• Bulletin of the Korean Space Science Society
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• 한국우주과학회 1993년도 한국우주과학회보 제2권2호
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• pp.17-17
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• 1993

The wall shear stress in the vicinity of end-to end anastomoses under steady flow conditions was measured using a flush-mounted hot-film anemometer(FMHFA) probe. The experimental measurements were in good agreement with numerical results except in flow with low Reynolds numbers. The wall shear stress increased proximal to the anastomosis in flow from the Penrose tubing (simulating an artery) to the PTFE: graft. In flow from the PTFE graft to the Penrose tubing, low wall shear stress was observed distal to the anastomosis. Abnormal distributions of wall shear stress in the vicinity of the anastomosis, resulting from the compliance mismatch between the graft and the host artery, might be an important factor of ANFH formation and the graft failure. The present study suggests a correlation between regions of the low wall shear stress and the development of anastomotic neointimal fibrous hyperplasia(ANPH) in end-to-end anastomoses. 30523 T00401030523 ^x Air pressure decay(APD) rate and ultrafiltration rate(UFR) tests were performed on new and saline rinsed dialyzers as well as those roused in patients several times. C-DAK 4000 (Cordis Dow) and CF IS-11 (Baxter Travenol) reused dialyzers obtained from the dialysis clinic were used in the present study. The new dialyzers exhibited a relatively flat APD, whereas saline rinsed and reused dialyzers showed considerable amount of decay. C-DAH dialyzers had a larger APD(11.70${\pm}$1.32mmHg/min)compared to CF dialyzers(4.32${\pm}$0.55mmHg/min)(p<0.05). However, there was no observable difference in the UFR between the two dialyzers. Neither APD nor UFR showed any significant increase with an increasing number of reuses for up to more than 20reuses. A substantial number of failures observed in APD(larger than 20mmHe/min)on the reused dialyzers(2 out of 40 CP and S out 26 C-DAK) were attributed to the Possible damage on the fibers. The CF 15-11 HFDs which failed APD test did not show changes in the UFR compared to normal dialyzers indicating that APD is a more sensitive test than UFR test to evaluate the integrity of the fibers. 30527 T00401030527 ^x For quantitative measurement of reflected light from a clinical diagnostic strip, a prototype old reflectance photometer was designed. The strip loader and cassette were made to obtain more accurate reflectance parameters. The strip was illuminated at 45˚c through optical fiber and the intensity of reflected light was determined at rectanguLat angle using a photodiode. The kubelka-munk coefficient and reflection optical density were determined ar four different wavelengths(500,550,570 and 610nm) for blood glucose strip. For higher concentration than 300mg/41 about glucose, a saturation state of abforbance was observed at 500,550 and 570nm. The correlation between glucose concentration and parameters was the best at 610nm. 30535 T00401030535 ^x Radiation-induced fibrosarcoma tumors were grown on the flanks of C3H mice. The mice were divided into two groups. One group was injected with Photofrin II, intravenously (2.5mg/kg body weight). The other group received no Photofrin II. Mice from both groups were irradialed for approximately 15 minutes at 100,300, or 500 mW/cm2 with the argon (488nm/514.5 nm), dye(628nm) and gold vapor (pulsed 628 nm) laser light. A photosensitizer behaved as an added absorber. Under our experimental conditions, the presence of Photolfrin II increased surface temperature by at least 40% and the temperature rise due to 300 mW/cm2 irradiation exceeded values for hyperthermia. Light and temperature distributions with depth were estimated by a computer model. The model demonstrated the influence of wavelength on the thermal process and proved to be a valuable tool to investigate internal temperature rise. 30536 T00401030536 ^x We investigated the structural geometry of thirty-eight Korean femurs. The purpose of this study is to identify major geometrical differences between Korean femurs 3nd others that we believe belong to Caucasians so that we would be able to get insights into the femoral component design that fits Asians including Koreans. We utilized computerized tomography (CT) images of femurs extracted from cadavers. The CT images were transformed into bitmap data by using a film scanner, and then analyzed by using a commercially available software called Image v.1.0 and a Macintosh IIci computer.The resulting data were compared with already published data. The major results show that the geometry of the Korean femurs is significantly different from that of Caucasians: (1) the anteversion angle and the canal flare index are greater by the amount of approximately 8˚ and 0.5, respectively, (2) the shape of the isthmus cross section is more round, and (3) the distance between the teaser trochanter and the proximal border of the isthmus is shelter by about 15 mm. The results suggested that the femoral component suitable for Asians should be different from the currently-used components designed and manufactured mostly by European or American companies. 