Search PubMed⌕ Search

Biomedical subjects

J Tsao

Publications and source records attributed to J Tsao.

At least 37 records · Page 2Linked to original sources
PubMed1993

In vitro activity of the herpes simplex virus type 1 protease with peptide substrates.

Herpes simplex virus type-1 (HSV-1) protease is responsible for proteolytic processing of itself and the virus assembly protein ICP35 (infected cell protein 35). Two proteolytic processing sites within the protease have recently been identified between Ala247 and Ser248 and between Ala610 and Ser611. In this report we demonstrate that peptides corresponding to each of these cleavage sites are substrates for recombinant HSV protease-glutathione S-transferase fusion protein in vitro by high performance liquid chromatography analysis of cleavage reactions. Analysis of the products by fast atom bombardment-mass spectrometry confirmed that cleavage occurred at the expected position between the Ala and Ser residues of the substrate. Peptide cleavage was linear with respect to time and enzyme concentration and proceeded optimally at pH 8.0. A peptide that spans Ala99/Ser100 of the protease but does not correspond to a naturally occurring cleavage site was not a substrate for the protease in vitro confirming that sequence elements outside the conserved dipeptide sequence are required for substrate recognition and cleavage. Identification of P5-P8' as the minimal substrate peptide for the Ala610/Ser611 cleavage site revealed a requirement for residues flanking the conserved core P4-LVNA/S-P1' in substrate recognition and hydrolysis. Kinetic analysis with peptide P5-P8' yielded a Km of 190 microM and kcat of 0.2 min-1. Experiments with a panel of class-specific protease inhibitors were consistent with the protease being a member of the general class of serine proteases.

Amino Acid Sequence↗
PubMed1992

Ab initio phase determination for viruses with high symmetry: a feasibility study.

Conditions that would permit the complete structure determination of spherical viruses that have high internal symmetry were examined starting only from an initial spherical shell model. Problems were considered that might arise due to the following. 1. Creation of centric phases due to the simple shell model and its position in the unit cell. The centric symmetry can generally be broken on averaging an initial electron density map based on observed structure amplitudes, provided that the internal molecular symmetry is sufficiently non-parallel to the crystallographic symmetry. 2. Choice of the average model shell radius. Some incorrect radii led to the Babinet opposite solution (electron density is negative instead of positive). Phases derived from other models with incorrect radii failed to converge to the correct solution. 3. Error in structure amplitude measurements. 4. Lack of a complete data set. 5. Error in positioning the initial spherical-shell model within the crystal unit cell. It was found that an error of 1.6 A caused noticeable phasing error at a resolution greater than 20 A.

Feasibility Studies↗
PubMed1992

Ab initio phase determination for spherical viruses: parameter determination for spherical-shell models.

The structure determination of canine parvovirus depended on the extension of phases calculated initially from a spherical-shell model [Tsao, Chapman, Wu, Agbandje, Keller & Rossmann (1992). Acta Cryst. B48, 75-88]. Such ab initio phasing holds the promise of obviating initial experimental phasing by isomorphous or molecular replacement, thereby expediting the structure determinations of spherical virus capsids. In this paper, it is shown how parameters such as radii, DNA density and particle positions may be determined and refined from diffraction data with sufficient precision to start phase extension from 20 A resolution for a virus of approximately 122 A radius.

Capsid↗
PubMed1992

Structure determination of monoclinic canine parvovirus.

