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Biomedical subjects

K Chao

Publications and source records attributed to K Chao.

At least 19 recordsLinked to original sources
PubMed2001

Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest.

We previously reported that the CdtB polypeptide of Escherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtB(H154A) lacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.

Bacterial Toxins↗
PubMed2000

Proteoglycan synthesis in porcine nasal cartilage grafts following Nd:YAG (lambda = 1.32 microns) laser-mediated reshaping.

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na2(35)SO4 into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, lambda = 1.32 microns) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time-temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.

Animals↗
PubMed1999

Granulocyte colony-stimulating factor on the growth of human ovarian carcinoma cells in vitro.

Granulocyte colony-stimulating factor (GCSF) is a member of a family of glycoprotein hormones that stimulates the proliferation and differentiation of hematopoietic cells in vivo and in vitro. In ovarian cancer chemotherapy, GCSF is used clinically to build up bone marrow function after severe cytotoxicity to granulocytes by chemotherapy. Little is known about the effects of these cytokines on the growth of cancer cells. To study the influence of GCSF on the proliferation of ovarian cancer cells in vitro, five established ovarian cancer cell lines (OC-89-VGH, OC-117-VGH, OCPC-2-VGH, NIH-OVCAR-3, and NIH-SK-OV-3) were treated with the concentration ranging from 0.1 pg to 10 ng GCSF for 5 days and compared with vehicle control. In addition, we also examined the effect of GCSF (0. 01-1.0 ng) on eight primary cultures of fresh ovarian cancer tissues. Cell viability after treatment was measured by Cell Titer AQueous assay and expressed as a percentage of untreated control cultures. A decrease in cell growth (75-85%) was observed in OC-89-VGH, OC-117-VGH and OCPC-2-VGH cell lines while NIH-OVCAR-3 and NIH-SK-OV-3 cells showed minimal growth stimulation. However, all dose-response curves were nonsignificant, suggesting indirect effects. In the eight fresh tumor primary cultures treated with GCSF, no statistical significant difference in growth was observed. In conclusion, our data suggest that GCSF has little or no growth-modulatory effect on human ovarian carcinoma in vitro. Therefore, the clinical use of GCSF is unlikely to have a direct effect on tumor growth.

Cell Division↗
PubMed1997

Diagnostic laparoscopy for chronic right iliac fossa pain: a pilot study.

BACKGROUND: The aim of this study was to determine the value of diagnostic laparoscopy in patients with chronic right iliac fossa pain. METHODS: A retrospective study at Echuca Hospital involving case-note review and telephone questionnaire of patients who had undergone diagnostic laparoscopy for chronic right iliac fossa pain at least 12 months earlier (September 1992 to August 1995) was carried out. RESULTS: Forty-one cases were identified and followed up 12-40 months postoperatively (median 21 months). Eleven cases had positive findings at laparoscopy, of whom eight obtained lasting relief after treatment. Of the remaining 30 patients 17 had a normal-looking appendix removed and 12 were cured; these were younger patients with episodic symptoms and localized signs. Of eight patients who had adhesions divided, four with adhesions beneath old scars obtained relief. Altogether 32 of the 41 patients considered the laparoscopy worthwhile even though in some cases it did not relieve their chronic pain. CONCLUSIONS: Diagnostic laparoscopy is worthwhile for patients with chronic right iliac fossa pain. Concurrent appendicectomy should be considered in young patients with episodic, well-localized symptoms associated with systemic malaise while adhesiolysis may be beneficial for viscero-parietal adhesions beneath abdominal wall scars.

Abdominal Pain↗
PubMed1997

Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells.