30537 T00401030537 ^x It is well known that nonlinear propagation characteristics of the wave in the tissue may give very useful information for the medical diagnoisis. In this paper, a new method to detect nonlinear propagation characteristics of the internal vibration in the tissue for the low frequency mechanical vibration by using bispectral analysis is proposed. In the method, low frequency vibration of f0( = 100Hz) is applied on the surface of the object, and the waveform of the internal vibration x (t) is measured from Doppler frequency modulation of silmultaneously transmitted probing ultrasonic waves. Then, the bispectra of the signal x (t) at the frequencies (f0, f0) and (f0, 2f0) are calculated to estimate the nonlinear propagation characteristics as their magnitude ratio, w here since bispectrum is free from the gaussian additive noise we can get the value with high S/N. Basic experimental system is constructed by using 3.0 MHz probing ultrasonic waves and the several experiments are carried out for some phantoms. Results show the superiority of the proposed method to the conventional method using power spectrum and also its usefulness for the tissue characterization. 30541 T00401030541 ^x This paper describes the implementation of a computerized radial pulse diagnosis by aids of a clinical expert. On this base, we composed of the radial pulse diagnosis system in korean traditional medicine. The system composed of a radial pulse wave detection system and a radial pulse diagnosis system. With a detection system, we detected Inyoung and Cheongu radial pulse wave and processed it. Then, we have got the characteristic parameters of radial pulse wave and also quantified that according to the method of Inyoung-Cheongu Comparison Radial Pulse Diagnosis. We defined the jugement standard of radial pulse diagnosis system and then we confirmed the possibility for realization of automatic radial pulse diagnosis in korean traditional medicine. 30545 T00401030545 ^x Microspheres are expected to be applied to biomedical areas such as solid-phase immunoassays, drug delivery systems, immunomagnetic cell separation. To synthesize microspheres for biomedical application, "two stage shot growth method" was developed. The uniformity ratio of synthesized microspheres was always smaller than 1.05. And the surface charge density (or the number of ionizable functional groups) of the microspheres synthesized by "two stage shot growth method" was 6~13 times higher than that of the microspheres synthesized by conventional seeded batch copolymerization. As a previous step for biomedical application, adsorption experiments of bovine albumin on microspheres were carried out under various conditions. The maximum adsorbed amount was obtained in the neighborhood of pH 4.5. Isoelectric point of bovine albumin is pH 5.0, so experimental result shows that it shifted to acid area. The adsorption isotherm was obtained, the plateau region was always reached at 2.Og/L (bulk concentration of bovine albumin).The effect of the kind and the amount of surface functional group was also examined. 30575 T00401030575 ^x A medical image workstation was developed using multimedia technique. The system based on PC-486DX was designed to acquire medical images produced by medical imaging instruments and related audio information, that is, doctors' reporting results. Input information was processed and analyzed, then the results were presented in the form of graph and animation. All the informations of the system were hierarchically related with the image as the apex. Processing and analysis algorithms were implemented so that the diagnostic accuracy could be improved. The diagnosed information can be transferred for patient diagnosis through LAN(local area network). 30592 T00401030592 ^x In the conventional infrared imaging system, complex infrared lens systems are usually used for directing collimated narrow infrared beams into the high speed 2-dimensional optic scanner. In this paper, a simple reflective infrared optic system with a 2-dimensional optic scanner is proposed for the realization of medical infrared thermography system. It has been experimentally proven that the intfrared thermography system composed of the proposed optic system has the temperature resolution of 0.1˚c under the spatial resolution of lmrad, the image matrix size of 256 X 240, and tile imaging time of 4 seconds. 