Detection of foot and mouth disease and porcine reproductive and respiratory syndrome viral genes using microarray chip.

Two viral pathogens, namely, porcine reproductive and respiratory syndrome virus (PRRSV) and foot and mouth disease virus (FMDV), were selected as models for multiple pathogen detection in a cDNA microarray. Two signature regions selected from ORF2 (around 500 bp) and ORF5 (around 600 bp) of PRRVS (America serotype), and one signature region from structural genes VP1 (around 500 bp) of FMDV type O were designed and spotted on a nylon membrane. For PCR sensitivity study, the cloned FMDV-VP1 template could be diluted to near one copy and its PCR product was still detectable in gel electrophoresis. In the microarray detection, the labelling FMDV probes (3 mg/ml) could be diluted 320 times and still maintained a visible colour when hybridized with the chip. Using the mixing primers, the microarray chip demonstrated rapid and accurate detection of the specific genes. To our knowledge, this preliminary study is the first example reported applying the long signature sequences to the multiple pathogen detection in cDNA microarray.

Genotoxicity evaluation of electromagnetic fields generated by 835-MHz mobile phone frequency band.

It is still unclear whether the exposure to electromagnetic fields (EMFs) generated by mobile phone radiation is directly linked to cancer. We examined the biological effects of an EMF at 835 MHz, the most widely used communication frequency band in Korean CDMA mobile phone networks, on bacterial reverse mutation (Ames assay) and DNA stability (in vitro DNA degradation). In the Ames assay, tester strains alone or combined with positive mutagen were applied in an artificial mobile phone frequency EMF generator with continuous waveform at a specific absorption rate (SAR) of 4 W/kg for 48 h. In the presence of the 835-MHz EMF radiation, incubation with positive mutagen 4-nitroquinoline-1-oxide and cumene hydroxide further increased the mutation rate in Escherichia coli WP2 and TA102, respectively, while the contrary results in Salmonella typhimurium TA98 and TA1535 treated with 4-nitroquinoline-1-oxide and sodium azide, respectively, were shown as antimutagenic. However, these mutagenic or co-mutagenic effects of 835-MHz radiation were not significantly repeated in other relevant strains with same mutation type. In the DNA degradation test, the exposure to 835-MHz EMF did not change the rate of degradation observed using plasmid pBluescript SK(+) as an indicator. Thus, we suggest that 835-MHz EMF under the conditions of our study neither affected the reverse mutation frequency nor accelerated DNA degradation in vitro.

Protection of tea melanin on hydrazine-induced liver injury.

The protective activity of melanin derived from tea (MDFT) was studied using hydrazine as a DNA-reactive chemical agent. Intra-peritoneal administration of MDFT at the doses of 5 or 20 mg/kg dose-dependently prevented liver toxicity induced by hydrazine in rats. It normalized rises in serum alanine transferase activity and a decrease in the glutathione level in the liver. It also reduced the hepatic malondialdehyde concentration. Monitoring the intensity of chemiluminescence showed that MDFT could prevent the production of free radicals that are generated owing to metabolic transformation of hydrazine. It also prevented the formation 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adducts. The results obtained in vivo and in vitro suggest that MDFT confers marked protection of the liver against hydrazine-induced oxidative toxicity.

Anatomical site-specific response to DNA damage is related to later tumor development in the rat azoxymethane colon carcinogenesis model.