Cytolethal distending toxin (CDT) is a newly described toxin produced by a number of enteropathogens, including Campylobacter jejuni, various Escherichia coli strains, and a few Shigella species. CDT induces distension and eventual death of a number of transformed cell lines. Here, we extend previous studies by demonstrating that morphological changes in CDT-treated Chinese hamster ovary cells are coincident with changes in cytoskeletal structure and an inhibition of cell proliferation. CDT-treated cells underwent a progressive accumulation of F-actin assemblies which microscopically resembled actin stress fibers. Accumulation of the stress fiber-like structures in CDT-treated cells was accompanied by an apparent blockage of cell division. Multinucleation was detected in some cells but did not constitute a significant feature of CDT action. Although toxin-treated cells failed to divide, cell viability remained high for the first 4 days following toxin treatment, as evidenced by trypan blue exclusion and neutral red uptake. [3H]thymidine incorporation studies on CDT-treated cells were consistent with a blockage of cell proliferation without a direct inhibition of DNA synthesis. Although the progression of toxin action developed slowly, a 2-min exposure to CDT resulted in an irreversible development of toxicity. Together, our data indicate that CDT affects F-actin assembly within target cells and may interrupt the regulation or function of cell cycle-dependent events leading to cytokinesis.

Actins↗
PubMed1996

Manipulation of pyramidal cell firing in the hippocampus of freely behaving rats by local application of K+ via microdialysis.

In this study, microdialysis was performed in the hippocampus of freely behaving rats, and the firing of pyramidal cells, including place cells, was recorded at the site of the microdialysis probe. For 10-min periods, the artificial cerebrospinal fluid (ACSF) in the microdialysis system was replaced with ACSF containing 50 mM K+ (high K+ solution). Complementary in vitro tests determined that microdialysis with such high K+ solution produced an outflow of 5% of the perfused K+ from the microdialysis probe. Application of K+ with this method into the CA1 region significantly increased the firing of the local pyramidal cells, including place cells, during both movement and sleep. On average, K+ exposures increased the firing rate of the neurons to 306% and 448% of the control firing rate during movement and sleep, respectively. After the termination of the K+ outflow, the cells continued to discharge for 5-30 min with a significantly higher frequency than before the K+ challenge. This phenomenon also occurred in both behavioral states. During the period of enhanced firing, the out-of-field firing rate of the recorded place cells was dramatically increased. It was also found that during the K+ applications, otherwise silent pyramidal cells often became electrically active. The K(+)-induced firing modifications were usually not accompanied by behavioral or EEG changes. The data raise the possibility that transient elevations in the extracellular K+ concentration contribute to the ionic/molecular processes which are responsible for plastic firing pattern modifications in hippocampus. Pharmacological manipulation of place cells with the described method offers a new strategy to understand the molecular bases of spatial memory.

Animals↗
PubMed1996

Transverse abdominis musculo-peritoneal (TRAMP) flap for the repair of large duodenal defects.

Definitive surgical management of major acute injuries to the second and third portions of the duodenum has been enigmatic. Sometimes, the defect is so large that it is unwise to do primary repair, and resection at this critical portion of the intestinal tract is technically hazardous or impossible. A serosal or mucosal patch technique has been used to repair this kind of duodenal defect with encouraging results. Since the use of this technique has proved effective, such a defect was not necessarily treated with the more complicated pancreaticoduodenectomy and was managed with less morbidity and mortality. But these techniques are still controversial. So, we tried a pedicle flap, called the transverse abdominis musculo-peritoneal (TRAMP) flap, for repair of large duodenal defect. We have used this flap in 25 rabbits, and the specimens were followed up to a period of 3 months. The flap showed satisfactory results and is presented as another option for repair of large duodenal defects.

Animals↗
PubMed1996

The role of core biopsy in the diagnosis of mammographically detected lesions.

BACKGROUND: Both stereotactic imaging and ultrasound have been used to localize non-palpable breast lesions for core biopsy analysis. In particular, stereotactic core breast biopsy with a dedicated recumbent mammographic unit is an accurate and reliable technique, and this has been substantiated in a number of recent prospective studies comparing percutaneous core biopsy with open excisional biopsy. METHODS: This article reviews biopsies of 521 lesions from 502 cases using both stereotactic localization (469 lesions) and ultrasound (33 lesions). The types of breast cancer and benign breast disease diagnosed with core sampling are reviewed. In this series it was not intended to compare all the results of core biopsies with those of open excisional biopsy, but a subset group, which comprised certain benign lesions and malignancies, as well as lesions diagnosed on core biopsy in which the radiology and pathology did not correlate, was further evaluated and compared to the findings with open biopsy. The cases where there were differences in core and excisional biopsy diagnosis are discussed. RESULTS: In this series the benign to malignant ratio was 2.7:1, in other words, 27% of the lesions biopsied were malignant. The sensitivity for detecting cancer was 97% with the positive predictive value being 99%. CONCLUSIONS: This series further substantiates the accuracy of core biopsy. It is recommended that certain lesions diagnosed as benign on core biopsy histology should also proceed to open biopsy. Correlation between clinical findings, radiology and pathology further improves the reliability of core biopsy under imaging guidance.

