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K Tang

Publications and source records attributed to K Tang.

At least 91 records · Page 5Linked to original sources
PubMed1995

Matrix-assisted laser desorption/ionization mass spectrometry of immobilized duplex DNA probes.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry was used to analyze short DNA duplex probes with one strand immobilized on solid supports (straptavidin-coated magnetic beads or controlled pore glass beads). Only the non-immobilized strand could be detected. Partial denaturation was found when the duplex probes were mixed with 3-hydroxypicolinic acid, ammonium citrate matrix. The strategy has several applications, such as fast DNA sequence analysis and DNA diagnostics.

Base Sequence↗
PubMed1995

Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in self-fertilized progenies. However, extensive genome change was detected by 89 nuclear DNA clones used as probes. Most genome changes involved loss and/or gain of parental restriction fragments and appearance of novel fragments. Genome changes occurred in each generation from F2 to F5, and the frequency of change was associated with divergence of the diploid parental genomes. Genetic divergence among the derivatives of synthetic polyploids was evident from variation in genome composition and phenotypes. Directional genome changes, possibly influenced by cytoplasmic-nuclear interactions, were observed in one pair of reciprocal synthetics. Our results demonstrate that polyploid species can generate extensive genetic diversity in a short period of time. The occurrence and impact of this process in the evolution of natural polyploids is unknown, but it may have contributed to the success and diversification of many polyploid lineages in both plants and animals.

Biological Evolution↗
PubMed1995

Lines of murine oligodendroglial precursor cells immortalized by an activated neu tyrosine kinase show distinct degrees of interaction with axons in vitro and in vivo.

Replication-defective retroviruses expressing the t-neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr-neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t-neu lines into myelin-associated glycoprotein (MAG)-positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr-neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t-neu line cells engaged with the demyelinated axons whereas the egfr-neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron-glia interaction.

Animals↗
PubMed1995

Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS.

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that 1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and 2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co-transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y-chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X-irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS.

Animals↗
PubMed1995

Detection of delta F508 mutation of the cystic fibrosis gene by matrix-assisted laser desorption/ionization mass spectrometry.

The most common mutation of the cystic fibrosis gene is characterized by the deletion of three nucleotides that code phenylalanine in the 508 position of the cystic fibrosis transmembrane conductance regulator. We report the first measurements by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry for the delta F508 mutation in cystic fibrosis carriers and patients. Furthermore, in a blind test, results from the normal and delta F508 mutant alleles in 30 clinical samples based on MALDI mass spectrometry and on conventional gel analysis of the DNA were in total agreement. These results demonstrate the utility of MALDI mass spectrometry in the molecular diagnosis of mutant alleles and point to its potential use for ultra-fast detection in large-scale screening of DNA mutations.

Base Sequence↗
PubMed1995

Revisit of MALDI for small proteins.

Matrix-assisted laser desorption/ionization (MALDI) was used for several small proteins (such as insulin) and for peptides. It was found that the detection efficiencies of MALDI for the insulin B chain and the insulin A chain are drastically different. Similar phenomena were also observed for various types of peptides. The positive-ion signal of MALDI in detecting proteins or peptides was found to be greatly enhanced by the presence of a basic amino acid in their chains. The experimental results indicate that this enhancement may arise from proton transfer in solution by an acid-base reaction between the protein/peptide and matrix molecule. This pre-protonated mechanism provides a low energy barrier for the ionization of peptides in a MALDI process, and greatly reduces the energy threshold of MALDI. Matrix effects on the ionization mechanism are discussed.

Amino Acid Sequence↗
PubMed1994

Hormone storage vesicle proteins. Transcriptional basis of the widespread neuroendocrine expression of chromogranin A, and evidence of its diverse biological actions, intracellular and extracellular.

