• Microbiology and Biotechnology Letters
• /
• v.33 no.4
• /
• pp.302-307
• /
• 2005

The inactivation efficiency of Campylobacter jejuni were assessed in vitro and in vivo using confocal laser microscopy and flow cytometry. C. jejuni cells were inactivated with $1\%$ (w/v) trisodium phosphate (TSP) and the live cells and inactivated cells were distinguished by staining with LIVE/DEAD BacLight Bacteria Viability fluorescent probe. After treatment of TSP for 5 min, most of C. jejuni cells turned to coccoid form from original spiral shape. C. jejuni cells lost total cell viability in the absence of organic nutrients but did not lost total cell viability in the presence of organic nutrients. In vivo test, C. jejuni cells turned to viable but non-culturable (VBNC) form after TSP treatment and remained alive on chicken skin. C. jejuni cells attached on chicken meat would transform to coccoid form by sanitizer treatment, but could possibly be alive by the benefits of organic nutrients present in chicken meat.

• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.5
• /
• pp.291-298
• /
• 2005

To characterize cytosolic $Ca^{2+}$ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to $200{\mu}M$ 2,4-dinitrophenol (DNP), and mitochondrial $Ca^{2+}$, mitochondrial membrane potential (${\Delta}{\Psi}m$), and cytosolic $Ca^{2+}$ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal $Na^+$/$Ca^{2+}$ exchange (NCX) in cytosolic $Ca^{2+}$ efflux was studied in KB-R7943 and $Na^+$-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by $70{\pm}10$% within $70{\pm}10$ s, and later by $400{\pm}200$% at $850{\pm}45$ s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) $Ca^{2+}$ was depleted by $1{\mu}M thapsigargin plus $10{\mu}M ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic $Ca^{2+}$ overload, while $Na^+$-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic $Ca^{2+}$ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive $Ca^{2+}$ release from SR; 3) NCX plays an important role in transient cytosolic $Ca^{2+}$ shifts under metabolic inhibition with DNP.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.26 no.1
• /
• pp.24-39
• /
• 2000

Various methods and graft materials have been used to fill in the defect adjacent to the implants and considered as clinically acceptable. But it is not clear whether the regenerated bone increases the implant-bone contact and supports the implant. The purpose of this study is to evaluate regenerated bone surrounding implants using bone morphogenetic protein(BMP) and demineralized freeze-dried bone(DFDB), and the interfaces between implants and regenerated bone. bBMP was extracted and partially purified from the bovine bone matrix using heparine chromatography. Demineralized freeze-dried bone was made from the dog. Inactive insoluble collagenous bone matrix(IBM) of dog was used as carrier of bBMP. Interfaces of titanium coated epoxy resin implants were processed for demineralized section for transmission electron microscopy(TEM) and those of screw type implants were for nondemineralized section for light and fluoromicroscopic examination. Implants were inserted in the inferior border of mandible of adult dogs and artificial bony defects($3{\times}3{\times}4mm$) were made at the mesial and distal side of implants. Defects were filled with BMP(BMP group) and DFDB(DFDB group). For the fluoromicroscopic examination, the fluorescent dyes(oxytetracycline, calcein green, alizarin red) were injected 2, 4, 6, 8, 12 weeks after implantation. The experimental animals were sacrificed at the 6th and the 12th week and their mandible were extirpated and processed for examination with light microscopy, fluoromicroscopy and TEM. The obtained results were as follows : 1. By the light microscopic findings, the defects were filled with woven bone at the 6th week and compact bone at the 12th week, and the osseointegrations were seen in both groups. There was no histological difference between them. 2. On the basis of the histomorphometric analysis, BMP group(6th week: 40.25%, 12th week: 56.04%) had higher bony contact ratio than DFDB group(38.37%, 42.63%). There was significant difference between two groups at the 12th week(p<0.05). 3. The amount of bone formation in BMP group was more prominent than in DFDB group. Significant difference was noted among two groups at the 6th and the 8th week(p<0.05). 4. By the transmission electron microscopic findings, $0.4-2{\mu}m$ soft tissue layer was found in adjacent to the interfaces and over the collagen fibrils of bone at the 6th week. However, about 100nm amorphous layer was noted at the interface or collagen fibrils directly extended to the titanium surface at the 12th week. There was no significant difference between two groups. 5. These results suggest that BMP and DFDB can be used as good graft materials in the regeneration of bone adjacent to implant, and BMP is more valuable as a bone inducer than DFDB.

