• Horticultural Science & Technology
• /
• v.33 no.1
• /
• pp.83-92
• /
• 2015

xBrassicoraphanus line BB#5, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L. var. rafiphera induced by N-methyl-N-nitroso-urethane mutagenesis in microspore culture, shows high seed fertility and morphological uniformity. Dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes and genomic in situ hybridization (GISH) using B. rapa genomic DNA probe were carried out to analyze the chromosome composition and the meiosis pairing pattern compared to its parental lines. The somatic chromosome complement is 2n = 38, which consists of 17 metacentric and two submetacentric chromosomes with lengths of 2.18 to $5.01{\mu}m$. FISH karyotype analysis showed five and eight pairs of 5S and 45S rDNA loci. GISH meiosis pairing analysis showed that 19 complete bivalents were most frequent and accounted for 42% of the 100 pollen mother cells examined. Based on chromosome number, size, morphology, rDNA distribution, and meiosis pairing pattern, both parental genomes of B. rapa and R. sativus appear to exist in xBrassicoraphanus line BB#5, demonstrating its genome integrity. Such stable chromosome constitutions and meiotic pairing patterns in somatic and meiotic cells are very rare in natural and synthetic intergeneric hybrids. Chromosomal studies and genetic and phenotypic changes in allopolyploids a re discussed. The results p resented h erein will b e usef ul f or f urther g enomic s tudy o f xBrassicoraphanus lines and their improvement as promising new breeding varieties.

• ALGAE
• /
• v.37 no.1
• /
• pp.75-84
• /
• 2022

Intercellular nutrient and signal transduction are essential to sustaining multicellular organisms and maximizing the benefits of multicellularity. It has long been believed that red algal intercellular transport of macromolecules is prevented by the protein-rich pit plug within pit-connections, the only physical connection between cells. Fluorescein isothiocyanate-dextran and recombinant green fluorescence protein (rGFP) of various molecular sizes were injected into vegetative cells of Griffithsia monilis using a micromanipulator, and intercellular transport of the fluorescent probes was examined. Pit-connections were found to provide intercellular transport of tracers at rates comparable to plasmodesmata in other organisms. The time necessary for the transport to an adjacent cell was dependent on the molecular size and the direction of the transport. Fluorescent dextran of 3 kDa was transported to adjacent cells in 1-2 h after injection and migrated to all cells of the filament within 24 h, but fluorescent dextran of 10-20 kDa took 24 h to transfer to neighboring cells. The migration occurred faster towards adjacent reproductive cells and to apical cells than basally. Fluorescent tracers above 40 kDa and rGFP was not transported to neighboring cells, but accumulated near the pit plug. Our results suggest that pit-connections are conduit for macromolecules between neighboring cells and that these size-specific conduits allow intercellular communication between the vegetative cells of red algae.

• Animal Bioscience
• /
• v.36 no.5
• /
• pp.704-709
• /
• 2023

Objective: In tropical, subtropical and arid zones, heat stress is the main cause of productivity reduction in cattle. When climate stressors occur, animals become thermal adapted through differential expression of some genes, including heat shock proteins (HSP) family. The aim of this study was to determine levels of expression of HSP60, HSP70, and HSP90 genes in Simmental cattle raised in tropical environments of Mexico. Methods: In this study, expression of HSP60, HSP70, and HSP90 genes was analyzed in 116 Simmental cattle from three farms with tropical climate located in western Mexico. Animals were sampled twice a day, in the morning and noon. Gene expression was evaluated by quantitative polymerase chain reaction using probes marked with fluorescence. The MIXED procedure of SAS with repeated measures was used for all statistical analysis. Results: HSP60 gene expression differences were found for sex (p = 0.0349). HSP70 gene differences were detected for sampling hour (p = 0.0042), farm (p<0.0001), sex (p = 0.0476), and the interaction sampling hour×farm (p = 0.0002). Gene expression differences for HSP90 were observed for farm (p<0.0001) and year (p = 0.0521). HSP70 gene showed to be a better marker of heat stress than HSP60 and HSP90 genes. Conclusion: Expression of HSP70 gene in Simmental herds of the tropical region of western México was different during early morning and noon, but the expression of the HSP60 and HSP90 genes was similar. Identification of resilient animals to heat stress will be useful in the genetic improvement of the Simmental breed.

