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T Ha

Publications and source records attributed to T Ha.

At least 37 records · Page 2Linked to original sources
PubMed1999

Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism.

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.

Base Sequence↗
PubMed1999

Intracellular Ca2+ and adrenergic responsiveness of cardiac myocytes in streptozotocin-induced diabetes.

1. The contractile function of diabetic hearts is impaired. In addition, the responsiveness of diabetic cardiac muscle to sympathetic stimulation is altered. Previous studies have revealed a depressed response to beta-adrenoceptor stimulation; however, the response to alpha-adrenoceptor activation remains controversial. Because alpha- and beta-adrenoceptor agonists increase cardiac contractility, largely through increased mobilization of intracellular Ca2+, the aim of the present study was to investigate the effects of alpha- and beta-adrenoceptor stimulation on intracellular Ca2+ handling in cardiac myocytes from streptozotocin-induced diabetic rats. 2. Intracellular Ca2+ was measured using fura-2. Under basal conditions (27 degrees C, 2.5 mmol/L extracellular [Ca2+], 0.3 Hz stimulation), there was no significant difference in resting or peak Ca2+ levels between control and diabetic cardiomyocytes. However, the time course of the intracellular Ca2+ transient was significantly prolonged in cells from diabetic hearts. 3. The beta-adrenoceptor agonist orciprenaline (at 10(-7) and 10(-6) mol/L) increased the amplitude of the Ca2+ transient in both groups; however, the extent of potentiation was less in diabetic compared with control cardiomyocytes. Orciprenaline decreased the duration of the transient to the same extent in both groups. 4. The alpha-adrenoceptor agonist phenylephrine (at 10(-7) and 10(-6) mol/L) had no effect on the Ca2+ transient in control myocytes but caused a significant concentration-dependent increase in its amplitude in diabetic cardiomyocytes. Phenylephrine had no effect on the time course of the transient in either group. 5. These results demonstrate differential effects of insulin-dependent diabetes on the responsiveness of cardiomyocytes to alpha- and beta-adrenoceptor stimulation. The heightened response to alpha-adrenoceptor stimulation observed in diabetic cardiomyocytes may partly compensate for the diminished myocardial beta-adrenoceptor response.

Adrenergic alpha-Agonists↗
PubMed1999

Inhibiting early activation of tissue nuclear factor-kappa B and nuclear factor interleukin 6 with (1-->3)-beta-D-glucan increases long-term survival in polymicrobial sepsis.

BACKGROUND: Recent data implicate the activation of nuclear factor-kappa B (NF-kappa B) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. METHODS: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. RESULTS: CLP increased liver and lung NF-kappa B and NF-IL6 nuclear binding activity as well as tumor necrosis factor-alpha and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-kappa B and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased (P < .001) long-term survival (20% CLP vs 65% glucan phosphate, 75% scleroglucan). Posttreatment with glucan phosphate also increased (P < .05) long-term survival (20% vs 65%). CONCLUSIONS: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival.

Animals↗
PubMed1999

Early activation of hepatic NFkappaB and NF-IL6 in polymicrobial sepsis correlates with bacteremia, cytokine expression, and mortality.

BACKGROUND: The role of transcription factor activation in the pathophysiology of sepsis syndrome has not been established. This study investigated the relation between tissue nuclear factor kappaB (NFkappaB) and nuclear factor interleukin 6 (NF-IL6 or C/EBP) activation and bacteremia, inflammatory cytokine expression, and mortality in a murine model of cecal ligation and puncture (CLP). METHODS: Transcription factor activation was assessed by the electrophoretic mobility shift assay. Cytokine mRNA levels were established by reverse transcription-polymerase chain reaction and quantified by scanning densitometry. Bacteremia was evaluated by standard aerobic and anaerobic microbiologic methods. RESULTS: CLP stimulated hepatic NFkappaB activation at 2, 3, 4, 5, 6, and 8 hours compared with control and sham-operated mice. Hepatic NFkappaB activation during CLP peaked at 4 hours (1114% vs. no surgery, 609% vs. sham). Hepatic NF-IL6 activation was observed at 3, 4, and 6 hours after CLP. Hepatic and splenic levels of tumor necrosis factor-alpha and IL-6 mRNA were also elevated after CLP. Bacteremia in CLP mice consisted of Bacteroides species and to a lesser extent facultative gram-negative bacilli and group D Enterococcus. CONCLUSIONS: Early activation of hepatic and splenic NFkappaB and NF-IL6 positively correlates with tissue cytokine mRNA expression and mortality in a surgical model of polymicrobial sepsis. The data suggest that transcription factor activation is an early event in the pathophysiology of sepsis.

Animals↗
PubMed1999

Early activation of pulmonary nuclear factor kappaB and nuclear factor interleukin-6 in polymicrobial sepsis.