Chromogranin A (CgA) is an acidic soluble protein found in the core of secretory vesicles throughout the neuroendocrine system, from which it is coreleased by exocytosis with a variety of amine and peptide hormones and neurotransmitters. Much has now been learned about the structure of CgA, and there is emerging evidence that it plays several biological roles, both within secretory granules and after release from neuroendocrine cells. Factors governing its gene's widespread yet restricted (neuroendocrine) pattern of expression are only now being explored. In an attempt to understand how cells throughout the neuroendocrine system (but not exocrine or other nonendocrine cells) turn on and control the expression of CgA, we have isolated and begun to characterize functional 5' promoter elements from the rodent CgA genes. Within the sympathoadrenal system, interest focuses on a recently proposed (though as yet incompletely investigated) function of CgA: its ability to suppress catecholamine release from adrenal chromaffin cells when such cells are stimulated by their usual physiologic secretagogue. We anticipate that such studies will contribute to an understanding of this abundant, yet previously mysterious protein's role in neuroendocrine function.

Adrenal Medulla↗
PubMed1994

Picolinic acid as a matrix for laser mass spectrometry of nucleic acids and proteins.

We found that picolinic acid is a very good matrix for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry of oligonucleotides, proteins and tRNA. Among the oligonucleotides, picolinic acid was shown to be effective for homo-oligonucleotides, d(G)40 and d(C)60, and for mixed-base oligonucleotides up to 190 bases. In the case of the single-stranded oligonucleotides, and of double-stranded ones as well, only parent ions corresponding to single-stranded DNA were observed. The efficiency for MALDI of oligonucleotides using the picolinic acid matrix was superior to that using 3-hydroxypicolinic acid. MALDI of transfer RNA phenylalanine-specific (tRNA(Phe)), a 76-base ribonucleic acid, was detected with a signal-to-noise ratio of > 10. By comparison with 3-hydroxypicolinic acid the results with picolinic acid are notably superior for all oligonucleotides.

Base Sequence↗
PubMed1994

Detection of 500-nucleotide DNA by laser desorption mass spectrometry.

We report the first detection of DNA segments as large as 500 nucleotides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, using picolinic acid and 3-hydroxypicolinic acid mixtures as desorption matrices. The successful detection of 500-nucleotide DNA indicates that laser mass spectrometry is now emerging as a new biotechnology tool for DNA-related research. It should be possible to utilize fast detection of large DNA segments by laser mass spectrometry for rapid human genome sequencing.

Base Sequence↗
PubMed1994

3-Aminopicolinic acid as a matrix for laser desorption mass spectrometry of biopolymers.

3-Aminopicolinic acid (3-APA) was tested and found to be a useful matrix for matrix-assisted laser desorption/ionization of DNA and protein. Single-stranded DNA segments of 150-mer and double-stranded DNA of 246 base pairs were successfully detected by using 3-APA as an ultraviolet-absorbing matrix in a linear time-of-flight mass spectrometer. In the case of the double-stranded DNA, only parent ions corresponding to single-stranded DNA were observed. The comparison with 3-hydroxypicolinic acid and picolinic acid matrices is discussed.

Base Sequence↗
PubMed1994

Glucocorticoid activation of chromogranin A gene expression. Identification and characterization of a novel glucocorticoid response element.

Glucocorticoids regulate catecholamine biosynthesis and storage at several sites. Chromogranin A, an abundant protein complexed with catecholamines in secretory vesicles of chromaffin cells and sympathetic axons, is also augmented by glucocorticoids. This study reports isolation of the rat chromogranin A promoter to elucidate transcriptional regulation of chromogranin A biosynthesis by glucocorticoids in neuroendocrine cells. Endogenous chromogranin A gene expression was activated up to 3.5-fold in chromaffin cells by glucocorticoid, in time-dependent fashion. Inhibition of new protein synthesis by cycloheximide did not alter the rise in chromogranin A mRNA, suggesting that glucocorticoids directly activate the chromogranin A promoter; nuclear runoff assays confirmed a 3.3-fold increased rate of initiation of new chromogranin A transcripts after glucocorticoid. Transfected rat chromogranin A promoter/luciferase reporter constructs were activated 2.6-3.1-fold by glucocorticoid, and selective agonist/antagonist studies determined that dexamethasone effects were mediated by glucocorticoid receptors. Both rat and mouse chromogranin A promoter/luciferase reporter constructs were activated by glucocorticoid. A series of promoter deletions narrowed the region of glucocorticoid action to a 93-bp section of the promoter, from position -526 to -619 bp upstream of the cap site. A 15-bp sequence ([-583 bp] 5'-ACATGAGTGTGTCCT-3' [-597 bp]) within this region showed partial homology to a glucocorticoid response element (GRE; half-site in italics) consensus sequence, and several lines of experimental evidence confirmed its function as a GRE: (a) site-directed mutation of this GRE prevented glucocorticoid activation of a chromogranin A promoter/reporter; (b) transfer of this GRE to a heterologous (thymidine kinase) promoter/reporter conferred activation by glucocorticoid, in copy number-dependent and orientation-independent fashion; and (c) electrophoretic gel mobility shifts demonstrated binding of this GRE by ligand-activated glucocorticoid receptor, though at 2.75-fold lower affinity than the glucocorticoid receptor interaction with a consensus GRE. The rat chromogranin A GRE showed functional and structural similarities to GREs in other genes proportionally regulated by glucocorticoids. We conclude that a discrete domain of the chromogranin A promoter is both necessary and sufficient to confer glucocorticoid regulation onto the gene, and that the activity of this region also explains the degree of activation of the endogenous gene by glucocorticoid.