• Journal of Radiation Protection and Research
• /
• v.25 no.4
• /
• pp.223-232
• /
• 2000

Comparative study was performed for the assessment of DNA damage and Chromosomal aberration in human lymphocyte exposed to low dose radiation using fluorescence in situ hybridization(FISH) and single cell gel electrophoresis(SCGE). Chromosomal aberrations in human lymphocytes exposed to radiation at doses of 5, 10, 30 and 50cGy were analysed with whole chromosome-specific probes by human chromosome 1, 2 and 4 according to PAINT system. FISH with chromosome-specific probe has been used to be a valid and rapid method fer detection of chromosome rearrangements induced by low dose radiation. The frequencies of stable translocation per cell equivalents were 0.0116, 0.0375, 0.040f, 0.0727 and 0.0814 for 0, 5, 10, 30 and 50cGy, respectively, and those of dicentric were 0.00, 0.0125, 0.174, 0.0291 and 0.0407 respectively. Radiation induced DNA damage in human lymphocyte in a dose-dependent manner at low doses from 5cGy to 50cGy, which were analysed by single tell gel electrophoresis(SCGE). From above results, FISH seemed to be useful for radiation biodosimetry by which the frequencies of stable aberrations in human lymphocyte can be observed more easily than by conventional method and SCGE also seemed to be sensitive method f9r detecting DNA damage by low dose radiation exposure, so that those methods will improve our technique to perform meaningful biodosimetry for radiation at low doses.

• Journal of Radiopharmaceuticals and Molecular Probes
• /
• v.9 no.1
• /
• pp.9-16
• /
• 2023

Liposomes as drug delivery system have proved useful carrier for various disease, including cancer. In addition, perfluorocarbon cored microbubbles are utilized in conjunction with high-intensity focused-ultrasound (HIFU) to enable simultaneous diagnosis and treatment. However, microbubbles generally exhibit lower drug loading efficiency, so the need for the development of a novel liposome-based drug delivery material that can efficiently load and deliver drugs to targeted areas via HIFU. This study aims to develop a liposome-based drug delivery material by introducing a substance that can burst liposomes using ultrasound energy and confirm the ability to target tumors using PET imaging. Liposomes (Lipo-DOX, Lipo-DOX-Au, Lipo-DOX-Au-RGD) were synthesized with gold nanoparticles using an avidin-biotin bond, and doxorubicin was mounted inside by pH gradient method. The size distribution was measured by DLS, and encapsulation efficiency of doxorubicin was analyzed by UV-vis spectrometer. The target specificity and cytotoxicity of liposomes were assessed in vitro by glioblastoma U87mg cells to HIFU treatment and analyzed using CCK-8 assay, and fluorescence microscopy at 6-hour intervals for up to 24 hours. For the in vivo study, U87mg model mouse were injected intravenously with 1.48 MBq of ⁶⁴Cu-labeled Lipo-DOX-Au and Lipo-DOX-Au-RGD, and PET images were taken at 0, 2, 4, 8, and 24 hours. As a result, the size of liposomes was 108.3 ± 5.0 nm at Lipo-DOX-Au and 94.1 ± 12.2 nm at Lipo-DOX-Au-RGD, and it was observed that doxorubicin was mounted inside the liposome up to 52%. After 6 hours of HIFU treatment, the viability of U87mg cells treated with Lipo-DOX-Au decreased by around 20% compared to Lipo-DOX, and Lipo-DOX-Au-RGD had a higher uptake rate than Lipo-DOX. In vivo study using PET images, it was confirmed that ⁶⁴Cu-Lipo-DOX-Au-RGD was taken up into the tumor immediately after injection and maintained for up to 4 hours. In this study, drugs released from liposomes-gold nanoparticles via ultrasound and RGD targeting were confirmed by non-invasive imaging. In cell-level experiments, HIFU treatment of gold nanoparticle-coupled liposomes significantly decreased tumor survival, while RGD-liposomes exhibited high tumor targeting and rapid release in vivo imaging. It is expected that the combination of these models with ultrasound is served as an effective drug delivery material with therapeutic outcomes.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.11
• /
• pp.5391-5397
• /
• 2012