The three-dimensional structure of the single-stranded DNA canine parvovirus has been determined to 3.25 A resolution. Monoclinic crystals belonging to space group P2(1) (a = 263.1, b = 348.9, c = 267.2 A, beta = 90.82 degrees) were selected for data collection using primarily the Cornell High Energy Synchrotron Source and oscillation photography. There was one icosahedral particle per crystallographic asymmetric unit, giving 60-fold redundancy. The particle orientations in the unit cell were determined with a rotation function. The rough positions of the particles in the unit cell were estimated by considering the packing of spheres into the P2(1) crystal cell. More accurate particle centers were determined from Harker peaks in a Patterson function. Hollow-shell models were used to compute phases to 20 A resolution. The radii of the models were based on packing considerations, the fit of spherical shells to the low-resolution X-ray data and low-angle solution scattering data. The phases were extended to 9 A resolution using molecular replacement real-space averaging. These were then used to determine the heavy-atom position of a K2PtBr6 derivative, for which only 5% of the theoretically observable reflections had been recorded. The center of gravity of the 60 independent heavy-atom sites gave an improved particle center position. Single isomorphous replacement phases to 8 A resolution were then calculated with the platinum derivative. These were used to initiate phase improvement and extension to 3.25 A resolution using density averaging and Fourier back-transformation in steps of one reciprocal lattice point at a time. The resulting electron density map was readily interpretable and an atomic model was built into the electron density map on a PS390 graphics system using the FRODO program. The R factor prior to structure refinement for data between 5.0 and 3.25 A was 36%.

Crystallization↗
PubMed1991

The three-dimensional structure of canine parvovirus and its functional implications.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Amino Acid Sequence↗
PubMed1991

Delayed ileal pouch-anal anastomosis. Complications and functional results.

In patients with chronic ulcerative colitis (CUC), ileal pouch-anal anastomosis (IPAA) can be performed either at the time of colectomy or as a delayed procedure after total abdominal colectomy and ileostomy. There has been debate as to whether delayed IPAA results in superior functional results, since patients are frequently steroid-free and have little evidence of active disease. To assess this, we analyzed 95 patients who had undergone total abdominal colectomy, either with ileostomy and Hartmann's procedure or with ileorectostomy, 2-183 months prior to IPAA. Postoperative complications and functional results were compared with those of 776 CUC patients who underwent IPAA at the time of abdominal colectomy. Indications for prior colectomy included toxic megacolon (40 percent), failed medical therapy (36 percent), other reasons (e.g., iatrogenic perforation, cancer) (6 percent), and reasons unclear (18 percent). Nineteen percent of delayed-IPAA patients were taking steroids at the time of pouch construction. Follow-ups were similar in the two groups. The incidence of septic and obstructive complications after delayed IPAA vs. IPAA at the time of colectomy were 10.5 percent vs. 5.4 percent and 6.5 percent vs. 14.5 percent, respectively. There were no significant differences in postoperative functional results between the two groups. Delayed IPAA confers no advantage over IPAA performed at the time of colectomy in terms of functional outcome. Delayed IPAA was associated with a significantly higher rate of septic complications but a lower incidence of postoperative obstruction.

Adolescent↗
PubMed1991

Agonist-induced desensitization of adenylyl cyclase in Y1 adrenocortical tumor cells.

Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouse beta 2-adrenergic receptor (beta 2-AR). Transfectants expressed beta 2-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. We demonstrate that beta-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DS cells whereas transfected Y1DR cells are resistant to desensitization by beta-adrenergic agonists. The fate of the beta 2-ARs during desensitization was evaluated by photoaffinity labelling with [125I]iodocyanopindolol diazerine. Desensitization of Y1DS transfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness to beta-adrenergic agonists. The extent of receptor loss induced by beta-adrenergic agonists in Y1DR transfectants exceeded that in the Y1DS transfectants indicating that the mutation which protects Y1DR cells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells.

Adenylyl Cyclases↗
PubMed1991

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.

Adenylyl Cyclases↗
PubMed1990

Decreased levels of guanyl nucleotide-binding regulatory protein alpha-subunits in Y1 adrenocortical tumor cell mutants resistant to forskolin.

Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane.

Adenylyl Cyclases↗
PubMed1989

Sindbis virus core protein crystals.

The core protein of Sindbis virus has been crystallized. Three different crystal forms have been observed. They diffract variously from 2.5 A to 3.5 A resolution.

Crystallization↗
PubMed1988

Preliminary X-ray crystallographic analysis of canine parvovirus crystals.

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).

Animals↗
PubMed1987

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs.

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.