Animals↗
PubMed1994

The impact of health care financing on family budgets.

Although businesses, federal and state governments, and insurance companies are major funding sources for health care, they are just intermediate sources. Ultimately, individuals and families pay all health care costs through out-of-pocket spending, insurance premiums, or federal, state, and local taxes. Using a microsimulation model with data from the 1987 National Medical Expenditure Survey, the Internal Revenue Service's Individual Tax Model, and the Consumer Expenditure Survey, the authors examine the distribution of health care spending, by decile, among families and individuals. They find that the distribution of health expenditures is very regressive, with low-income families paying twice the share of income paid by high-income families. The distribution of out-of-pocket expenditures, which comprise 24 percent of total spending, is the most regressive, with low-income families paying 8.5 times the share of income paid by high-income families. Spending on premiums is also regressive, and the regressivity would increase if everyone had private insurance. Expenditures through the public sector are progressive. Regressivity is greater among the elderly than the nonelderly. Out-of-pocket expenditures account for 41 percent of all health care spending by the elderly. A more equitably financed health care system would increase the share of funding raised through progressive taxes, and decrease reliance on expenditures made out of pocket and on premiums.

Adolescent↗
PubMed1994

Neuroendocrine and reproductive consequences of overexpression of growth hormone in transgenic mice.

Availability of recombinant growth hormone (GH) and development of long-acting formulations of this material will undoubtedly lead to widespread use of GH in animal industry and in medicine. GH can act, directly or indirectly, on multiple targets, but its influence on the reproductive system and on the hormonal control of reproduction is poorly understood. Overexpression of GH genes in transgenic animals provides a unique opportunity to study the effects of long-term GH excess. Transgenic mice overexpressing bovine, ovine, or rat GH (hormones with actions closely resembling, if not identical to, those of endogenous [mouse] GH), exhibit enhancement of growth, increased adult body size, and reduced life-span as well as a number of endocrine and reproductive abnormalities. Ectopic overexpression of bovine GH (bGH) driven by metallothionein or phosphoenolpyruvate carboxykinase promoters is associated with altered activity of hypothalamic neurons which produce somatostatin, loss of adenohypophyseal GH releasing hormone (GHRH) receptors, and suppression of endogenous (mouse) GH release. Elevation of plasma levels of GH (primarily bGH) and insulin-like growth factor (IGF-I) in these transgenic mice leads to increases in the number of hepatic GH and prolactin (PRL) receptors, in the serum levels of GH-binding protein (GHBP), in the percent of GHBP complexed with GH, and in the circulating insulin levels. In addition, plasma adrenocorticotropic hormone (ACTH) and corticosterone levels are elevated. Plasma levels of luteinizing hormone (LH), as well as its synthesis and release, are not consistently affected, but follicle-stimulating hormone (FSH) levels are suppressed, apparently due to pre- and post-translational effects. Pituitary lactotrophs exhibit characteristics of chronic enhancement of secretory activity, and plasma PRL levels are elevated. Prolactin responses to mating or to pharmacological blockade of dopamine synthesis are abnormal. Reproductive life span and efficiency are reduced in both sexes, with the severity and frequency of reproductive deficits being related to plasma bGH levels. Most transgenic females expressing high levels of bGH are sterile due to luteal failure. Overexpression of human GH which, in the mouse, interacts with both GH and PRL receptors leads to additional endocrine and reproductive abnormalities including stimulation of LH beta mRNA levels and LH secretion, loss of responsiveness to testosterone feedback, overstimulation of mammary glands, enhanced mammary tumorigenesis, and hypertrophy of accessory reproductive glands in males.

