Differential Dot-blot Hybridization Research Articles

Isolation and Characterization of a Defensin-Like Peptide (Coprisin) from the Dung Beetle,Copris tripartitus

The antibacterial activity of immune-related peptides, identified by a differential gene expression analysis, was investigated to suggest novel antibacterial peptides. A cDNA encoding a defensin-like peptide, Coprisin, was isolated from bacteria-immunized dung beetle, Copris tripartitus, by using differential dot blot hybridization. Northern blot analysis showed that Coprisin mRNA was up-regulated from 4 hours after bacteria injection and its expression level was reached a peak at 16 hours. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with a predicted molecular weight of 8.6 kDa and a pI of 8.7. The amino acid sequence of mature Coprisin was found to be 79.1% and 67.4% identical to those of defensin-like peptides of Anomala cuprea and Allomyrina dichotoma, respectively. We also investigated active sequences of Coprisin by using amino acid modification. The result showed that the 9-mer peptide, LLCIALRKK-NH2, exhibited potent antibacterial activities against Escherichia coli and Staphylococcus aureus.

Maternal nutrient restriction alters gene expression in the ovine fetal heart.

Adequate maternal nutrient supply is critical for normal fetal organogenesis. We previously demonstrated that a global 50% nutrient restriction during the first half of gestation causes compensatory growth of both the left and right ventricles of the fetal heart by day 78 of gestation. Thus, it was hypothesized that maternal nutrient restriction significantly altered gene expression in the fetal cardiac left ventricle (LV). Pregnant ewes were randomly grouped into control (100% national research council (NRC) requirements) or nutrient-restricted groups (50% NRC requirements) from day 28 to day 78 of gestation, at which time fetal LV were collected. Fetal LV mRNA was used to construct a suppression subtraction cDNA library from which 11 cDNA clones were found by differential dot blot hybridization and virtual Northern analysis to be up-regulated by maternal nutrient restriction: caveolin, stathmin, G-1 cyclin, alpha-actin, titin, cardiac ankyrin repeat protein (CARP), cardiac-specific RNA-helicase activated by MEF2C (CHAMP), endothelial and smooth muscle derived neuropilin (ESDN), prostatic binding protein, NADH dehydrogenase subunit 2, and an unknown protein. Six of these clones (cardiac alpha-actin, cyclin G1, stathmin, NADH dehydrogenase subunit 2, titin and prostatic binding protein) have been linked to cardiac hypertrophy in other species including humans. Of the remaining clones, caveolin, CARP and CHAMP have been shown to inhibit remodelling of hypertrophic tissue. Compensatory growth of fetal LV in response to maternal undernutrition is concluded to be associated with increased transcription of genes related to cardiac hypertrophy, compensatory growth or remodelling. Counter-regulatory gene transcription may be increased, in part, as a response to moderating the degree of cardiac remodelling. The short- and long-term consequences of these changes in fetal heart gene expression and induction of specific homeostatic mechanisms in response to maternal undernutrition remain to be determined.

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10 −7 to 1×10 −5 M, base substitution mutations at K- ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10 −8 M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua R) phenotypic mutation was also found in cells treated with 1×10 −7 and 1×10 −5 M of bisphenol A. The induction of K- ras codon 12 mutations and Oua R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10 −6 M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.

Detection of UV-induced K-ras codon 12 mutation by PCR and differential dot-blot hybridization in cells from Down syndrome and Cockayne syndrome.

By means of the polymerase chain reaction (PCR) and differential dot-blot hybridization, base substitution mutations of K-ras codon 12 were investigated in skin fibroblast cells from Down syndrome (DS) patients. Mutations were identified in DS cells after UV irradiation, predominantly in cells from younger patients. In contrast, no mutation was detected in cells from Cockayne syndrome (CS) patients who had the same features of premature aging as in DS but were not prone to cancer. This association of DS cells, but not CS cells, with inducibility of the K-ras codon 12 mutation may imply the proneness of DS patients to cancer development but a lack of proneness of CS patients.

Detection of serum factors enhancing cell mutability from lung cancer patients by application of hypermutable human RS cells.