30593 T00401030593 ^x In this paper, MIIS (Medical Image Information System) has been designed and implemented using INGRES RDBMS, which is based on a client/server architecture. The implemented system allows users to register and retrieve patient information, medical images and diagnostic reports. It also provides the function to display these information on workstation windows simultaneously by using the designed menu-driven graphic user interface. The medical image compression/decompression techniques are implemented and integrated into the medical image database system for the efficient data storage and the fast access through the network. 30594 T00401030594 ^x In this paper, computerized BEAM was implemented for the space domain analysis of EEG. Trans-formation from temporal summation to two-dimensional mappings is formed by 4 nearest point inter-polaton method. Methods of representation of BEAM are two. One is dot density method which classify brain electrical potential 9 levels by dot density of gray levels and the other is colour method which classify brain electrical 12 levels by red-green colours. In this BEAM, instantaneous change and average energy distribution over any arbitrary time interval of brain electrical activity could be observed and analyzed easily. In the frequency domain, the distribution of energy spectrum of a special band can easily be distinguished normality and abnormality. 30608 T00401030608 ^x Laboratory information system (LIS) is a key tool to manage laboratory data in clinical pathology. Our department has developed an information system for routine hematology using down-sized computer system. We have used an IBM 486 compatible PC with 16MB main memory, 210 MB hard disk drive, 9 RS-232C port and 24 pin dot printer. The operating system and database management system were SCO UNIX and SCO foxbase, respectively. For program development, we used Xbase language provided by SCO foxbase. The C language was used for interface purpose. To make the system use friendly, pull-down menu was used. The system connected to our hospital information system via application program interface (API), so the information related to patient and request details is automatically transmitted to our computer. Our system interfaced with fwd complete blood count analyzers(Sysmex NE-8000 and Coulter STKS) for unidirectional data tansmission from analyzer to computer. The authors suggests that this system based on down-sized computer could provide a progressive approach to total LIS based on local area network, and the implemented system could serve as a model for other hospital's LIS for routine hematology. 30609 T00401030609 ^x To develop an artificial bone substitute that is gradually degraded and replaced by the regenerated natural bone, the authors designed a composite that is consisted of calcium phosphate and collagen. To use as the structural matrix of the composite, collagen was purified from human umbilical cord. The obtained collagen was treated by pepsin to remove telopeptides, and finally, the immune-free atelocollagen was produced: The cross linked atelocollagen was highly resistant to the collagenase induced collagenolysis. The cross linked collagen demonstrated an improved tensile strength. 30618 T00401030618 ^x This paper is a study on the design of adptive filter for QRS complex detection. We propose a simple adaptive algorithm to increase capability of noise cancelation in QRS complex detection with two stage adaptive filter. At the first stage, background noise is removed and at the next stage, only spectrum of QRS complex components is passed. Two adaptive filters can afford to keep track of the changes of both noise and QRS complex. Each adaptive filter consists of prediction error filter and FIR filter The impulse response of FIR filter uses coefficients of prediction error filter. The detection rates for 105 and 108 of MIT/BIH data base were 99.3% and 97.4% respectively. 30619 T00401030619 ^x To develop an artificial bone substitute that is gradually degraded and replaced by the regenerated natural bone, the authors designed and produced a composite that is consisted of calcium phosphate and collagen. Human umbilical cord origin pepsin treated type I atelocollagen was used as the structural matrix, by which sintered or non-sintered carbonate apatite was encapsulated to form an inorganic-organic composite. With cross linking atelocollagen by UV ray irradiation, the resistance to both compressive and tensile strength was increased. Collagen degradation by the collagenase induced collagenolysis was also decreased. 