BACKGROUND: Transcription factor activation may be a pivotal step in the pathophysiology of sepsis syndrome and adult respiratory distress syndrome. This study investigated the activation of lung nuclear factor kappaB (NFkappaB) and nuclear factor interleukin-6 (NF-IL6) and how they correlate to proinflammatory cytokine expression and mortality in a murine model of cecal ligation and puncture (CLP). METHODS: Polymicrobial sepsis was induced by CLP. Transcription factor activation was assessed at 0, 1, 2, 3, 4, 5, 6, 8, and 24 hours after CLP by the electrophoretic mobility-shift assay. Lung cytokine mRNA levels were established by reverse transcriptase-polymerase chain reaction. RESULTS: CLP induced pulmonary NFkappaB activation at 3, 4, and 8 hours (p < 0.05). Lung NFkappaB activation peaked at 3 hours (533% vs. no surgery, 2,900% vs. sham treatment) after CLP. Supershift analysis revealed a predominance of p50 subunits in the lung nuclear extracts of septic mice 3 hours after CLP, indicating the presence of p50 homodimer. In contrast, liver nuclear extracts from septic mice indicated the presence of both p65 and p50 subunits at 3 hours. Lung NF-IL6 activation (p < 0.05) was observed at 4 hours (649% vs. no surgery, 296% vs. sham treatment) and 6 hours after CLP. Lung tumor necrosis factor-alpha mRNA levels were increased (p < 0.05) at all time intervals after CLP. Lung IL-6 mRNA levels were increased at 3, 6, and 8 hours after CLP. CONCLUSION: Early activation of lung NFkappaB and NF-IL6 and lung cytokine mRNA expression correlated with mortality in polymicrobial sepsis. Although IL-6 mRNA levels correlated with NFkappaB and NF-IL6 activation, tumor necrosis factor-alpha mRNA levels did not, in that they preceded transcription factor activation. These data suggest a potential role for NFkappaB and NF-IL6 activation in the initiation and propagation of acute lung injury.

Animals↗
PubMed1998

Ligand binding to the (1 --> 3)-beta-D-glucan receptor stimulates NFkappaB activation, but not apoptosis in U937 cells.

Recent data suggest that sepsis stimulates macrophage apoptosis (Ao) with subsequent induction of macrophage dysfunction. Nuclear factor-kappaB (NFkappaB) activation has been linked to Ao in either a pro- or antiapoptotic role. Glucans are biological response modifiers which exert antisepsis activity. This investigation examined the effect of (1-3)-beta-D-glucan receptor binding by a high affinity ligand on Ao and NFkappaB activation in U937 cells in the presence or absence of LPS. A high affinity glucan ligand (IC50 = 23 nM) activated NFkappaB, but did not induce Ao or significantly alter LPS induced U937 Ao. These data indicate that: i) modulation of the macrophage (1-3)-beta-D-glucan receptor stimulates NFkappaB; ii) does not induce Ao or significantly diminish LPS induced Ao and iii) activation of the U937 FAS receptor does not alter the relative Ao responses in glucan and LPS treated cells.

Antibodies↗
PubMed1998

Near-field fluorescence microscopy of cells.

We demonstrate the surface sensitivity of near-field scanning optical microscopy by fluorescence imaging of membrane and bulk proteins in cells. We discuss instrument design considerations for successful cell-biology work with NSOM and show that the technique is most suited for studying membrane proteins.

Animals↗
PubMed1998

Identification of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe.

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.

DNA Fingerprinting↗
PubMed1998

Necrotizing fasciitis complicating disseminated cutaneous herpes zoster.

The association of necrotizing fasciitis, often due to group A streptococcus and primary varicella (chicken pox), is unusual but recognized in children. The association in adults is rare but one report in the literature describes a previously healthy man with the two disorders. We now describe a case of disseminated cutaneous herpes zoster complicated by subacute necrotizing fasciitis in an elderly woman taking low dose methotrexate and prednisone for rheumatoid arthritis. Staphylococcus aureus was isolated. Localized debridement and split skin grafting were required.

Aged↗
PubMed1997

A sensitive ELISPOT assay to detect low-frequency human T lymphocytes.

We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 10(5) PBMC with the standard assay compared to 19 SFC per 2 x 10(5) PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocyte cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.

Amino Acid Sequence↗
PubMed1997

Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy.

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA) (parasite)/protein4.1(host) and P. falciparum histidine rich protein (PfHRP1) (parasite)/protein4.1(host) showed good real-space correlation for the MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.

Cells, Cultured↗
PubMed1997

Interpreting a sequenced genome: toward a cosmid transgenic library of Caenorhabditis elegans.

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.]

Animals↗
PubMed1997

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.

Bacterial Typing Techniques↗
PubMed1997

Therapeutic modification of nuclear factor kappa B binding activity and tumor necrosis factor-alpha gene expression during acute biliary pancreatitis.

The role of cytokines has been well documented in the pathogenesis of acute pancreatitis. Antibodies against specific cytokines have been used to treat pancreatitis, with mixed results. The transcription factor nuclear factor (NF)-kappa B is a pleiotropic regulator of many genes involved in stress and inflammatory responses. The aim of this study was to prevent the NF-kappa B binding activity and tumor necrosis factor (TNF)-alpha gene overexpression as a possible therapeutic intervention for acute pancreatitis. Reversible acute biliary pancreatitis was induced in male Sprague Dawley rats as established in this laboratory. The animals were sacrificed at 0, 5, 15, 30 min and 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours after the induction of pancreatitis. NF-kappa B binding activity was determined by electrophoretic mobility shift assay, and TNF-alpha gene expression was assayed by reverse transcription-PCR. NF-kappa B binding activity was markedly higher around 4 hours and persisted up to 24 hours after pancreatitis induction in animals with acute pancreatitis, whereas TNF-alpha mRNA levels peaked at 24 hours. When amobarbital (to block NF-kappa B activation) was given (60 mg/kg body weight, I.P.) 3 hours before induction of pancreatitis, the activation of NF-kappa B and the overexpression of TNF-alpha gene was prevented, with significantly decreased severity of pancreatitis as assessed by amylase and clinical recovery. We conclude that 1) preventing the activation of NF-kappa B eliminates the induced overexpression of inflammatory cytokines (TNF-alpha) in acute pancreatitis, 2) such intervention correlates with clinical improvement in pancreatitis, and 3) this genetic modification offers a possible therapeutic intervention in acute pancreatitis.

Acute Disease↗
PubMed1996

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.

Base Sequence↗
PubMed1996

Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines.

Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta-D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D-glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells.

Animals↗