Failure of anti-lambda immunofixation reagent mimics alpha heavy-chain disease.
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Aged↗
Biomedical subjects
L Su
Publications and source records attributed to L Su.
[The effect of transforming growth factor-beta on collagen expression by human embryonic fibroblasts].
Transforming growth factor-beta (TGF-beta) is a cytokine with diverse biological activity. It can regulate the metabolic function of extracellular matrix (ECM) and play an important role in the development of pulmonary fibrosis. Collagen are major ECM components that are responsible for normal lung structure and function. Collagen accumulation is a major feature of pulmonary fibrosis. In order to study the mechanism of pulmonary fibrosis, we examined the effects of TGF-beta on the production, mRNA expression of type I collagen and type IV collagen by cultures of human embryonic lung fibroblasts. The results indicated that TGF-beta can induce collagen formation and type I collagen, type IV collagen mRNA expression without affecting cell proliferation. We concluded that TGF-beta plays a role in collagen accumulation of pulmonary fibrosis and the changes in collagen production appear to be mediated on collagen mRNA level.
Collagen↗
Construction and characterization of a normalized cDNA library.
We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.
Base Sequence↗
Transgenic gpt+ V79 cell lines differ in their mutagenic response to clastogens.
Several gpt+ transgenic cell lines were derived from hprt V79 cells to study mutagenesis mechanisms in mammalian cells. The G12 cell line was previously shown to be hypermutable by X-rays and UV at the gpt locus compared to the endogenous hprt gene of the parental V79 cells (Klein and Rossman, 1990), and is now shown to be highly mutable by the clastogenic anti-tumor agent bleomycin sulfate. A second transgenic cell line G10, which has a different gpt insertion site, was studied in comparison with G12. Both G12 and G10 cell lines carry the stable gpt locus at a single integration site in the Chinese hamster genome, and neither spontaneously deletes the integrated gpt sequence at a high frequency. Although spontaneous mutation to 6-thioguanine resistance in G10 cells is 3-4 times higher than in G12 cells, the cell lines differ to a much greater extent when mutated by clastogens. In comparison to G12 cells, the gpt locus in G10 cells is up to 13 times more sensitive to bleomycin mutagenesis and 5 times more responsive to X-ray mutagenesis. In contrast, there is much less difference in UV-induced mutagenesis and no differences in mutagenesis induced by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The dose-dependent decrease in survival of the transgenic cells is the same for all mutagens tested, and does not differ from that of V79 cells. Neither transgenic cell line is generally hypermutable, since mutagenesis at an endogenous gene, Na+K+/ATPase, is similar to that of the parental V79 cell line. Although both cell lines can be induced to delete the transgene following clastogen exposure, deletions are not the only recovered mutations, and the cell lines can also be used to study mutations within the PCR recoverable gpt gene. The utility of these transgenic cells to investigate genome position effects related to mammalian mutagenesis mechanisms is discussed.
Animals↗
Intermediate filaments in oral neoplasia. I. Oral cancer and epithelial dysplasia.
The major value of intermediate filaments (IFs) in biological and applied research lies in their high order of cell and tissue specificity. This is particularly well illustrated in keratin (K) expression in various oral epithelia. Although the original class of IF is usually conserved in tissues after neoplastic transformation, epithelia show a tendency to shift their pattern of keratin expression in a manner which, while not predictable with precision, may sometimes be of diagnostic or prognostic significance. This review compares the keratins in normal oral epithelia, which show a mainly site-dependent expression, with those in squamous cell carcinoma. Key changes in the latter are the presence of simple epithelial keratins, K8 and K18 (occasional K7), reduced expression of differentiation-linked keratins (K1, K10, K4 and K13) and a tendency for down-regulation of primary keratins, K5 and K14. Moderate and severe dysplasias also tend to exhibit K8 and K18 with concomitant disordered expression of differentiation-linked keratins. There are reports of similar changes after neoplastic transformation in other mucosal sites and skin. Before this information can be applied diagnostically in immunocytochemical studies, the anti-keratin antibodies must be fully characterised and their interaction with the relevant tissue, both frozen and conventionally processed, should be evaluated.
Carcinoma, Squamous Cell↗
Selection of cDNAs using chromosome-specific genomic clones: application to human chromosome 13.