There is now general agreement that the etiology of proximal and distal colon cancers may differ, thus prompting renewed interest in understanding anatomical site-specific molecular mechanisms of tumor development. Using a 2x2x2 factorial design with male Sprague-Dawley rats (corn oil, fish oil; pectin, cellulose; plus or minus azoxymethane injection) we found a greater than 2-fold difference (P < 0.001) in tumor incidence proximally versus distally (prox/dist ratio: corn oil, 2.25; fish oil, 2.61). The purpose of the present study was to determine if the higher degree of proximal versus distal tumors in our model system could be accounted for by differences between these two sites in initial DNA damage, response to that damage or an effect of diet at one site but not the other. DNA damage was assessed by quantitative immunohistochemistry of O(6)-methylguanine adducts; repair by measurement of O(6)-methylguanine-DNA alkyltransferase and removal was determined by measurement of targeted apoptosis. Although overall initial DNA damage was similar at both sites, in the distal colon there was a greater expression of repair protein (P < 0.001) and a greater degree of targeted apoptosis (P < 0.0001). There was also a reduction in DNA damage in the distal colon of rats consuming fish oil. Together, these results suggest that the lower tumor incidence in the distal colon may be a result of the capacity to deal with initial DNA damage by the distal colon, as compared with the proximal colon. Therefore, the determination of site-specific mechanisms in tumor development is important because distinct strategies may be required to protect against cancer at different sites.

Controlling self-incompatibility by CO2 gas treatment in Brassica campestris: structural alteration of papillae cell and differential gene expression by increased CO2 gas.

Self-imcompatibility is a controlling genetic mechanism to prevent self-pollination for Chinese cabbage (Brassica campestris), one of the major vegetable crops in Korea. To maintain inbred lines of the crop plant, a method in that high CO2 gas is treated to the pistils to overcome the self-incompatibility and thereby self-pollens can successfully make germination and fertilization has been widely used in seed companies. Despite the common utilization of this method, any molecular and cellular studies on how the self-incompatibility is removed from the Chinese cabbage plant have not been done. In this study, we show that the increased CO2 gas causes a structural alteration of the papillae cell and thereby the self-incompatibility is removed from the Chinese cabbage plant, allowing the self-pollens to germinate and penetrate the papillae cell. Also, gene expression in the pistil treated with CO2 gas was studied by DD/RT-PCR and reverse northern hybridization experiments. The results suggest that the failure in self-incompatible reaction resulted not only from the structural alteration of the papillae cell but also from change in the pistil component production that is either positively or negatively regulated by the environmental stimulation.

Differential gene expression of livers from ApoE deficient mice.

A genomic survey for differentially expressed genes was performed to livers of ApoE deficient mice using human cDNA microarray containing approximately 9,000 human cDNA clones. Due to the homology between mouse and human, hybridization was performed at lower stringency condition, 10 degrees below the regular hybridizing temperature. Gene expression profiles of livers corresponding to high levels of blood cholesterol were generated at genomic scale. Thirty-seven genes were randomly selected from a pool of differentially expressed genes and subjected to semi-quantitative RT-PCR, further confirmed the result from microarray hybridization. These included genes associated with atherosclerosis, and novel genes that implied novel pathways correlated to high levels of blood cholesterol. It is promising using human cDNA microarray, the most complete collection among all species, to study other mammalian systems with satisfying speed and accuracy.

Functionalization of a poly(amidoamine) dendrimer with ferrocenyls and its application to the construction of a reagentless enzyme electrode.

Poly(amidoamine) dendrimers having various degrees of modification with the redox-active ferrocenyls were prepared by controlling the molar ratio of ferrocenecarboxaldehyde to amine groups of dendrimers. By alternate layer-by-layer depositions of partial ferrocenyl-tethered dendrimers (Fc-D) with periodate-oxidized glucose oxidase (GOx) on a Au surface, an electrochemically and enzymatically active multilayered assembly of enzyme was constructed. The resulting GOx/Fc-D multilayer-associated electrodes were electrochemically analyzed, and the surface concentration of ferrocenyl groups, active enzyme coverage, and sensitivity were estimated. A 32% dendrimer modification level of surface amines to ferrocenyls was found to be an optimum in terms of enzyme-dendrimer network formation, electrochemical interconnectivity of ferrocenyls, and electrode sensitivity. With the prepared Fc(32%)-tethered dendrimers, mono- and multilayered GOx/Fc-D electrodes were constructed, and their electrochemical and catalytic properties were characterized. The bioelectrocatalytic signals from the multilayered GOx/Fc-D electrodes were shown to be directly correlated to the number of deposited bilayers. From this result, it seems that the electrode sensitivity is directly controllable, and the multilayer-forming strategy with partial ferrocenyl-tethered dendrimers is useful for the construction of reagentless biosensors.

