Biomedical subjects
Publications and source records attributed to W Chen.
A simple method for isolating DNA from human peripheral blood was compared with the routine method which involves SDS-proteinase K digestion. Denaturation of cellular proteins and release of DNA were achieved by means of the addition of guanidium hydrochloride. The amounts of intact high molecular weight DNA and total yields were similar.
BACKGROUND: The pathogenesis of nasopharyngeal carcinoma (NPC) is not well defined yet. To evaluate oncosuppressor genes in NPC, two NPC cell lines were investigated for expression of the retinoblastoma (RB) gene. EXPERIMENTAL DESIGN: We used Western blotting to identify RB protein species, and checked the RB gene conformation by Southern blotting. We also used immunohistochemistry, in situ nucleic acid hybridization, and in situ extraction of RB protein to observe RB protein in NPC culture cells. RESULTS: Both cell lines as well as sublines could synthesize normal RB proteins of 110, 113 and 114 kilodaltons. These cells showed no RB DNA rearrangement. Most interphase cells showed variable amounts of anti-RB reaction product in their nuclei when immunostained by 13 monoclonal antibodies. However, all mitotic cells contained RB protein either in the cytosol or the chromosomes, depending upon the mitotic phase. The level of RB mRNA increased slightly in mitotic cells as compared with interphase cells. Double localization of bromodeoxyuridine and RB protein in NPC cells and localization of RB protein in synchronized NPC cells in different phases of the cell cycle revealed random RB protein expression in each individual tumor cell. Co-localization of RB mRNA and RB protein in interphase cells showed a different degree of RB message expression in each cell. Low-salt hypotonic buffer could remove a fraction of RB protein from stained interphase nuclei. A similar finding with slight variations was observed in 16 other cancer cell lines. CONCLUSIONS: These data indicate that our NPC cell lines contain no obvious RB gene rearrangement, but each cancer cell may have an abnormal expression of RB mRNA and protein. This phenomenon may also be true in other cancer cell lines with a normal RB gene.
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Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C. The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca(2+)-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710]. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-helix content, and hydrophobic exposure. Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of alpha-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels. Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin I and relief of inhibition of the actomyosin S1 ATPase. While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+. dSEEE differs in conformation from wild type, but it is normal in functional assays. This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.
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The therapeutic efficacy of adoptive immunotherapy of cancer has been shown to positively correlate with the dose of tumor-immune T cells transferred. Therefore, the success of this therapy is critically dependent on the ability to procure large numbers of functionally active T cells. Previous studies in animal models have shown that the limited therapeutic efficacy of a small number of immune T cells can be greatly enhanced by expansion of T cells in vitro to greater numbers before transfer in vivo. Optimal regimens for T cell expansion in vitro have generally employed the use of intermittent stimulation of the TCR with specific Ag followed by exogenous IL-2. The use of IL-2 alone does not provide for requisite episodic up-regulation of IL-2R. Stimulation of the invariant CD3 portion of the TCR/CD3 complex with antibody to CD3 (anti-CD3) represents an alternative method of up-regulating IL-2R and has been used to nonspecifically induce the growth of Ag-specific T cell lines and clones long-term in vitro with maintenance of function and specificity. The current study examined whether resting T cell populations containing small numbers of memory tumor-specific T cells could be rendered more effective in tumor therapy by nonspecific expansion in vitro with anti-CD3 plus IL-2. Spleens from C57BL/6 mice previously immunized to FBL-3, a syngeneic virus-induced leukemia, were nonspecifically stimulated with anti-CD3 plus IL-2. The resultant T cells were expanded in number, were nonlytic to FBL-3 but retained the ability to become lytic upon specific stimulation by FBL-3, and were effective in specific tumor therapy. The Ag-specific anti-tumor immune function declined on a per cell basis after each cycle of anti-CD3-induced T cell expansion. However, the approach resulted in a substantial increase in total T cell number and an overall net increase in the function of the effector T cell population. Thus, stimulation of tumor-immune T cell populations with anti-CD3 plus IL-2 represents a nonspecific method for expanding the number of specific effector T cells for cancer therapy.