30620 T00401030620 ^x We have developed a monoleaflet polymer valve as an inexpensive and viable alternative, especially for short-term use in the ventricular assist device or total artificial heart. The frame and leaflet of the polymer valve were made from polyurethane, To evaluate the hemodynamic performance of the polymer valve a comparative study of flow dynamics past a polymer valve and a St. Jude Medical prosthetic valve under physiological pulsatile flow conditions in vitro was made. Comparisons between the valves were made on the transvalvular pressure drop, regurgitation volume and maximum valve opening area. The polymer valve showed smaller regurgitation volume and transvalvular pressure drop compared to the mechanical valve at higher heart rate. The results showed that the functional characteristics of the polymer valve compared favorably with those of the mechanical valve at higher heart rate. 30621 T00401030621 ^x Explosive evaporative removal process of biological tissue by absorption of a CW laser has been simulated by using gelatin and a multimode Nd:YAG laser. Because the point of maximun temperature of laser-irradiated gelatin exists below the surface due to surface cooling, evaporation at the boiling temperature is made explosively from below the surface. The important parameters of this process are the conduction loss to laser power absorption (defined as the conduction-to-laser power parameter, Nk), the convection heat transfer at the surface to conduction loss (defined as Bi), dimensionless extinction coefficient (defined as Br.), and dimensionless irradiation time (defined as Fo). Dependence of Fo on Nk and Bi has been observed by experiment, and the results have been compared with the numerical results obtained by solving a 2-dimensional conduction equation. Fo and explosion depth (from the surface to the point of maximun temperature) are increased when Nk and Bi are increased.To find out the minimum laser power for explosive evaporative removal process, steady state analysis has been also made. The limit of Nk to induce evaporative removal, which is proportional to the inverse of the laser power, has been obtained. 30622 T00401030622 ^x N1 and N2 gross neural action potentials were measured from the round window of the guinea pig cochlea at the onset of the acoustic stimuli. N1-N2 audiograms were made by means of regulating stimulant intensities in order to produce constant N1-N2 potentials as criteria for different input tone pip frequencies. The lowest threshold was measured with an input tone pip I5 dB SPL in intensity and 12 KHz in frequency when the animal was in normal physiological condition. The procedure of experimental measurements is explained in detail. This experimental approach is very useful for the investigation of the Cochlear function. Both noN1inear and active functions of the Cochlea can be monitored by N1-N2 audiograms. 30623 T00401030623 ^x In electrical impedance tomography(EIT), we use boundary current and voltage measurements toprovide the information about the cross-sectional distribution of electrical impedance or resistivity. One of the major problems in EIT has been the inaccessibility of internal voltage or current data in finding the internal impedance values. We propose a new image reconstruction method using internal current density data measured by NMR. We obtained a two-dimensional current density distribution within a phantom by processing the real and imaginary MR images from a 4.77 NMR machine. We implemented a resistivity mage reconstruction algorithm using the finite element method and sensitivity matrix. We presented computer simulation results of the mage reconstruction algorithm and furture direction of the research. 30624 T00401030624 ^x A new method of digital image analysis technique for discrimination of cancer cell was presented in this paper. The object image was the Thyroid eland cells image that was diagnosed as normal and abnormal (two types of abnormal: follicular neoplastic cell, and papillary neoplastic cell), respectively. By using the proposed region segmentation algorithm, the cells were segmented into nucleus. The 16 feature parameters were used to calculate the features of each nucleus. A9 a consequence of using dominant feature parameters method proposed in this paper, discrimination rate of 91.11% was obtained for Thyroid Gland cells. 30625 T00401030625 ^x An electrical stimulator was designed to induce locomotion for paraplegic patients caused by central nervous system injury. Optimal stimulus parameters, which can minimize muscle fatigue and can achieve effective muscle contraction were determined in slow and fast muscles in Sprague-Dawley rats. Stimulus patterns of our stimulator were designed to simulate electromyographic activity monitored during locomotion of normal subjects. Muscle types of the lower extremity were classified according to their mechanical property of contraction, which are slow muscle (msoleus m.) and fast muscle (medial gastrocneminus m., rectus femoris m., vastus lateralis m.). Optimal parameters of electrical stimulation for slow muscles were 20 Hz, 0.2 ms square pulse. For fast muscle, 40 Hz, 0.3 ms square pulse was optimal to produce repeated contraction. Higher stimulus intensity was required when synergistic muscles were stimulated simultaneously than when they were stimulated individually. Electrical stimulation for each muscle was designed to generate bipedal locomotion, so that individual muscles alternate contraction and relaxation to simulate stance and swing phases. Portable electrical stimulator with 16 channels built in microprocessor was constructed and applied to paraplegic patients due to lumbar cord injury. The electrical stimulator restored partially gait function in paraplegic patients. 