Adenosine Diphosphate Ribose↗
PubMed1984

Isolation of forskolin-resistant adrenal cells defective in the adenylate cyclase system.

Forskolin is an activator of adenylate cyclase in many cell types. In order to determine the mechanism of forskolin's action and to determine if this mechanism is shared by hormones and other agonists of the adenylate cyclase system, we isolated and partially characterized several spontaneous, forskolin-resistant mutants from the Y1 mouse adrenocortical tumor cell line. Forskolin increased adenylate cyclase activity in Y1 cell homogenates approximately 30-fold. By virtue of its effect on cAMP accumulation, forskolin (10 microM) also inhibited the growth of Y1 cells in monolayer culture. Using forskolin as a selective agent, spontaneous mutants capable of growth in the presence of 10 microM forskolin were isolated from the Y1 cell line at a frequency of 1-2/10(6) cells. In these mutants, resistance was stable, resulting from a defect in cAMP accumulation rather than cAMP action, and was associated with a reduced ability of forskolin to stimulate adenylate cyclase activity in cell homogenates. Whereas corticotropin stimulated adenylate cyclase activity over 35-fold in cell homogenates from the Y1 parent, ACTH had only marginal effects on the enzyme's activity in the mutant clones. Fluoride-stimulated adenylate cyclase activity seemed unimpaired. These results suggest that the resistance to forskolin resulted from a mutation in the adenylate cyclase system, not in the catalytic subunit, but at a locus related to the ACTH.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

A 68 000 dalton protein genetically associated with corticotropin-sensitive adenylate cyclase activity. Purification and preliminary characterization using a specific antiserum.

Previous work from this laboratory described an association, based on genetic evidence, between a 68 000 dalton protein (p68) and corticotropin (ACTH) sensitive adenylate cyclase activity among variants of the Y1 mouse adrenocortical tumor cell line. To study the nature of this association further, we have purified p68 and raised a polyclonal anti-p68 serum in rabbits. A variant subclone of the Y1 line, in which p68 comprised approximately 10% of total soluble protein, was used as starting material. Purification of p68 was achieved by passage of a 100 000 X g supernatant fraction over DEAE-cellulose, fractionation with ammonium sulfate, and chromatography on hydroxylapatite. The purified protein had an isoelectric point of 7.3, a polarity value of 46%, and a blocked amino terminal end group. A rabbit antiserum raised against the purified p68 had a titer of 1:16 000 and specifically precipitated p68 from extracts of Y1 cells labeled with L-[35S]methionine. Using this antiserum, p68 also was detected in other cell lines including mouse erythroleukemia and Sertoli cells; rat Leydig, ovary, and glioma cells; and Chinese hamster ovary cells. The presence of p68 in a variety of cell types suggests that the function of p68 is not restricted to adrenal cells or to specific actions of ACTH.

Adenylyl Cyclases↗
PubMed1984

Association of an interleukin abnormality with the T cell defect in Hodgkin's disease.

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.

Adolescent↗
PubMed1983

Enterococci from Bangkok, Thailand, with high-level resistance to currently available aminoglycosides.

Enterococcal endocarditis is usually treated with a combination of a penicillin and an aminoglycoside. Recent reports have documented the emergence of enterococci in France with high-level resistance to gentamicin, tobramycin, and kanamycin and the emergence of strains in Houston, Tex. with high-level resistance to all of these drugs and streptomycin. In this study, we examined strains from a geographic area where newer aminoglycosides have been less commonly used. Of 125 distinct patient isolates, 18 (14%) were resistant to greater than 2,000 micrograms of gentamicin and most other aminoglycosides per ml. Four of these strains transferred gentamicin resistance to a laboratory recipient. One strain, chosen for further study, was resistant to synergism between penicillin and gentamicin, tobramycin, kanamycin, streptomycin, and amikacin and demonstrated the following enzymatic activities: 3'- and 2"-aminoglycoside phosphotransferases, 6'-aminoglycoside acetyltransferase, and adenylylation of streptomycin. Optimal therapy for endocarditis caused by such highly resistant strains is currently unknown.

Aminoglycosides↗