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Biomedical subjects

S H Cheng

Publications and source records attributed to S H Cheng.

At least 127 records · Page 7Linked to original sources
PubMed1993

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

Alanine↗
PubMed1993

BK virus infection of the human urinary tract.

By screening consecutive autopsy cases with an antibody that recognizes human polyomaviruses, we found a case of malignant lymphoma in which the virus infection was confined to epithelia of the renal calyces, renal pelvis, ureter, and urinary bladder. The virus was confirmed as BK virus by a specific monoclonal antibody against BK virus T antigen, and numerous virus particles were identified by electron microscopy. The results showed that BK virus is a human urotheliotrophic virus.

Antibodies, Monoclonal↗
PubMed1993

Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.

Monocytes from purified protein derivative S Mantoux-negative children and young adults inhibited intracellular growth of Mycobacterium microti more in Chingleput than in London. Mycobacterium bovis BCG vaccination did not enhance bacteriostasis with the Indians but did so with the Londoners. No evidence was found for involvement of cytokines such as macrophage-activating factor and granulocyte macrophage colony-stimulating factor in the differences.

BCG Vaccine↗
PubMed1993

Effect of deletion mutations on the function of CFTR chloride channels.

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Intracellular protein trafficking defects in human disease.

Secretory proteins and integral membrane proteins travel through the secretory pathway to a variety of destinations. Their targets are often specified by signals in the amino acid sequence or signals added post-translationally. The KDEL sequence that retains soluble proteins in the endoplasmic reticulum and the mannose 6-phosphate group of lysosomal enzymes are well-characterized examples of targeting signals; other signals are less well understood. Given the complexity and importance of the intracellular trafficking pathways, it is perhaps not surprising that mutations that affect the trafficking of proteins are associated with some human genetic diseases.

Journal Article↗
PubMed1992

Production of cystic fibrosis transmembrane conductance regulator in the milk of transgenic mice.

Here we describe the production of cystic fibrosis transmembrane conductance regulator (CFTR), the product of the gene associated with cystic fibrosis, in the milk of transgenic mice. Mammary specific expression was achieved by placing the CFTR cDNA under the control of the goat beta-casein gene promoter. By fractionation, CFTR was shown to be associated with the membranes that envelop milk fat globules as they are discharged from the apical surface of the mammary epithelia. Since milk fat globules may comprise up to 10% of whole milk, this represents a novel, inexpensive and efficient approach to produce CFTR and possibly other membrane-associated proteins. The availability of large quantities of CFTR could have important implications for the development of new therapies for cystic fibrosis.

Animals↗
PubMed1992

The pattern of mycobacterial antigen recognition in sera from Mantoux-negative individuals is essentially unaffected by bacille Calmette-Guérin (BCG) vaccination in either south India or London.

Paired sera were obtained before and 8 weeks after routine BCG vaccination from 20 PPD-S Mantoux-negative individuals who were living adjacent to the Chingleput BCG vaccine trial area in Tamil Nadu and from seven Mantoux-negative school-children in London, UK. Most subjects became Mantoux-positive after vaccination. In ELISA tests against soluble extracts of BCG or Mycobacterium tuberculosis H37Rv or against PPD-S, pre-vaccination antibody titres of South Indian subjects were about twice those of British subjects but there was no increase in titre of antibodies after vaccination of either population. Western blotting showed that even before vaccination, and even in British subjects, antibodies were present that recognized numerous antigenic components in extracts of BCG and M. tuberculosis. There was no consistent difference between band patterns with South Indian and British subjects and any effect of vaccination on the patterns was minimal.

Adolescent↗
PubMed1992

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.

Antibodies, Monoclonal↗
PubMed1992

The amino-terminal half of pp59c-fyn contains sequences necessary for formation of a 75 kDa form and also repressive elements absent in pp60c-src.

Members of the src family of tyrosine kinases are composed of amino acid sequences which can be divided into three regions: the unique amino-terminal 80 residues; the next 180 residues of conserved but non-catalytic sequence; and the catalytic carboxy-terminal half of the molecule. To characterize the elements that regulate the catalytic and transforming activities of two members of this family, pp59c-fyn and pp60c-src, we generated six chimeric proteins by interchanging the three regions of the 531F mutant of pp59c-fyn and of the 527F mutant of pp60c-src. Our data indicate that substituting all or part of the amino-terminal 263 residues of pp59c-fyn for those of 527F inhibited the kinase and transforming activities of 527F. Conversely, substituting the amino-terminal half of pp60c-src for that of 531F resulted in an increase in the transforming potential of 531F. These results suggest that the amino-terminal half of pp59c-fyn contains elements which act to suppress the catalytic and transforming activities of the enzyme and that these suppressive elements are either absent or inactive in pp60c-src. These differences argue that the src family of tyrosine kinases are regulated differently in the cell. In vitro translation of some of the chimeras in rabbit reticulocyte lysates generated a 75 kDa protein in addition to the expected 59 kDa product. This 75 kDa species is analogous to the p75 protein previously detected in wild-type pp59c-fyn translation products. Interestingly, formation of p75 required the presence of DNA sequences encoding the unique amino-terminal residues of pp59c-fyn.

Animals↗
PubMed1991

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.

Chloride Channels↗
PubMed1991

Structural elements that regulate pp59c-fyn catalytic activity, transforming potential, and ability to associate with polyomavirus middle-T antigen.