Animals↗
PubMed1994

Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung.

Adenoviral vectors have an important role as in vivo gene delivery vehicles in developing human gene therapy for the fatal pulmonary component of cystic fibrosis. In this study we evaluated the immune responses to wild-type adenovirus and replication-deficient, first generation adenoviral (Av1) vectors in the cotton rat (Sigmodon hispidus) and then quantitatively evaluated the efficiency of gene delivery and expression of single and repeated in vivo administration of Av1 vectors to the respiratory tract. Av1 vector reporter gene expression was quantitatively evaluated by employing a luciferase expression vector (Av1Luc1) and measuring luciferase activity in whole lung tissue homogenates by routine luminometry. Gene transfer and expression in naive animals (e.g. first Av1 vector dose) was efficient. A repeat dose also resulted in successful gene transfer and expression, although at a significantly reduced level (p < 0.01) compared with naive animals. This reduction inversely correlated with serum human adenovirus neutralizing antibody (HANA) titers. Importantly, increasing doses of Av1Cf2, an Av1 vector expressing the human CFTR cDNA, resulted in a graded HANA response consistent with a lack of in vivo replication. These observations have significant implications for repeated administration of adenoviral vectors to the lungs of individuals with cystic fibrosis.

Adenoviruses, Human↗
PubMed1993

Brefeldin-A inhibits the delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells.

We have studied the effects of brefeldin A (BFA) on the polarized delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells. Unlike the delivery of several other basolateral membrane and secretory proteins, the delivery of the polymeric immunoglobulin receptor from the trans-Golgi network to the cell surface was inhibited by BFA. The effect of BFA treatment was apparent at 1.0 microgram/ml (36% inhibition), and maximal inhibition was observed at 10 micrograms/ml (70% inhibition). The delivery of the receptor from the endoplasmic reticulum to the basolateral surface was even more sensitive to the effect of BFA; delivery was inhibited 95% in cells treated with 1 micrograms/ml BFA. The selective action of BFA on the basolateral delivery of the polymeric immunoglobulin receptor suggests that there may be multiple pathways for delivery of proteins to the basolateral cell surface of MDCK cells.

Animals↗
PubMed1993

DIDS inhibition of deformation-induced cation flux in human erythrocytes.

The permeability of human erythrocytes to sodium, potassium and calcium increases when the cells are deformed by shear. We now report that the anion-exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) inhibited 55-60% of the deformation-induced flux with an apparent K1/2 of 1 microM. Covalently bound DIDS was also effective. In cells partially derivatized at 0 degrees C (pH 7.4), anion exchange and the deformation flux were inhibited in parallel, implying that lysine a is the site of inhibition for both fluxes. Ektacytometry showed that DIDS does not inhibit by lowering the cell's ability to deform. Crosslinking of lysines in Band 3 was not required for inhibition of the stress flux, as demonstrated by electrophoretic analysis of chymotrypsin-cleaved Band 3 after DIDS treatment. Chymotrypsin cleavage itself did not affect the cation flux rates. DNDS, an anion exchange inhibitor that binds to the chloride site on Band 3 but is unable to derivatize lysine a, is an ineffective inhibitor of the deformation flux. Other high-affinity inhibitors of anion exchange were also relatively ineffective against the deformation flux, and anion exchange itself was unchanged by shear. These results suggest that 55-60% of the deformation-induced cation movement traverses a route that includes Band 3, but is distinct from the pathway utilized by anion exchange. Chloride-dependent cation pathways do not participate in the stress induced cation flux, since complete exchange of intracellular chloride for sulfate had no effect on the rates. Deformation of erythrocytes by laminar shear appears to increase the non-specific cation permeability.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