Background: Neuroblastoma (NB), like most human cancers, is characterized by genomic instability, manifested at the chromosomal level as allelic gain, loss or rearrangement. Genetics methods, as well as conventional and molecular cytogenetics may provide valuable clues for the identification of target loci and successful search for major genes in neuroblastoma. We aimed to investigate AURKA and MYCN gene rearrangements and the chromosomal aberrations (CAs) to determine the prognosis of neuroblastoma. Methods: We performed cytogenetic analysis by G-banding in 25 cases [11 girls (44%) and 14 boys (66%)] and in 25 controls. Fluorescence in situ hybridization (FISH) with AURKA and MYCN gene probes was also used on interphase nuclei to screen for alterations. Results: Some 18.4% of patient cells exhibited CAs., with a significant difference between patient and control groups in the frequencies (P<0.0001). Some 72% of the cells had structural aberrations, and only 28% had numerical chnages in patients. Structural aberrations consisted of deletions, translocations, breaks and fragility in various chromosomes, 84% and 52% of the patients having deletions and translocations, respectively. Among these expressed CAs, there was a higher frequency at 1q21, 1q32, 2q21, 2q31, 2p24, 4q31, 9q11, 9q22, 13q14, 14q11.2, 14q24, and 15q22 in patients. 32% of the patients had chromosome breaks, most frequently in chromosomes 1, 2, 3, 4, 5, 8, 9, 11, 12, 19 and X. The number of cells with breaks and the genomic damage frequencies were higher in patients (p<0.001). Aneuploidies in chromosomes X, 22, 3, 17 and 18 were most frequently observed. Numerical chromosome abnormalities were distinctive in 10.7% of sex chromosomes. Fragile sites were observed in 16% of our patients. Conclusion: Our data confirmed that there is a close correlation between amplification of the two genes, amplification of MYCN possibly contributing significantly to the oncogenic properties of AURKA. The high frequencies of chromosomal aberrations and amplifications of AURKA and MYCN genes indicate prognostic value in children with neuroblastomas and may point to contributing factors in their development.

• Archives of Pharmacal Research
• /
• v.20 no.1
• /
• pp.1-6
• /
• 1997

We determined the microviscosity of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex and liposomes of total lipids (SPMTL) and phospholipids (SPMPL) extracted from SPMV. Changes in the microviscosity induced by the range and rate of lateral diffusion were measured by the intramolecular excimerization of 1, 3-di(1-pyrenyl)propane (Py-3-Py). The microviscosity values of the direct probe environment in SPMV, SPMTL and SPMPL were 38.17, 31.11 and 27.64 cP, respectively, at$37^{\circ}C$and the activation energies $(E_a)$ of the excimer formation of Py-3-Py in SPMV, SPMTL and SPMPL were 8.236, 7.448 amd 7.025 kcal/mol, respectively. Probe location was measured by polarity and polarizability parameters of the probe Py-3-Py and probe analogues, pyrene, 1-pyrenenonanol and 1-pyrenemethyl-3${\beta}$-hydroxy-22, 23-bisnor-5-cholenate (PMC), incorporated into membranes or solubilized in reference solvents. There existed a good linear relationship between the first absorption peak of the $^1_a$ band and the polarizability parameter $(n^{2}-1)/(2n^{2}+1)$.The calculated refractive index values for SPMV, SPMTL and SPMPL were close to 1.50, which is higher than that of liquid paraffin (n=l.475). The probe location was also determined by using a polarity parameter $(f-1/2f^{I})$. Here f=$({\varepsilon}-1)/(2{\varepsilon}+1)$ is the dielectric constant function and $f^I=(n^2-1)/(2n^2+1)$ is the refractive index function. A correlation existed between the monomer fluorescence intensity ratio and the solvent polarity parameter. The probes incorporated in SPMV, SPMTL, and SPMPL report a polarity value close to that of 1-hexanol $({\varepsilon}=13.29)$. In conclusion, Py-3-Py is located completely inside the membrane, not in the very hydrophobic core, but displaced toward the polar head groups of phospholipid molecules, e.g., central methylene region of aliphatic chains of phospholipid molecules.