Recombinant human choriogonadotropin and selenomethionyl human choriogonadotropin (rhCG and SehCG) were expressed in baculovirus expression system by coinfection of SF9 insect cells by recombinant viruses, AcMNPV-hCG alpha and AcMNPV-hCG beta containing hCG alpha and hCG beta cDNAs. The expression efficiency of both rhCG and SehCG was quite high. The association of the alpha and beta subunits into a dimer was apparently complete since no detectable amount of rhCG beta was found in the rhCG eluate from the monoclonal hCG beta antibody immunoaffinity column. Both rhCG and SehCG preparations were homogeneous as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography. The apparent molecular mass of rhCG and SehCG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was about 38 kDa while under reducing conditions the heterodimer dissociated to yield beta and alpha subunits with molecular masses of 22.5 and 18 kDa, respectively. The carbohydrate analysis of rhCG showing the presence of 2.1, 3.3, 7.38, 4.2, and 27.8 residues of Fuc, GalNAC, GlcNAC, Gal, and Man, respectively, per mole of the hormone was consistent with the presence of 4 N-linked high mannose type carbohydrate hydrate and 4 O-linked simple carbohydrate chains, probably made up of Gal-GalNAC. Despite the altered glycosylation, rhCG demonstrated close similarity to the native urinary hCG in amino acid composition, receptor binding, and in its ability to stimulate cAMP and steroidogenesis. This indicates that there is no specificity of carbohydrate required for biological activity. Furthermore, it implies that the alteration from the complex to high mannose type carbohydrates in rhCG does not affect its proper folding. Finally, amino acid analysis of SehCG showed that 84% of methionine residues in rhCG were replaced by selenomethionine.
Activated ras proto-oncogenes contribute to the pathogenesis of many animal and human malignancies. ras proto-oncogenes are generally activated by point mutations within codons 12 or 61, which result in the expression of ras protein (p21) bearing characteristic single amino acid substitutions at the corresponding residues. The purpose of the current study was to determine whether the presence of single transforming amino acid substitutions can render normal ras protein immunogenic and, thus, a possible target for T cell-mediated tumor therapy. In initial experiments, C57BL/6 mice were immunized with a synthetic peptide corresponding to residues 5 through 16 of p21 containing the transforming substitution of arginine for normal glycine at residue 12. The results demonstrated that class II MHC-restricted T cells which were specific for the peptide could be elicited, and that the peptide-induced T cells could specifically recognize the corresponding intact p21 ras protein. Recognition of p21 ras protein by peptide-specific T cells implies that C57BL/6 APC can process the activated ras protein in a fashion that allows presentation of digested protein by class II MHC molecules in a configuration similar to the configuration with synthetic peptide. Evaluation of the immunogenicity of peptides containing alternative transforming amino acid substitutions of ras protein demonstrated that some, but not all, were immunogenic in individual strains of mice. Therefore, although ras protein-specific T cells can be elicited by immunization with synthetic peptides, not all of the potential ras mutations commonly associated with malignancy may be recognizable by T cells from all individuals.
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The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.
Charge movements were measured in frog cut twitch fibers with the double Vaseline-gap voltage-clamp technique. In most fibers, when a depolarizing pulse to -60 to -40 mV was applied at 13-14 degrees C, the ON segment of a charge movement trace showed an early I beta component and a late I gamma hump component. An ongoing controversy is whether the I gamma hump component triggers calcium release from the sarcoplasmic reticulum or arises as a consequence of the release. Interestingly, a number of cut fibers showed normal I gamma components but greatly diminished, or unresolvable, I beta components. When the amount of charge associated with the current transient was plotted against the membrane potential, the steeply voltage-dependent Q gamma component appeared normal whereas the less steeply voltage-dependent Q beta component was also greatly diminished or unresolvable. These results suggest that I gamma can flow in the absence of I beta, thereby ruling out the possibility that Q beta triggers calcium release which, in turn, causes Q gamma to move. The results, however, do not rule out the positive feedback of calcium release to activate Q gamma, if calcium release is not triggered by Q beta but by Q gamma itself or by some other signal.
The mucosal epithelium of the pharyngeal tonsil was studied in 10 conventionally raised sheep by scanning and transmission electron microscopy. The surface of the tonsil was covered by two types of topographically and ultrastructurally distinct epithelium. The epithelium overlying the lymphoid follicle region (follicle-associated epithelium) was compressed by adjacent ciliated epithelium and consisted of predominantly non-ciliated cells of varying height. The majority of these cells possessed microvilli or microfolds of varying number, height and density on their surface. Small numbers of squamous cells and, occasionally, intermediate cells were also present in these areas. Ultrastructurally, the microvillous cells showed features similar to M cells which occur in other mucosal sites. They contained cytoplasmic vesicles and vacuoles and formed intercellular digital junctions. Between these cells was a heavy infiltration of lymphoid cells. Focal disintegration of the mucosal epithelium was occasionally seen. The epithelium covering non-follicle areas consisted of mainly ciliated cells interspersed with some goblet cells and squamous cells, morphologically resembling those elsewhere in the respiratory tract. These findings have provided evidence which confirms that the ovine pharyngeal tonsil is part of the respiratory tract-associated lymphoid tissue.