Except for its unique amino-terminal region (residues 1 through 83), which possibly dictates substrate recognition, pp59c-fyn bears a high degree of homology with other members of the src family of tyrosine kinases. Here we show that the carboxy terminus of pp59c-fyn is necessary for stable middle-T-antigen association, that pp59c-fyn is normally phosphorylated on both serine and tyrosine residues, and that Tyr-531 and Tyr-420 are phosphorylation sites in vivo and in vitro, respectively. Analysis of a spontaneously generated mutant encoding a truncated form of pp59c-fyn and of variants specifically mutated at the Tyr-531 and Tyr-420 phosphorylation sites indicates that pp59c-fyn has regulatory elements analogous to those that have already been identified for other src-like tyrosine kinases. However, further examination of the pp59c-fyn variants suggests the likelihood of additional means by which its activities might be regulated. Although alteration of Tyr-531 to phenylalanine (531F) in pp59c-fyn results in a protein which is more active enzymatically that the wild type, the enhancement is much less than that for the analogous variant of pp60c-src. Furthermore, contrary to results of similar experiments on other src-like proto-oncogene products, 531F did not induce transformation of NIH 3T3 cells. Studies involving pp59c-fyn-pp60c-src chimeras in which the unique amino-terminal sequences (residues 1 through 83) of the two kinases were precisely interchanged implied that the inability of 531F to induce transformation is probably not caused by the absence of substrates for pp59c-fyn in NIH 3T3 cells but rather by the insufficient enhancement of pp59c-fyn kinase activity. It is therefore probable that the kinase and transforming activities of pp59c-fyn are repressed by additional regulatory elements possibly located in the amino-terminal half of the molecule.

Amino Acid Sequence↗
PubMed1991

The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence.

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal approximately 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.

Animals↗
PubMed1991

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibody specific for BK virus large-T antigen allows discrimination among the different papovaviral large-T antigens.

The human papovaviruses BK virus (BKV) and JC virus (JCV) both encode a large-tumor (T) antigen which are highly homologous to each other as well as to simian virus 40 (SV40) large-T antigen. This high conservation of amino acid sequence has resulted in overlapping antigenicity such that immunological differentiation of the large-T antigens from the three different viruses has been difficult. Here we describe the generation and characterization of a new monoclonal antibody (BK-T.1) which is specific for BKV large-T antigen and which does not cross-react with the other papovaviral T antigens. We show that when BK-T.1 is used in conjunction with preexisting monoclonal antibodies against SV40 large-T antigen, it is possible to identify the origin of papovavirus T antigens.

3T3 Cells↗
PubMed1991

Biology of murine gamma delta T cells.

Murine lymphocytes express either a T-cell receptor alpha beta or a gamma delta heterodimer. The function of alpha beta cells are well characterized, while gamma delta cells remain an enigmatic population. In the mouse, gamma delta cells appear in significant proportions in the epithelia of various nonlymphoid tissues such as the skin, intestine, tongue, lung, and reproductive organs. While gamma delta T-cell subsets with distinct antigen receptor repertoires are associated with certain organs, diversified populations of gamma delta cells showing heterogeneous TCR phenotypes, as a result of junctional region diversification and usage of different V chains, can be found in the lymphoid organs and in the intestinal epithelia. Recent evidence has shown that gamma delta cells might recognize heat shock proteins, possibly in association with classical and nonpolymorphic MHC molecules. Together with their tissue distribution, gamma delta cells may represent the first line of defense of the immune system. gamma delta Cells are the first T cells to colonize the thymus. Intriguingly, there is more evidence to support the hypothesis that they might also affect the development of alpha beta cells and other hematopoietic stem cells.

Animals↗
PubMed1991

Efficacy of ivermectin for control of microfilaremia recurring after treatment with diethylcarbamazine. I. Clinical and parasitologic observations.

We compared the efficacy of a single dose of ivermectin with that of a standard course of diethylcarbamazine (DEC) for the control of microfilaremia in 60 patients with bancroftian filariasis who had developed recurrent microfilaremia after each of three or more prior treatments with DEC. The study was done as a randomized, double-blind trial. Complete, but in some cases, transient clearance of microfilaremia was observed in both treatment groups. At one year, recurrent microfilaremia was present in seven patients treated with ivermectin and in five treated with DEC. Pretreatment levels of microfilaremia were significantly higher in patients who relapsed within one year after treatment than in those who remained amicrofilaremic. Side effects with both treatments were common, but mild. Febrile reactions were more frequent in the ivermectin group; localized reactions consistent with a flare-up of acute filarial disease occurred mostly in the DEC group. We conclude that ivermectin is an effective and practical alternative to DEC for treatment of recurrent microfilaremia due to bancroftian filariasis.

Adolescent↗
PubMed1991

Efficacy of ivermectin for control of microfilaremia recurring after treatment with diethylcarbamazine. II. Immunologic changes following treatment.

We compared the effect of a single dose of ivermectin with that of a standard course of diethylcarbamazine (DEC) on several parameters of the host's antifilarial immune response in 60 patients with bancroftian filariasis enrolled in a double-blind drug trial. All participants had measurable serum levels of antifilarial antibodies and parasite antigens at the onset of the study. Drug-induced clearance of microfilaremia was associated with a temporary increase in HC 11 antigenemia and a decrease in serum levels of antibodies to soluble filarial antigens. Antigenemia progressively declined in patients who remained amicrofilaremic after treatment, but declined and then increased in persons with recurrent microfilaremia. Treatment triggered a sustained increase in serum levels of interleukin-1, tumor necrosis factor, and interleukin-6 in all patients studied. Although ivermectin and DEC are believed to exert their antiparasite activity via different mechanisms, the same pattern of serologic changes was observed in patients treated with either drug.

Adolescent↗