A search for serum factors that modulate the mutability of human cells has been attempted in the peripheral blood of lung cancer patients. Factors were separated by dye-ligand chromatography and first identified as those exhibiting the ability to enhance the frequency of drug-resistance mutations in human RSa cells. The frequency was assessed by estimation of the cloning efficiency of mutant cells resistant to ouabain-mediated cell killing (OuaR) after irradiation with far-ultraviolet light (UV, mainly 254-nm wavelength). Pre-culture of cells with medium containing the factors prior to UV irradiation led to about a 19- to 37-fold increase in the OuaR mutation frequency compared with that of cells irradiated but not treated with the factors. The enhancing activity was detected in the serum of all 7 lung cancer patients, although the serum itself, which had not been treated with chromatography, had little or no enhancing activity in all patients. No enhancing activity was detected in serum preparations from healthy donors. The enhancing activity of lung cancer serum factors on UV-induced mutagenicity was next confirmed by detecting an enhancement of K-ras codon 12 base substitution mutations in human RSb cells, as analyzed by polymerase chain reaction (PCR) and differential dot-blot hybridization. Our results, together with previous findings on suppression of mutagen-induced mutagenicity by human interferons, suggest the existence of extracellular factors that modulate the mutability of human cells.

Mutagenicity of microcystin-LR in human RSa cells.

Microcystin-LR (MCLR) is a potent cyclic heptapeptidic hepatotoxin produced by bloom-forming cyanobacteria. The mutagenicity induced by this compound in cultured human RSa cells was found by determination of ouabain-resistant (OuaR) mutation, with the highest frequency at the concentration of 15 microgram/ml. Moreover, base substitution mutations at K-ras codon 12 in genomic DNA, assessed by polymerase chain reaction (PCR) and differential dot-blot hybridization using digoxigenin-labeled probes, were detected in RSa cells 6 days after exposure to MCLR (7.5-15 microgram/ml).

UV-induced mutations affecting codon 12 of the K- ras gene are suppressed by interferon-α in human RSa cells

K- ras gene sequences mutant at codon 12 were recovered following differential dot-blot hybridization of genomic DNA from human RSa cells up to 12 days after the cells had been irradiated with far-UV (principally 254 nm). By contrast, no mutant codon 12 sequences were recovered from cells which had been treated with 50 IU/ml human interferon (HuIFN)-α for 24 h prior to their UV exposure. HuIFN-α treatment in combination with anti-HuIFN-α antibody did not lead to the loss of mutant sequences. However, culture of interferon-pretreated cells with medium containing the protease inhibitor antipain (0.01 mM) for 6 h immediately after UV irradiation led to the recovery of mutant codon 12 sequences. Thus, while treatment with HuIFN-α appeared to prevent any UV-induced mutations affecting codon 12 of the K- ras gene from being recovered, the putative antipain-sensitive protease responsible for this suppressive affect appeared to be significantly affected by the protease inhibitor antipain.

Hypomutable potential accompanying interferon-alpha resistance in a human cell line, IFr, derived from interferon-alpha-sensitive and hypermutable RSa cells.

A human cell line, IFr, established from RSa cells, is a variant with increased resistance to cell proliferation inhibition (CPI) by human interferon (HuIFN)-alpha. The parent RSa cells are also hypermutable after irradiation with far-ultraviolet light (UV), as assessed by two different methods: cloning efficiency of ouabain-resistant (OuaR) mutants and K-ras codon 12 mutation in genomic DNA identified by polymerase chain reaction (PCR) following differential dot-blot hybridization. In the present study, IFr cells were found to be hypomutable: Less than 1 OuaR mutant per 10(4) surviving cells after UV (0-12 J/m2), in contrast to 1-53 OuaR mutants per 10(4) survivors in RSa cells, and no-detectable K-ras codon 12 mutation at any doses tested. However, IFr cells, when cultured with medium containing the protease inhibitor antipain after UV irradiation showed hypermutability to almost the same extent as RSa cells, as determined by both phenotypic and genetic mutation analyses. These results, together with the previous finding of antipain-sensitive protease induction in UV-irradiated or HuIFN-alpha-treated IFr cells, suggest that antipain-sensitive proteases or cellular functions or both may be involved in not only HuIFN-alpha resistance but also hypomutability of IFr cells.

