventilator-dependent days with AVCO(2)R were 2.4 versus 6.2 (P <.01) for the 3 sham survivors. All 8 AVCO(2)R animals and 3 of 8 sham animals survived 7 days after randomization. CONCLUSIONS: Percutaneous AVCO(2)R achieved significant reduction in airway pressures, increased ventilator-free days, decreased ventilator-dependent days, and improved survival in a sheep model of respiratory distress syndrome.

Animals↗
PubMed2001

[Magnetic resonance imaging of pituitary hyperplasia in a child with growth arrest and primary hypothyroidism].

We report a case of pituitary hyperplasia in a child presented with significant growth arrest and laboratory evidence of hypothyroidism. Magnetic resonance imaging (MRI) revealed symmetrical pituitary enlargement seemed to be macroadenoma. After thyroid hormone replacement therapy, the child's height increased and pituitary enlargement regressed. Awareness of MRI appearance of pituitary hyperplasia in children with laboratory evidence of hypothyoidism might avoid misdiagnosis for pituitary tumor, which may also manifest as growth disorder, obviating unnecessary surgery.

Child↗
PubMed2000

In vitro nuclear reconstitution could be induced in a plant cell-free system.

A cell-free system derived from carrot cell cytosol extract has been developed for reassembling nuclear structure around the added demembranated sperm chromatin of Xenopus. Morphological evidence suggests that reassembled nuclei display the typical characteristics of normal eukaryotic nuclei, such as double-layered nuclear membrane and nuclear pores. Micrococcal nuclease treatment indicates that remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution. These data indicate that the nuclear reconstitution can be induced in cell-free systems from plants, and the self-assembly of the nucleus is ubiquitous in both animal and plant cells.

Animals↗
PubMed2000

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

3T3 Cells↗
PubMed2000

Mammalian-cell-produced neurturin (NTN) is more potent than purified Escherichia coli-produced NTN.

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor. Both factors promote the survival of dopaminergic (DA) neurons. We investigated the biological activity of mammalian-cell-produced NTN versus purified Escherichia coli-produced NTN. Baby hamster kidney cells were engineered to stably secrete mature human NTN. Mammalian-cell-derived NTN enhanced the activity of embryonic DA neurons in vitro, with greater potency (maximum effect achieved in the picogram range) than purified E. coli-produced NTN. Cell-based delivery of NTN (less than 10 ng/day) was also shown to be biologically active in vivo. These results suggest that mammalian-cell-derived NTN, synthesized de novo and delivered in small quantities to the parenchyma at the target site, may be as active as much larger quantities of purified, E. coli-produced NTN, delivered by other means.

Animals↗
PubMed2000

Venovenous perfusion-induced systemic hyperthermia: hemodynamics, blood flow, and thermal gradients.

BACKGROUND: Thermal events during extracorporeal venovenous perfusion-induced systemic hyperthermia (VV-PISH) were studied and related to determination of whole-body and regional thermal isoeffect doses. METHODS: Swine (n = 6, 77+/-4.5 kg) were heated to a target temperature of 43 degrees C for 120 minutes using VV-PISH. Colored microspheres were injected during preheat, heat induction, maintenance, cool down, and after decannulation. The esophageal, tympanic, rectal, pulmonary artery, bladder, bone marrow, kidney, brain, blood, lung, and airway temperatures were recorded continuously. The thermal dose, thermal exchange, metabolic heat production, heat loss to the environment, the change in body heat, and the thermal isoeffect dose were studied at 15-minute intervals. RESULTS: VV-PISH increased heart rate and cardiac output and caused a redistribution of blood flow favoring the thoracoabdominal organs. Greatest thermal exchange occurred during the heating phase (total 2,162+/-143 kJ), metabolic heat production contributed in all phases (274+/-9 kJ), the greatest change in body heat occurred during heating (1,310+/-309 kJ) with a total delivered thermal dose of 298+/-21 kJ, and the total whole body thermal isoeffect dose at 100+/-5 minutes. CONCLUSIONS: VV-PISH is feasible, is capable of transferring sufficient heat, causes a redistribution of blood flow favoring the thoracoabdominal organs, and facilitates calculation of whole-body and regional thermal isoeffect doses.

Animals↗
PubMed2000

Rac2 stimulates Akt activation affecting BAD/Bcl-XL expression while mediating survival and actin function in primary mast cells.

Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.

Actins↗
PubMed2000

New clinically relevant sheep model of severe respiratory failure secondary to combined smoke inhalation/cutaneous flame burn injury.

OBJECTIVES: To develop a predictable, dose-dependent, clinically relevant model of severe respiratory failure associated with a 40% total body surface area, full-thickness (third-degree) cutaneous flame burn and smoke inhalation injury in adult sheep. DESIGN: Model development. SETTING: Research laboratory. SUBJECTS: Adult female sheep (n = 22). INTERVENTIONS: Animals were divided into three groups, determined by the number of smoke breaths administered (24, 36, 48) for a graded inhalation injury. The smoke was insufflated into a tracheostomy with a modified bee smoker at airway temperatures <40 degrees C. All animals concurrently received a 40% total body surface area (third-degree) cutaneous flame burn to the body (flanks). After injury, the animals were placed on volume-controlled ventilation to achieve PaO2 >60 mm Hg and PaCO2 <40 mm Hg. Arterial blood gases and ventilator settings were monitored every 6 hrs postinjury for up to 7 days. MEASUREMENTS AND MAIN RESULTS: All animals survived the induction of injury. In the 24 smoke breath/40% total body surface area burn (24/40) group, PaO2/F(IO2) never decreased below 300, and peak inspiratory pressure was consistently <14 cm H2O with normal arterial blood gases throughout the observation period. With 36 smoke breaths/40% total body surface area burn (36/40) (n = 7), all animals had PaO2/F(IO2) of <200 and peak inspiratory pressure of 26 cm H2O within 40-48 hrs, as 30% died during the study period. With 48 smoke breaths/40% total body surface area burn (48/40) (n = 12), all animals developed respiratory distress syndrome (RDS) in 24-30 hrs, but none survived the experimental period. CONCLUSIONS: Development of RDS by smoke and cutaneous flame bum injury depends on smoke inhalation dose. A combination of 36 breaths of smoke and a 40% total body surface area (third-degree) cutaneous flame burn injury can induce severe RDS (PaO2/F(IO2) <200) within 40-48 hrs to allow evaluation of various treatment modalities of RDS.

Algorithms↗
PubMed2000

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions.

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

Aerobiosis↗