Adenocarcinoma↗
PubMed1995

NMR study of the reconstitution of the beta-sheet of thioredoxin by fragment complementation.

The study of complementary protein fragments is thought to be generally useful to identify early folding intermediates. A prerequisite for these studies is the reconstitution of the native-like structure by fragment complementation. Structural analysis of the complementation of the domain-sized proteolytic fragments of E. coli thioredoxin, using a combination of H-exchange and 2D NMR experiments as a fingerprint technique, provide evidence for the extensive reconstitution of a native beta-sheet, with local conformational adjustments near the cleavage site. Remarkably, the antiparallel beta-strand between the fragments shows a native-like protection of the amide protons to solvent exchange. Our results indicate that these fragments can be useful to study the early events in the still little understood formation of beta-sheets.

Escherichia coli↗
PubMed1993

Familial medullary thyroid carcinoma: report of three cases in a family.

Familial medullary thyroid carcinoma is a distinct clinical entity in which early diagnosis by screening of family members for elevated calcitonin levels can be useful. A 33-year-old female was hospitalized because of an enlarging left thyroid mass. Resected thyroid showed medullary thyroid carcinoma. Her mother had received a total thyroidectomy due to the same disease 10 years previously, and her younger brother had an elevated basal calcitonin level of 468 pg/mL when the index case was diagnosed. The younger brother was advised to undergo surgery; a total thyroidectomy also revealed medullary thyroid carcinoma. There was no evidence of multiple endocrine neoplasia in the family. Recognition of new cases by screening with a basal or provocative test in families of patients with medullary thyroid carcinoma should be mandatory.

Adult↗
PubMed1992

Influence of the Coanda effect on color Doppler jet area and color encoding. In vitro studies using color Doppler flow mapping.

We studied surface adherence and its effects on color Doppler jet areas and color encoding in an in vitro model with a noncompliant receiving chamber into which a steady flow jet was directed parallel to either a straight or a curved surface adjacent to and 4 mm away from the inflow orifice (1.50 mm2) with the control condition being a free jet matched for flow rates and driving pressures. Jets were imaged perpendicular to the plane of the surface, the plane in which most clinical images of jet-surface interactions are obtained. Ten different flow rates ranging from 0.13 to 0.30 l/min were used. Surface-adherent jet areas were smaller than control jets for every driving pressure-volume combination (paired t test, p less than 0.01). Computer analysis of color Doppler images showed more green and blue (reverse flow) pixels on the surface side of the adherent jets than the control jets (p less than 0.05), suggesting that viscous energy loss and flow deceleration and reversal play a role in the jet-surface interaction. Analysis of variance demonstrated that linear regression slopes of flow rate versus jet area for surface jets were lower (slopes, 11-21 cm2/l/min; r = 0.95-0.97) than those for the control (slope, 33 cm2/l/min; r = 0.97) (p less than 0.0001). Surface adherence (Coanda effect) influences jet size and color encoding, causing smaller color Doppler jet areas and greater variance and reverse velocity encoding.

Coronary Circulation↗
PubMed1990

DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins.

The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin. Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S). The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities. When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities. This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA. This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA. The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides. Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N. & Kornberg, A. (1981) J. Biol. Chem. 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities. However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein. Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions. This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein. The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin. Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1989

Large-scale purification and characterization of the Escherichia coli rep gene product.

We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.

Adenosine Triphosphatases↗