Dietary fish oil reduces O6-methylguanine DNA adduct levels in rat colon in part by increasing apoptosis during tumor initiation.

There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode.

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.

Isolation and characterization of a class I SLG gene from Chinese cabbage (Brassica campestris).

In Brassica species, self-incompatibility in the recognition reaction between self and non-self pollens is determined by two genes, SLG and SRK, at the S locus. We have cloned and characterized a genomic DNA fragment containing a complete open reading frame of the SLG gene from Chinese cabbage. The genomic clone, named BcSLG2, was found to possess the region that shares a homology of 77% in amino acid identity with the SLG46 gene of Brassica campestris. Northern blot analysis revealed that the BcSLG2 gene expression is restricted to the pistil of Chinese cabbage flower. In situ hybridization showed that in the pistil, the gene is expressed predominantly in the stigmatic tissue. Much lower expression in the tapetum was also detectable at an immature stage of the flower development. Southern blot hybridization with the BcSLG2 DNA probe showed polymorphism in the SLG gene organization of the Chinese cabbage plants. These results will provide valuable information in understanding the S gene complex of the Chinese cabbage plants.

Relationship between DNA adduct levels, repair enzyme, and apoptosis as a function of DNA methylation by azoxymethane.

DNA alkylating agent exposure results in the formation of a number of DNA adducts, with O6-methyl-deoxyguanosine (O6-medG) being the major mutagenic and cytotoxic DNA lesion. Critical to the prevention of colon cancer is the removal of O6-medG DNA adducts, either through repair, for example, by O6-alkylguanine-DNA alkyltransferase (ATase) or targeted apoptosis. We report how rat colonocytes respond to administration of azoxymethane (a well-characterized experimental colon carcinogen and DNA-methylating agent) in terms of O6-medG DNA adduct formation and adduct removal by ATase and apoptosis. Our results are: (a) DNA damage is greater in actively proliferating cells than in the differentiated cell compartment; (b) expression of the DNA repair enzyme ATase was not targeted to the proliferating cells or stem cells but rather is confined primarily to the upper portion of the crypt; (c) apoptosis is primarily targeted to the stem cell and proliferative compartments; and (d) the increase in DNA repair enzyme expression over time in the bottom one-third of the crypt corresponds with the decrease in apoptosis in this same crypt region.

Ectopic expression of tobacco MADS genes modulates flowering time and plant architecture.

MADS genes encode regulatory factors that are involved in various developmental steps of the plant life cycle. During flower development, they regulate the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here, we report the isolation of two cDNA clones, NsMADS2 and NsMADS3, from a long-day tobacco species, Nicotiana sylvestris, which encode MADS domain-containing proteins. The NsMADS2 amino acid sequence showed 66% identity to SQUA and 64% to AP1. NsMADS3 showed a high degree of amino acid identity with FBP2 (98%), DEFH 72 (89%), DEFH 200 (88%), and AGL9 (76%). RNA blot analyses of NsMADS2 and NsMADS3 revealed that both transcripts were present in floral organs, but not in vegetative organs such as the leaf, root, stem, and 10 d old seedlings. The NsMADS2 transcript was localized in all four whorls and the NsMADS3 transcript was restricted in the three inner whorls of floral organs. The ectopic expression of NsMADS2 using the CaMV 35S promoter caused early flowering and lengthened internode length in transgenic tobacco plants. The ectopic expression of NsMADS3 also caused early flowering phenotype in transgenic tobacco plants, but the plants exhibited reduced apical dominance. Possible implications of these results in relation to the functions of NsMADS2 and NsMADS3 are discussed.

Relationship among colonocyte proliferation, differentiation, and apoptosis as a function of diet and carcinogen.