30626 T00401030626 ^x Two-Dimensional modelling of the Cochlear biomechanics is presented in this paper. The Laplace partial differential equation which represents the fluid mechanics of the Cochlea has been transformed into two-dimensional electrical transmission line. The procedure of this transformation is explained in detail. The comparison between one and two dimensional models is also presented. This electrical modelling of the basilar membrane (BM) is clearly useful for the next approach to the further. Development of active elements which are essential in the producing of the sharp tuning of the BM. This paper shows that two-dimension model is qualitatively better than one-dimensional model both in amplitude and phase responses of the BM displacement. The present model is only for frequency response. However because the model is electrical, the two-dimensional transmission line model can be extended to time response without any difficult. 30627 T00401030627 ^x A method has been proposed for the fully automatic detection of left ventricular endocardial boundary in 2D short axis echocardiogram using geometric model. The procedure has the following three distinct stages. First, the initial center is estimated by the initial center estimation algorithm which is applied to decimated image. Second, the center estimation algorithm is applied to original image and then best-fit elliptic model estimation is processed. Third, best-fit boundary is detected by the cost function which is based on the best-fit elliptic model. The proposed method shows effective result without manual intervention by a human operator. 30628 T00401030628 ^x The intelligent trajectory control method that controls moving direction and average velocity for a prosthetic arm is proposed by pattern recognition and force estimations using EMG signals. Also, we propose the real time trajectory planning method which generates continuous accelleration paths using 3 stage linear filters to minimize the impact to human body induced by arm motions and to reduce the muscle fatigue. We use combination of MLP and fuzzy filter for pattern recognition to estimate the direction of a muscle and Hogan's method for the force estimation. EMG signals are acquired by using a amputation simulator and 2 dimensional joystick motion. The simulation results of proposed prosthetic arm control system using the EMf signals show that the arm is effectively followed the desired trajectory depended on estimated force and direction of muscle movements. 30638 T00401030638 ^x A new neural network architecture for the recognition of patterns from images is proposed, which is partially based on the results of physiological studies. The proposed network is composed of multi-layers and the nerve cells in each layer are connected by spatial filters which approximate receptive fields in optic nerve fields. In the proposed method, patterns recognition for complicated images is carried out using global features as well as local features such as lines and end-points. A new generating method of matched filers representing global features is proposed in this network. 30659 T00401030659 ^x An implementation scheme of the magnetic nerve stimulator using a switching mode power supply is proposed. By using a switching mode power supply rather than a conventional linear power supply for charging high voltage capacitors, the weight and size of the magnetic nerve stimulator can be considerably reduced. Maximum output voltage of the developed magnetic nerve stimulator using the switching mode power supply is 3,000 volts and switching time is about 100 msec. Experimental results or human nerve stimulations using the developed stimulator are presented. 30768 T00401030768 ^x In this paper, we describe the design methodology and specifications of the developed module-based bedside monitors for patient monitoring. The bedside monitor consists of a main unit and module cases with various parameter modules. The main unit includes a 12.1" TFT color LCD, a main CPU board, and peripherals such as a module controller, Ethernet LAN card, video card, rotate/push button controller, etc. The main unit can connect at maximum three module cases each of which can accommodate up to 7 parameter modules. They include the modules for electrocardiograph, respiration, invasive blood pressure, noninvasive blood pressure, temperature, and SpO2 with Plethysmograph.