Correlated susceptibility between interferon-beta and UV in human cell lines, F-IFr and RSa

The human cell line F-IFr is a variant with an increased resistance to cell proliferation inhibition (CPI) by human interferon (HuLFN)-beta, established from RSa cells with high sensitivity to CPI. The parent RSa cells were recently found to be unusually hypermutable after irradiation with far-ultraviolet light (UV), as assessed by two different methods; estimation of the cloning efficiency of ouabain-resistant (Oua(R)) mutants and detection of K-ras codon 12 mutation in genomic DNA identified by polymerase chain reaction following differential dot blot hybridization. In the present study, F-IFr cells were found to be hypomutable; less than one Oua(R) mutant per 10(4) survival cells after UV (0-12 J/m(2)), in contrast to 0.51-85 Oua(R) mutants per 10(4) survivors in RSa cells, and no detectable K-ras codon 12 mutation at any UV dose tested. However, F-IFr cells, when cultured with medium containing the protease inhibitor antipain immediately and transiently after UV irradiation displayed hypermutability to almost the same extent as RSa cells by both phenotypic and genetic mutation analyses. The refractoriness of F-IFr cells to HuIFN-beta CPI was also suppressed by culture with medium containing antipain during HuIFN-beta exposure. Moreover, F-IFr cells irradiated with UV or treated with HuIFN-beta showed elevation of antipain-sensitive protease activity, but not the irradiated or treated RSa cells. UV- and HuIFN-beta-susceptibility were not modulated by antipain in RSa cells. These antipodal characteristics between the two cell lines suggested that antipain-sensitive proteases and/or cellular functions may be involved in increased resistance of F-IFr cells to both Wand HuIFN-beta refractoriness.

Piperonyl butoxide mutagenicity in human RSa cells

Piperonyl butoxide (PB) is used as a pesticide synergist and food additive. Its chemically induced mutagenicity was found in cultured human RSa cells by determination of ouabain-resistant (Oua r) phenotypic mutation, with the highest frequency at the concentration of 0.2 μg/ml. Moreover, K- ras codon 12 mutations in genomic DNA, analyzed by polymerase chain reaction (PCR) and differential dot-blot hybridization using digoxigenin-labeled probes, were detected in RSa cells 6 days after exposure to PB (0.03–0.40 μg/ml).

Detection of K- ras Codon 12 Mutation by Polymerase Chain Reaction and Differential Dot-Blot Hybridization in Sodium Saccharin-Treated Human RSa Cell

Using polymerase chain reaction and differential dot-blot hybridization, point mutations of K- ras codon 12 were investigated in hypermutable human RSa cells treated with sodium saccharin. Cells carried mutational changes after 6 days of treatment at sodium saccharin concentrations of 15 to 30 mg/ml. Results imply that sodium saccharin is a mutagen which induces genetic mutation and, furthermore, that the assay system applied here will be beneficial in identifying mutagen-induced K- ras oncogene mutations in human cells in vitro.

Molecular cloning of gene sequences from rat heart rapidly responsive to pressure overload.

Although pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes, the molecular mechanism of both is not well understood. We demonstrated the early change in the translational activity of specific cardiac messenger RNA by two-dimensional gel electrophoresis of in vitro translational products. A total of over 400 translational products were detected by fluorography, and the relative predominance of eight species was increased by pressure overload whereas that of two translational products was decreased. We cloned four pressure-overload-responsive complementary DNA clones, pPO-1, -4, -5, and -6, by differential dot blot hybridization. Two clones were increased from the early period after the imposition of pressure overload; the other two were decreased. The expression pattern of each complementary DNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. Pressure overload also induced the additional messenger RNA, which hybridized with pPO-4. This mRNA was also recognized in fetal, neonatal, and adult spontaneously hypertensive rat hearts. The induction of this transcript by pressure overload was not suppressed but, rather, stimulated by cycloheximide. These results suggest that there are some genes that rapidly respond to pressure overload and that may play some role in the development of cardiac hypertrophy.

Molecular mechanism of cardiac hypertrophy.

Pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes. To ascertain the molecular mechanism of these events, we examined the expression of cellular oncogenes and the early change in the translational activity of specific cardiac mRNA by two-dimensional gel electrophoresis of in vitro translational products. Pressure overload increased the expression levels of c-fos, c-myc, and c-Ha-ras genes. The relative predominance of 8 species out of over 400 translational products was increased by pressure overload while that of 2 translational products was decreased. We cloned four pressure-overload-responsive cDNA clones by differential dot blot hybridization. The expression pattern of each cDNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocytes stretching stimulated amino acid uptake and expression of the c-fos gene, which was blocked by protein kinase C inhibitors. These results suggest that there are some early responsive genes in cardiac hypertrophy and that mechanical loading directly stimulates gene expression possibly via protein kinase C activation.