• Journal of Ginseng Research
• /
• v.41 no.2
• /
• pp.169-179
• /
• 2017

Background: Ginsenosides are the main pharmacological components of Panax ginseng root, which are thought to be primarily responsible for the suppressing effect on oxidative stress. Methods: 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and oxygen radical absorption capacity were applied to evaluate the antioxidant activities of the ginsenosides. Human embryonic kidney 293 (HEK-293) cells were incubated with ginsenosides extracted by pulsed electric field (PEF) and solvent cold soak extraction (SCSE) for 24 h and then the injury was induced by $40{\mu}M$ $H_2O_2$. The cell viability and surface morphology of HEK-293 cells were studied using MTS assay and scanning electron microscopy, respectively. Dichloro-dihydro-fluorescein diacetate fluorescent probe assay was used to measure the level of intracellular reactive oxygen species. The intracellular antioxidant activities of ginsenosides were evaluated by cellular antioxidant activity assay in HepG2 cells. Results: The PEF extracts displayed the higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and stronger oxygen radical absorption capacity (with an oxygen radical absorption capacity value of $14.48{\pm}4.04{\mu}M\;TE\;per\;{\mu}g/mL$). The HEK-293 cell model also suggested that the protective effect of PEF extracts was dose-dependently greater than SCSE extracts. Dichloro-dihydro-fluorescein diacetate assay further proved that PEF extracts are more active (8% higher than SCSE extracts) in reducing intracellular reactive oxygen species accumulation. In addition, scanning electron microscopy images showed that the HEK-293 cells, which were treated with PEF extracts, maintained more intact surface morphology. Cellular antioxidant activity values indicated that ginsenosides extracted by PEF had stronger cellular antioxidant activity than SCSE ginsenosides extracts. Conclusion: The present study demonstrated the antioxidative effect of ginsenosides extracted by PEF in vitro. Furthermore, rather than SCSE, PEF may be more useful as an alternative extraction technique for the extraction of ginsenosides with enhanced antioxidant activity.

• Korean Journal of Clinical Laboratory Science
• /
• v.51 no.2
• /
• pp.125-133
• /
• 2019

A microscope is the fundamental research and diagnostic apparatus for clinical investigation of signaling transduction, morphological changes and physiological tracking of cells and intact tissues from patients in the biomedical laboratory science. Proper use, care and maintenance of microscope with comprehensive understanding in mechanism are fully requested for reliable image data and accurate interpretation for diagnosis in the clinical laboratory. The standard operating procedure (SOP) for light microscopes includes performance procedure, brief information of all mechanical parts of microscopes with systematic troubleshooting mechanism depending on the laboratory capacity. Maintenance program encompasses cleaning objective, ocular lenses and inner optics; replacement and calibration of light source; XY sample stage management; point spread function (PSF) measurement for confocal laser scanning microscope (CLSM); quality control (QC) program in fluorescent microscopy; and systematic troubleshooting. Laser safety is one of the concern for medical technologists engaged in CLSM laboratory. Laser safety guideline based on the laser classification and risk level, and advisory lab wear for CLSM users are also expatiated in this overview. Since acquired image data presents a wide range of information at the moment of acquisition, well-maintained microscopes with proper microscopic maintenance program are impulsive for its interpretation and diagnosis in the clinical laboratory.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.9 no.2
• /
• pp.137-142
• /
• 2004

In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (CP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene of Streptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of $\mu$CP and cSA-fused proteins. is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications. one such being the use in protein-protein assays.

• Korean Journal of Materials Research
• /
• v.22 no.3
• /
• pp.140-144
• /
• 2012

Compared with bulk material, quantum dots have received increasing attention due to their fascinating physical properties, including optical and electronic properties, which are due to the quantum confinement effect. Especially, Luminescent CdSe quantum dots have been highly investigated due to their tunable size-dependent photoluminescence across the visible spectrum. They are of great interest for technical applications such as light-emitting devices, lasers, and fluorescent labels. In particular, quantum dot-based light-emitting diodes emit high luminance. Quantum dots have very high luminescence properties because of their absorption coefficient and quantum efficiency, which are higher than those of typical dyes. CdSe quantum dots were synthesized as a function of the synthesis time and synthesis temperature. The photoluminescence properties were found strongly to depend on the reaction time and the temperature due to the core size changing. It was also observed that the photoluminescence intensity is decreased with the synthesis time due to the temperature dependence of the band gap. The wavelength of the synthesized quantum dots was about 550-700 nm and the intensity of the photoluminescence increased about 22~70%. After the CdSe quantum dots were synthesized, the particles were found to have grown until reaching a saturated concentration as time increased. Red shift occurred because of the particle growth. The microstructure and phase developments were measured by transmission electron microscopy (TEM) and X-ray diffractometry (XRD), respectively.

• Parasites, Hosts and Diseases
• /
• v.45 no.1 s.141
• /
• pp.11-18
• /
• 2007

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within free-living amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multi-plied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Trasnmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.2
• /
• pp.144-151
• /
• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• Journal of Applied Biological Chemistry
• /
• v.54 no.2
• /
• pp.120-125
• /
• 2011

In order to exploit Methylobacterium oryzae CBMB20 as of plant growth promoting agent, different inoculation methods have been evaluated. The present study aimed to evaluate soil, foliar, and soil+foliar inoculations of M. oryzae CBMB20 to improve the growth, fruit yield, and nutrient uptake of red pepper (Capsicum annuum L.) under greenhouse conditions. The population range of green fluorescent protein (gfp)-tagged M. oryzae CBMB20 using the three inoculation methods was 2.5-2.9 ${\log}_{10}$ cfu/g in the rhizosphere and 4.5-6.0 ${\log}_{10}$ cfu/g in the phyllosphere of red pepper plants. Confocal laser scanning microscopy results confirmed the colonization of M. oryzae CBMB20 endophytically on leaf surface. Plant height, fruit dry weight, and total biomass were significantly higher ($p{\leq}0.05$) in all M. oryzae CBMB20 inoculation methods as compared to non-inoculated control. Furthermore, uptake of mineral nutrients such as N, P, K, Ca, and Mg in red pepper plants in all M. oryzae CBMB20 inoculation methods was higher than in non-inoculated control. Comparative results of inoculation methods clearly demonstrated that soil+foliar inoculation of M. oryzae CBMB20 lead to the highest biomass accumulation and nutrient uptake which may be due to its efficient colonization in the red pepper rhizosphere and phyllosphere.