• Journal of Periodontal and Implant Science
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• 제32권3호
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• pp.457-473
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• 2002

The purposes of this study is to evaluate the combination effects of TGF-${\beta}_1$ and PDGF-BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/100% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Authors measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis according to the concentration of TGF-${\beta}_1$,(1,5ng/ml) and PDGF-BB (1,10 ng/ml) in combination. To explore further this delayed effect of TGF-${\beta}_1$, we preincubated human periodontal ligament cells with TGF-${\beta}_1$ for 4 or 24 hours before PDGF-BB stimulation. The results were as follows: The DNA synthetic activity was increased dose dependently by TGF-${\beta}_1$, PDGF-BB. The combination of TGF-${\beta}_1$ and PDGF-BB consistently enhanced the DNA synthetic activity to PDGF-BB alone. The ability of TGF-${\beta}_1$ to enhance DNA synthetic activity in PDGF-BB treated periodontal ligament cells was dose dependent. The maximum mitogenic effect was at the 5ng/ml of TGF-${\beta}_1$ and l0ng/ml of PDGF-BB. Preincubation of cell with TGF-${\beta}_1$ resulted in significantly greater response to PDGF-BB at all TGF-${\beta}_1$ concentration studied, and may be useful for clinical application in periodontal regenerative procedures. The total protein, collagen and noncollagen synthesis was increased dose pendently by TGF-${\beta}_1$, PDGF-BB. The % of collagen was slightly decreased according to the concentration of TGF-${\beta}_1$, PDGF-BB. The effect of TGF-${\beta}_1$, PDGF-BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. This study demonstrates that PDGF-BB is major mitogens for human periodontal ligament cells in vitro, and supports a role for TGF-${\beta}_1$ as a regulation of the mitogenic and total protein formation to PDGF-BB in these cells.

• Tuberculosis and Respiratory Diseases
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• 제41권3호
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• pp.231-238
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• 1994

Backgroud: Since the demonstration of the fact that vascular relaxation by acetylcholine(Ach) results from the release of relaxing factor from the endothelium, the identity and physiology of this endothelium-derived relaxing factor(EDRF) has been the target for many researches. EDRF has been identified as nitric oxide(NO). With the recent evidences that EDRF is an important mediator of vascular tone, there have been increasing interests in defining the role of the EDRF as a potential mediator of hypoxic pulmonary vasoconstriction. But the role of EDRF in modulating the pulmonary circulation is not compeletely clarified. To investigate the endothelium-dependent pulmonary vasodilation and the role of EDRF during hypoxic pulmonary vasoconstriction, we studied the effects of $N^G$-monomethyl-L-arginine(L-NMMA) and L-arginine on the precontracted pulmonary arterial rings of the rat in normoxia and hypoxia. Mothods: The pulmonary arteries of male Sprague Dawley(300~350g) were dissected free of surrounding tissue, and cut into rings. Rings were mounted over fine rigid wires, in organ chambers filled with 20ml of Krebs solution bubbled with 95 percent oxygen and 5 percent carbon dioxide and maintained at $37^{\circ}C$. Changes in isometric tension were recorded with a force transducer(FT.03 Grass, Quincy, USA) Results: 1) Precontraction of rat pulmonry artery with intact endothelium by phenylephrine(PE, $10^{-6}M$) was relaxed completely by acetylcholine(Ach, $10^{-9}-10^{-5}M$) and sodium nitroprusside(SN, $10^{-9}-10^{-5}M$), but relaxing response by Ach in rat pulmonary artery with denuded endothelium was significantly decreased. 2) L-NMMA($10^{-4}M$) pretreatment inhibited Ach($10^{-9}-10^{-5}M$)-induced relaxation, but L-NMMA ($10^{-4}M$) had no effect on relaxation induced by SN($10^{-9}-10^{-5}M$). 3) Pretreatment of the L-arginine($10^{-4}M$) significantly reversed the inhibition of the Ach ($10^{-9}-10^{-5}M$)-induced relaxation caused by L-NMMA($10^{-4}M$) 4) Pulmonary arterial contraction by PE($10^{-6}M$) was stronger in hypoxia than normoxia but relaxing response by Ach($10^{-9}-10^{-5}M$) was decreased, 5) With pretreatment of L-arginine($10^{-4}M$), pulmonary arterial relaxation by Ach($10^{-9}-10^{-5}M$) in hypoxia was reversed to the level of relaxation in normoxia. Conclusion: It is concluded that rat pulmonary arterial relaxation by Ach is dependent on the intact endothelium and is largely mediated by NO. Acute hypoxic pulmonary vasoconstriction is related to the suppression on NO formation in the vascular endothelium.