We have developed a general method for en masse isolation of cDNAs present in a normalized library by hybridization to arrayed chromosome-specific phage lambda clones; we have used this approach to initiate exon-mapping of human chromosome 13. An advantage of the simultaneous isolation of cDNA/lambda pairs is that it allows cytogenetic assignment of a bona fide genomic clone by in situ hybridization, which also verifies that the corresponding cDNA or a homologous expressed sequence resides on chromosome 13. This information is enriched by partial sequencing of a selected cDNA from both ends. The sequence of the 3' noncoding region provides an 'identifier' that is used to develop STSs, while the sequence from the 5' end, often corresponding to a coding region, is used for homology searches in databases that occasionally reveal gene functions.
Amino Acid Sequence↗
Ligand regulation of bovine protein kinase C alpha response via either cysteine-rich repeat of conserved region C1.
Based on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC alpha responses. We have prepared two amino terminal truncation mutants of bovine PKC alpha, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC alpha. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C alpha responses.
Animals↗
Rapid-high, syncytium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse.
Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) disease is associated with an increased viral burden in the peripheral blood and a loss of circulating CD4+ T cells. HIV-1 isolates obtained prior to this stage of disease often have a "slow-low," non-syncytium-inducing (NSI) phenotype, whereas those obtained afterwards are often characterized as "rapid-high" and syncytium inducing (SI). Paired NSI and SI isolates from two different patients were inoculated into the human thymus implants of SCID-hu mice. The two slow-low, NSI isolates replicated to minimal levels in the grafts and did not induce thymocyte depletion. In contrast, the two SI isolates from the same patients showed high levels of viral replication and induced a marked degree of thymocyte depletion, accompanied by evidence of programmed cell death. These observations reveal a correlation between the replicative and cytopathic patterns of HIV-1 isolates in vitro and in the SCID-hu mouse in vivo and provide direct evidence that the biological phenotype of HIV-1 switch may be a causal and not a derivative correlate of HIV-1 disease progression.
Acquired Immunodeficiency Syndrome↗
Metal mutagenesis in transgenic Chinese hamster cell lines.
Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium.
Animals↗
A study on patterns in the average life expectancies and mortality rates of 56 nationalities in China in 1990.
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Asia↗
Protein and mRNA expression of simple epithelial keratins in normal, dysplastic, and malignant oral epithelia.
Simple epithelial keratins K7, K8, and K18 are present in no more than trace amounts in normal stratified squamous epithelial but have been reported in squamous cell carcinomas. With the aim of determining the level at which keratin synthesis is regulated in vivo, we have compared the expression of mRNA by in situ hybridization and protein by immunohistochemistry for K7, K8, and K18 in a series of normal, dysplastic, and malignant oral epithelia. In normal epithelia mRNAs for K7, K8, and K18 were present in basal and lower spinous cells but adjacent sections were generally negative for the respective proteins. In severe dysplasia there was irregular suprabasal extension of K8 and K18 mRNAs in all cases and their proteins were expressed in more than half of the cases. The carcinomas expressed K8 and K18 mRNAs homogeneously and were strongly reactive for these keratin proteins but K7 expression appeared reduced in malignancy. These results are consistent with the post-transcriptional regulation of K7, K8, and K18 expression in normal epithelia and the presence of their proteins in dysplastic and malignant epithelia suggests the release of these epithelial cells from a post-transcriptional block on K8 and K18 translation. Alternatively, rapid degradation of K8 and K18 protein might be occurring in normal epithelia but be suppressed in dysplasia and malignancy.
Base Sequence↗
[Advances in the study of laminin receptors].
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Animals↗
[Guanfu base A inhibit the post-infarction ventricular arrhythmias in dogs].
Harris tow-stage left anterior coronary ligation was performed in anesthetized dogs. After twenty hours, severe ventricular arrhythmias were recorded under conscious state. Cumulative intravenous injections of guanfu base A, a new alkaloid isolated from the tuber of Aconitum coreanum in China, from 10 to 40 mg.kg-1 produced remarkable antiarrhythmic effect lasting over 30 min and a moderate reduction of heart rate. The P-R interval, QRS complex and Q-T interval of the sinus rhythm were not significantly altered. The antiarrhythmic potency of guanfu base A in this model was found to be about one third that of quinidine.
Alkaloids↗
Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha.
Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism. To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro. Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology. This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms. These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A. J., Milner, Y., Saville, S. P., Dvir, A., Mochly-Rosen, D., and Orr, E. (1991) Proc. R. Soc. Lond. B 243, 165-171). Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Amino Acid Sequence↗
Phorbol ester activation of functional rat protein kinase C beta-1 causes phenotype in yeast.
