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S H Cheng

Publications and source records attributed to S H Cheng.

At least 145 records · Page 8Linked to original sources
PubMed1990

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Animals↗
PubMed1990

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Cell Membrane Permeability↗
PubMed1990

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

High yields of soluble, biologically active pp60c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60c-src was not phosphorylated on either Tyr-527 or Tyr-416 in insect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60c-src was phosphorylated on tyrosine residues both in vivo and in vitro.

Animals↗
PubMed1990

Association of p62c-yes with polyomavirus middle T-antigen mutants correlates with transforming ability.

A number of mutants of polyomavirus middle T antigen (MTag) were constructed into replication-competent avian retroviruses. To assess the ability of these MTag variants to transform and to associate with the avian p60c-src and p62c-yes proto-oncogene products, we used these viruses to infect chicken embryo fibroblasts. We found that the ability of individual mutant MTags to associate with p62c-yes correlated well with the ability of these mutants to transform, as has been previously shown for the association of MTag with p60c-src. All transformation-competent mutant MTags retained the ability to complex with p62c-yes. Two transformation-defective mutants, RX67 and RX68, which could weakly associate with p60c-src, were unable to associate with p62c-yes.dl1015, a transformation-defective mutant which could associate with p60c-src and with a phosphatidylinositol kinase activity, was also able to associate with p62c-yes. Therefore, some as yet unmeasured biochemical property is defective in this mutant.

Animals↗
PubMed1990

Stoichiometry of cellular and viral components in the polyomavirus middle-T antigen-tyrosine kinase complex.

Our results indicate that only one type of tyrosine kinase is present within each middle-T antigen-tyrosine kinase complex, suggesting that middle-T antigen forms separate complexes with different tyrosine kinases. Furthermore, we determined that there is only one molecule of middle-T antigen within any one of these complexes. We interpret this to mean that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation. Finally, we also demonstrate that the separate middle-T:pp60c-src and middle-T:pp59c-fyn complexes are each able to interact with the same cellular p81/85-kDa phosphoprotein, a possible component of the phosphatidylinositol kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

The amino-terminal region of pp60c-src has a modulatory role and contains multiple sites of tyrosine phosphorylation.

The c-src gene product from either platelet-derived growth factor treated cells or from polyomavirus-infected cells migrates anomalously on gels, displays enhanced kinase activity, and contains additional sites of tyrosine phosphorylation within its amino-terminus. To probe the importance of these post-translational modifications, each of the five amino-terminal tyrosine residues (residues 90, 92, 131, 136, and 149) was altered to phenylalanine using site-directed mutagenesis. Except for the 136F variant, the mutants displayed enhanced kinase activity, albeit at a low level shown insufficient to induce focus formation in NIH3T3 cells. Mutagenesis of residues adjacent to tyrosines 90 and 92 also resulted in variants with enhanced kinase activity indicating that the region and not the tyrosines per se, may be involved in regulating this activity. Fingerprinting analysis demonstrated that the enhanced kinase activity brought about by these mutations occurred through a mechanism which appears to be independent of the phosphorylation state of Tyr 416 and possibly Tyr 527. Upon treatment of cells with vanadate both amino-terminal variants and transforming mutants of pp60c-src displayed a slower migrating form, designated p60+. Hence, the appearance of these p60+ proteins may be elicited either by mutations that enhance the transforming activity of pp60c-src or by perturbations within the amino-terminal region of the enzyme. The retarded mobility of p60+ was shown to be due in part to additional tyrosine phosphorylations residing at its amino-terminus. The demonstration that p60+ could be resolved into multiple bands and that amino-terminal fragments containing phosphorylated tyrosine residues were obtained regardless of which of the five tyrosines in the region was altered to phenylalanine indicates that there are multiple sites of tyrosine phosphorylation within the amino-terminal region of pp60c-src.

Amino Acid Sequence↗
PubMed1989

An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular protein, p120.

In addition to Rb and p53, a third cellular protein, p120 in monkey and p118 in human cells, forms a specific complex with SV40 large T antigen (T). p118/120 is not a product of the Rb-gene. As was shown with T/Rb complex formation, the interaction between T and p120 is dependent on the intact nature of a ten residue, transformation-controlling domain in T (residues 105-114). In mouse cells, a readily detectable protein of 115 kd was detected, which, like murine Rb, also forms a stable complex with T. Like p118/120, p115 binding is also dependent on the intact nature of the 105-114 sequence. Given their similar size and T antigen binding sequence dependence, p115 and p118/120 may be products of the same gene in different species. These results suggest that interactions between T and p115/118/120, as well as T and Rb, contribute to the SV40 transforming mechanism.

Adenovirus Early Proteins↗
PubMed1989

Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis.

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO(2) compensation point and the inhibition of photosynthesis by 21% O(2) were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C(4) cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO(2) compensation point and the degree of O(2) inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO(2) between Rubisco of the C(3) pathway and phosphoenolpyruvate carboxylase of the C(4) cycle was determined by exposing leaves to (14)CO(2) for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that approximately 94% of the CO(2) was fixed by the C(4) cycle in high light grown plants, versus approximately 78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C(4) photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C(4)-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.