• Journal of Life Science
• /
• v.30 no.6
• /
• pp.563-569
• /
• 2020

Oncoplastic surgery (OPS) combines oncologically safe tumor resection with aesthetically satisfying reconstruction of defects using established plastic surgery techniques. OPS is characterized by initial excision as extensive as is beneficial for oncological safety, and, once sufficient resection is complete, displacement or replacement techniques are selected based on remnant volume. The size of the lesion and the individual patient are important factors when considering the appropriate approach, and when pre-operative imaging, including MRI, is used to determine the technique, the complete removal of cancer cells by permanent pathology is essential. A frozen section is used during the operation to reduce the reoperation rate, but it is difficult to cover the entire margin surface theoretically and even harder in practice. A recent report about adequate margins has empowered OPS in its oncological safety. Considering the patients to whom each modality could be applied, basic breast volume is an important factor, and this is influenced by ethnic differences. In Europe or the US, for example, the average breast size is 36D (600 ㎤) and reduction mammoplasty is predominantly used. However, the average size of patients in our institution is 33A (300 ㎤), and so quite different approaches are selected in most cases. New techniques involving radiofrequency and fluorescence have been proposed as safe and easily accessible ways of reducing complications.

• Journal of Ginseng Research
• /
• v.41 no.4
• /
• pp.469-476
• /
• 2017

Background: Panax ginseng Meyer (Asian ginseng) has a large nuclear genome size of > 3.5 Gbp in haploid genome equivalent of 24 chromosomes. Tandem repeats (TRs) occupy significant portions of the genome in many plants and are often found in specific genomic loci, making them a valuable molecular cytogenetic tool in discriminating chromosomes. In an effort to understand the P. ginseng genome structure, we characterized an ultrahigh copy 167-bp TR (Pg167TR) and explored its chromosomal distribution as well as its utility for chromosome identification. Methods: Polymerase chain reaction amplicons of Pg167TR were labeled, along with 5S and 45S rDNA amplicons, using a direct nick-translation method. Direct fluorescence in situ hybridization (FISH) was used to analyze the chromosomal distribution of Pg167TR. Results: Recently, we reported a method of karyotyping the 24 chromosome pairs of P. ginseng using rDNA and DAPI (4',6-diamidino-2-phenylindole) bands. Here, a unique distribution of Pg167TR in all 24 P. ginseng chromosomes was observed, allowing easy identification of individual homologous chromosomes. Additionally, direct labeling of 5S and 45S rDNA probes allowed the identification of two additional 5S rDNA loci not previously reported, enabling the refinement of the P. ginseng karyotype. Conclusion: Identification of individual P. ginseng chromosomes was achieved using Pg167TR-FISH. Chromosome identification is important in understanding the P. ginseng genome structure, and our method will be useful for future integration of genetic linkage maps and genome scaffold anchoring. Additionally, it is a good tool for comparative studies with related species in efforts to understand the evolution of P. ginseng.

• Tuberculosis and Respiratory Diseases
• /
• v.56 no.3
• /
• pp.261-268
• /
• 2004

Background : The resurgence of tuberculosis and outbreaks of multidrug resistant (MDR) tuberculosis have increased the emphasis for the development of new susceptibility testing of the Mycobacterium tuberculosis for the effective treatment and control of the disease. Conventional drug susceptibility testings, such as those using egg-based or agar-based media have some limits, such as the time required and difficulties in determining critical inhibitory concentrations, but these are still being used in many diagnostic laboratories because of no better lternatives, considering cost and accuracy. To overcome these limits, a rapid and simple method for new susceptibility testing, using live and dead assays, was applied for a bacterial cell viability assay to distinguish dead from live bacterial cells based on two-color fluorescence. Materials and Methods Strains : Forty strains were used in this study, 20 susceptible to all antituberculosis drugs and the other 20 resistant to the four first line antituberculosis drugs isoniazid, rifampicin, streptomycin and ethambutol. Antibiotics : The four antibiotics were dissolved in 7H9 broth to make the following solutions: $0.1{\mu}g\;isoniazid(INH)/m{\ell}$, $0.4{\mu}g\;rifampicin(RMP)/m{\ell}$, $4.0{\mu}g\;streptomycin(SM)/m{\ell}$ and $4.0{\mu}g\;ethambutol(EMB)/m{\ell}$. Results : Live and dead Mycobacterium tuberculosis cells fluoresced green and red with the acridin (Syto 9) and propidium treatments, respectively. These results are very well accorded with conventional drug susceptibility testing by proportional method on Lowensen-Jensen media (L-J) containing 4 drugs (INH, RMP, EMB and SM), showing a 93.7 % accordance rate in susceptible strains and 95% in resistant strains. Conclusion : The results of the drug susceptibility testing using the live and dead bacterial cell assay showed high accordance rates compared with the conventional proportion method on L-J. This finding suggests that the live and dead bacterial cell assay can be used as an alternative to conventional drug susceptibility testing for M. tuberculosis strains.