The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC beta-1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC beta-1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC beta-1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC beta-1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Amino Acid Sequence↗
Expression of keratin mRNAs and proteins in normal salivary epithelia and pleomorphic adenomas.
Control of keratin (K) gene expression may be important for cell differentiation in complex epithelia such as salivary gland. To investigate differences in distribution between keratin mRNAs and their respective proteins, a combined in situ hybridization (ISH) and immunohistochemical study was undertaken on nine normal salivary glands and seven pleomorphic adenomas. ISH employed riboprobes to K7, K8, K14, K18, and K19. Immunohistochemistry was performed on adjacent sections using monoclonal antibodies (MAbs) to the same keratins. Normal luminal cells showed abundant hybridization with probes for K7, K8, K18, and K19. Keratin 14 mRNA was present in basal and myoepithelial cells at a low level of expression. Proteins of their keratins were strongly stained. Neoplastic cells showed variable expression of mRNA and protein for K7, K8, K18, and K19. There was a high level of K14 mRNA but variable protein. The findings provide evidence that expression of these keratins in normal salivary epithelia is regulated transcriptionally and that in neoplasia this system is in considerable disarray.
Adenoma, Pleomorphic↗
Reconstitution of protein kinase C alpha function by the protein kinase C beta-I carboxy terminus.
The Ca(2+)- and phospholipid-dependent Ser/Thr kinase protein kinase C (PKC) plays important roles in the transduction of cellular signals. Various PKC isoforms exist in mammalian cells which share conserved and variable regions as defined by cDNA sequence comparisons. To test whether carboxyl (C) terminal sequences of distinct isoforms can complement each other to yield functional chimeric molecules, we have constructed a PKC chimera in which amino acids 595-672 at the C-terminus of bovine PKC alpha (a) were replaced with the corresponding C-terminal amino acids (598-671) of rat PKC beta-I (b) to yield the chimera alpha/beta-I (ab). The chimera was then characterized biochemically and functionally, and compared with the parental isoforms. Since structure/function analysis of PKC in mammalian experimental systems is complicated by multiple PKC isoforms and by cellular complexity, we stably introduced the PKC constructs into the yeast Saccharomyces cerevisiae, a simple, lower eukaryote with a short doubling time and well established molecular genetics. In yeast, the faithfully expressed PKCab chimera and normal PKC isoforms bound radiolabelled phorbol ester and were recognized on immunoblots by PKC-specific antibodies. The chimera phosphorylated substrate peptides in a PMA- and Ca(2+)-dependent manner, and, upon activation, increased the cell doubling time and the rate of Ca2+ uptake into cells. In addition, PKCab displayed characteristics distinct from normal PKCb, but virtually indistinguishable from normal PKCa. Our findings indicate the reconstitution of PKCa function by the PKCb carboxyl terminus.
Animals↗
LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation.
A cDNA encoding a novel zinc finger protein has been isolated from a mouse T-cell leukemia line on the basis of its expression of a Ly-1 epitope in a lambda gt11 library. The putative gene was mapped on mouse chromosome 1, closely linked to Idh-1, but not linked to the Ly-1 (CD5) gene. The cDNA is therefore named Ly-1 antibody reactive clone (LYAR). The putative polypeptide encoded by the cDNA consists of 388 amino acids with a zinc finger motif and three copies of nuclear localization signals. Antibodies raised against a LYAR fusion protein reacted with a protein of 45 kD on Western blots and by immunoprecipitation. Immunolocalization indicated that LYAR was present predominantly in the nucleoli. The LYAR mRNA was not detected in brain, thymus, bone marrow, liver, heart, and muscle. Low levels of LYAR mRNA were detected in kidney and spleen. However, the LYAR gene was expressed at very high levels in immature spermatocytes in testis. The LYAR mRNA is present at high levels in early embryos and preferentially in fetal liver and fetal thymus. A number of B- and T-cell leukemic lines expressed LYAR at high levels, although it was not detectable in bone marrow and thymus. During radiation-induced T-cell leukemogenesis, high levels of LYAR were expressed in preleukemic thymocytes and in acute T leukemia cells. Fibroblast cells overexpressing the LYAR cDNA from a retrovirus vector, though not phenotypically transformed in vitro, had increased ability to form tumors in nu/nu mice. Therefore, LYAR may function as a novel nucleolar oncoprotein to regulate cell growth.
Amino Acid Sequence↗