To determine the relationship among colonocyte proliferation, differentiation, and apoptosis as a function of fiber and carcinogen, we conducted a 2 x 2 factorial design study with two fibers (pectin or cellulose) and two injection protocols (azoxymethane or saline) in male Sprague-Dawley rats. Rats were killed six weeks after the injections, and in vivo cell proliferation was measured by incorporation of bromodeoxyuridine into DNA, differentiation by binding of the lectin Dolichos biflorus agglutinin, and apoptosis by immunoperoxidase detection of digoxigenin-labeled genomic DNA. In the proximal colon, pectin decreased differentiation and apoptosis, resulting in a greater number of cells per crypt column. In the distal colon, pectin increased cell proliferation, resulting in more crypts per millimeter of colon and a greater number of surface cells. Azoxymethane increased cell proliferation and decreased differentiation and apoptosis in the proximal and the distal colon. This resulted in a greater number of surface cells proximally and more crypts per millimeter of colon distally. These results illustrate the importance of considering all three parameters (proliferation, differentiation, and apoptosis) when evaluating neoplastic growth.

[Relapsing polychondritis: report of a case and review of the literature].

Relapsing polychondritis (RP) is a rare disease of unknown etiology, characterized by episodic and progressive chondritis, ocular and audiovestibular involvement, and occasional cardiovascular abnormalities. The inflammation typically involves the cartilage of the ears, nose, trachea, larynx, ribs, joints, and Eustachian tubes. The major clinical features include auricular chondritis, arthritis, nasal chondritis, ocular inflammation, chondritis of the respiratory tract, audiovestibular damage, and cardiovascular disorders. RP occurs predominantly in Caucasians and is uncommon in Oriental patients. Two cases of RP have previously been reported in Taiwan. We report one Taiwanese patient who was diagnosed with RP according to the criteria proposed by Damiani and Levine. The patient has bilateral auricular chondritis, episcleritis, and uveitis. Successful treatment was accomplished with systemic corticosteroid and topical corticosteroid eye drops. At an outpatient visit one year and ten months after discharge, there was no evidence of recurrence.

[Congenital nasopharyngeal teratoma: report of a case and review of the literature].

Teratomas are the most common congenital tumors, but teratomas of the nasopharynx are rare and seen almost exclusively in infants, usually in neonates. An unusual case of a neonate with respiratory distress is presented and a nasopharyngeal mass protruding into the oral cavity. After successful removal of the mass, pathological examination revealed a mature teratoma. The management and differential diagnosis are discussed, accompanied by a review of the literature.

Time- and spectral-domain evolution of subpicosecond pulses in semiconductor optical amplifiers.

The evolution of ultrashort pulses in semiconductor laser amplifiers is modeled, including the effects of gain dispersion, slow and fast carrier dynamics, and self-phase modulation, which includes an instantaneous response (n(2)). The temporal and spectral evolution of 2-ps and 500-fs pulses differ considerably. This is due mainly to saturation caused by the fast (heating) dynamics and to the phase modulation by n(2).

[Surgical treatment of diffuse scrotal epidermal cysts: case report].

Epidermal cysts are common, benign skin tumors that are found mainly on the face, neck, and trunk. This case was a 53-year-old male bus driver who had suffered from multiple papules on his scrotum for 7 years. This affliction had caused intractable itching and responded poorly to medical treatment. The patient received an excision of these papules (total 32) 3 years ago. According to the pathological reports, these papules were epidermal cysts; unfortunately, these papules recurred within 3 months after the operation and then resulted in itching more severe than before. The patient was again admitted to the hospital ward and received an operation in Oct. 1990. The diseased tissue (14cm x 8cm) was then excised and the defective scrotal skin was covered with a meshed, split-thickness skin graft. The cosmetic result was enhanced by the rugate appearance of the healed meshed graft. The patient was satisfied with both the functional and cosmetic results. The itching sensation on the scrotum improved and there was no recurrence of epidermal cysts within the graft area during the 9 month follow-up period. With this simple and safe operation, the result gave a satisfying improvement in both symptoms and appearance; so this case is offered for reference.