Journal Article↗
PubMed1989

pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.

Animals↗
PubMed1988

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.

Animals↗
PubMed1988

Efficacy of a mass hepatitis B vaccination program in Taiwan. Studies on 3464 infants of hepatitis B surface antigen-carrier mothers.

To evaluate the efficacy of the mass hepatitis B vaccination program in Taiwan in interrupting perinatal hepatitis B virus transmission, 3464 randomly selected 18-month-old infant vaccinees born to hepatitis B surface antigen-carrier mothers were recruited from 9697 eligible infants during a six-month period of the program. They were divided into ten groups according to maternal infectivity and compliance with the vaccination schedule. Serum samples were tested for hepatitis B surface antigen, antibody to hepatitis B surface antigen, and antibody to hepatitis B core antigen. In 786 infants who had highly infectious mothers and who received hepatitis B immune globulin and vaccine on schedule, the protective efficacy was about 85%. The efficacy seemed to be slightly lower in those immunized off schedule. Overall, 11% of infants still carried hepatitis B surface antigen, and 81% of the infants had antibody to hepatitis B surface antigen that exceeded 10 mIU/mL in more than 90% of them. The geometric mean titers of antibody to hepatitis B surface antigen were more than 200 mIU/mL in every group of infants. We conclude that the mass vaccination program is efficacious in preventing perinatal hepatitis B virus transmission and the chronic carrier state; most infant vaccinees have adequate levels of protective antibody at 18 months of age. This program is extremely significant in the control of hepatitis B virus infection in Taiwan.

Carrier State↗
PubMed1988

The pigmented epithelium sustains cell growth and tissue differentiation of chicken retinal explants in vitro.

Embryonic retinae from 5-6-day-old chicks (E5-E6) were cut into stripes either in close contact with (RPE stripes) or in absence of the neighboring retinal pigmented epithelium (R stripes). The stripes were explanted and cultivated in vitro for up to 6 days, during which time they show the following differences in their characteristics of growth and differentiation. Compared with R stripes, RPE stripes morphologically showed a significant increase in size during the first 2 days in culture. Using E5 tissue, this is also demonstrated by a higher rate of cell proliferation (as measured by uptake of radioactive thymidine as well as by DNA contents). In contrast, R stripes after two days in culture show a much stronger neurite growth. After longer periods of culturing (5-6 days) we can show by cholinesterase histochemistry (AChE and BChE) and by PNA-lectin binding that the RPE stripes have started to form all major layers of the in vivo retina, whereas R stripes remain unstratified and start to degenerate earlier. We conclude that the pigment epithelium might exert a specific stimulus on growth and tissue differentiation of the neural retina not only during in vitro, but possibly also during in vivo development. The in vitro methods introduced here could become useful model systems to further investigate the significance of the RPE for developmental, regenerative and even adult processes of the neural retina. Their future applicability in ophthalmologic research is briefly discussed.

Animals↗
PubMed1988

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2).

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO(2) compensation points at different O(2) levels, which is typical of C(4) plants, yet it does show about 4% inhibition of net photosynthesis by 21% O(2) at 30 degrees C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C(4) pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C(3) cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial (14)C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO(2) is fixed into C(4) acids (malate and aspartate), whereas about 20% of the CO(2) directly enters the C(3) cycle. This is consistent with the high activity of enzymes for CO(2) fixation by the C(4) pathway and the substantial activity of enzymes of the C(3) cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C(3) photosynthesis in the mesophyll cells and should be considered a C(4)-like species.

Journal Article↗
PubMed1988

The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.

A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.

Amino Acid Sequence↗
PubMed1988

Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.

A blood sample was taken from children aged 13-15 years immediately before BCG vaccination and 8 weeks after. The children were tuberculin skin-test negative to PPD-S before vaccination and positive after. Mononuclear cells were separated from the blood, infected with Mycobacterium microti at a low bacterium/monocyte ratio and allowed to form monolayers in microtitre wells. The infected monolayers were rinsed daily and the change in number of live bacteria in monolayers and supernatants was monitored by colony counts on agar. The cells were bacteriostatic during the first day, thereafter growth accelerated in pre-vaccination monolayers. When monolayers received pulsed exposures to autologous lymphocytes that had been incubated with whole dead tubercle bacilli the growth rates of M. microti were increased. However, growth rates in lymphocyte-pulsed monolayers were significantly lower after vaccination than before. It is proposed that this difference reflects the protective effect of vaccination.

Adolescent↗
PubMed1987

Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src.

To investigate the importance of tyrosine phosphorylation in the regulation of pp60c-src, we have substituted phenylalanine for tyrosine at positions 416, 519, and 527. Cells expressing the 527 or the 519/527 mutant but not the 416 or the 519 mutant were morphologically transformed, grew in soft agar, and formed foci. In addition, the 527 and 519/527 mutants had elevated kinase activities in vitro. Modifying Tyr 416 to phenylalanine in the 527 or the 519/527 mutants only partially inhibited their kinase activities yet abolished their ability to induce focus formation and promote growth in soft agar. These results suggest that two events must occur to activate the full transforming potential of pp60c-src: hypophosphorylation at Tyr 527 and hyperphosphorylation at Tyr 416.

Animals↗