Thirty-six mice were inoculated intranasally with Bordetella parapertussis organisms (isolated from sheep with chronic non-progressive pneumonia) to study their deposition and clearance in the lower respiratory tract. The deposition of the organism was greater in the trachea than in the lungs. At 48 hours after inoculation, almost 100% of organisms were cleared from the trachea but only 55% of organisms were cleared from the lungs. This result correlates well with the morphological changes seen previously in the pulmonary parenchyma and airways of mice and lambs experimentally infected with this organism.
1. The effect of nifedipine on charge movement was studied in cut twitch fibres of Rana temporaria with a double Vaseline-gap voltage-clamp technique. The steady-state charge-voltage (Q-V) plot, in the absence or presence of nifedipine, was separated into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. 2. When a fibre was held at -90 mV, low concentrations (around 20 nM) of nifedipine suppressed a large fraction of Q beta than Q gamma. Higher concentrations of nifedipine suppressed Q gamma more effectively than Q beta, but even 2 microM-nifedipine did not suppress Q beta and Q gamma completely. Ten micromolar was required for complete suppression of Q gamma. Nifedipine thus suppressed Q beta and Q gamma with different dose dependencies. 3. When the holding potential was changed to -70 mV, some Q beta and Q gamma were inactivated. Low concentrations (around 20 nM) of nifedipine still suppressed a larger fraction of the mobile Q beta than the mobile Q gamma. Higher concentrations of nifedipine also suppressed Q gamma more effectively than Q beta, but 2 microM-nifedipine was sufficient to suppress Q gamma completely. Hence, at this slightly depolarized holding potential, nifedipine also suppressed Q beta and Q gamma with different dose dependencies. 4. A portion of Q beta appeared to be resistant to the action of nifedipine. At -70 mV, the blockage of the nifedipine-sensitive portion of Q beta appeared to saturate at 2 microM of the drug. At -90 mV, the nifedipine-resistant portion of Q beta was more difficult to identify, because the blockage of the nifedipine-sensitive portion of Q beta did not saturate at 2 microM. 5. Based on double-reciprocal plots for the dose-response relationships, the half-blocking concentration of nifedipine for Q beta was found to be 14-19 nM at -90 mV and less than 13 nM at -70 mV, whereas that for Q gamma was approximately 1.6 microM at -90 mV and 120 nM at -70 mV. Thus, nifedipine suppressed Q gamma in a voltage-dependent manner, but its suppression of Q beta was much less voltage dependent. 6. It was demonstrated that the enhancement in the blockage of charge movement by maintained depolarization could not be achieved by depolarizing pulses lasting up to hundreds of milliseconds. 7. The difference in the half-blocking concentrations of nifedipine for Q beta and Q gamma implies that Q beta and Q gamma cannot be tightly coupled.
In order to investigate the relationship between airway inflammation and the development of naturally occurring allergic airway hypersensitivity, the lower respiratory tract was studied in eighteen sheep 14 days after challenge with inhaled Ascaris suum antigen. Sheep could be classed into three groups on the basis of their response: group A (n = 6) had significant changes in both airway resistance and dynamic lung compliance; group B (n = 6) had changes in only dynamic lung compliance, and group C (n = 6) were nonresponders. The results showed that the volume density of secretory granules in lung mast cells was greater in hypersensitive sheep than in nonreacting sheep (p less than 0.01). This high volume density was due to a high numerical density of granules in the mast cells. There was, however, no significant difference between groups in the numerical density of mast cells and eosinophils, or the observed degree of degranulation of mast cells. Although lymphocytes were commonly seen within the airway epithelium and subepithelial regions, infiltration of neutrophils or aggregation of dense and nodular lymphoid tissues were not common in the airways of any sheep. There was no significant difference in the infiltration of neutrophils or the aggregation of lymphocytes and lymphoid tissue in airways between the three groups. These findings indicate that there is an inherent difference in the volume density of lung mast cell granules between hypersensitive and nonreacting sheep but that other inflammatory cells are probably not directly involved in the initial development of allergic airway hypersensitivity in this species.