• The Journal of Natural Sciences
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• 제4권
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

• Journal of Korean Society of Forest Science
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• 제21권1호
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• pp.1-34
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• 1974

The author intended to investigate external and internal changes in the cone structure, changes in water content, sugar, fat and protein during the period of seed maturation which bears a proper germinability. The experimental results can be summarized as in the following. 1. Male flowers 1) Pollen-mother cells occur as a mass from late in April to early in May, and form pollen tetrads through meiosis early and middle of May. Pollen with simple nucleus reach maturity late in May. 2) Stamen number of a male flower is almost same as the scale number of cone and is 69-102 stamens. One stamen includes 5800-7300 pollen. 3) The shape is round and elliptical, both of a pollen has air-sac with $80-91{\mu}$ in length, and has cuticlar exine and cellulose intine. 4) Pollen germinate in 68 hours at $25^{\circ}C$ with distilled water of pH 6.0, 2% sugar and 0.8% agar. 2. Female flowers 1) Ovuliferous scales grow rapidly in late April, and differentiation of ovules begins early in May. Embryo-sac-mother cells produce pollen tetrads through meiosis in the middle of May, and flower in late May. 2) The pollinated female flowers show repeated divisions of embryo-sac nucleus, and a great number of free nuclei form a mass for overwintering. Morphogenesis of isolation in the mass structure takes place from the middle of March, and that forms albuminous bodies of aivealus in early May. 3. Formation of pollinators and embryos. 1) Archegonia produce archegonial initial cells in the middle and late April, and pollinators are produced in the late April and late in early May. 2) After pollination, Oespore nuclei are seen to divide in the late May forming a layer of suspensor from the diaphragm in early June and in the middle of June. Thus this happens to show 4 pro-embryos. The organ of embryos begins to differentiate 1 pro-embryo and reachs perfect maturation in late August. 4. The growth of cones 1) In the year of flowering, strobiles grow during the period from the middle of June to the middle of July, and do not grow after the middle of August. Strobiles grow 1.6 times more in length 3.3 times short in diameter and about 22 times more weight than those of female flower in the year of flowering. 2) The cones at the adult stage grow 7 times longer in diameter, 12-15 times shorter diameter than those of strobiles after flowering. 3) Cone has 96-133 scales with the ratio of scale to be 69-80% and the length of cone is 11-13cm. Diameter is 5-8cm with 160-190g weight, and the seed number of it is 90-150 having empty seed ratio of 8-15%. 5. Formation of seed-coats 1) The layers of outer seed-coat become most for the width of $703{\mu}$ in the middle of July. At the adult stage of seed, it becomes $550-580{\mu}$ in size by decreasing moisture content. Then a horny and the cortical tissue of outer coats become differentiated. 2) The outer seed-coat of mature seeds forms epidermal cells of 3-4 layers and the stone cells of 16-21 layers. The interior part of it becomes parenchyma layer of 1 or 2 rows. 3) Inner seed-coat is formed 2 months earlier than the outer seed-coat in the middle of May, having the most width of inner seed-coat $667{\mu}$. At the adult stage it loses to $80-90{\mu}$. 6. Change in moisture content After pollination moisture content becomes gradually increased at the top in the early June and becomes markedly decreased in the middle of August. At the adult stage it shows 43~48% in cone, 23~25% in the outer seed-coat, 32~37% in the inner seed-coat, 23~26% in the inner seed-coat and endosperm and embryo, 21~24% in the embryo and endosperm, 36~40% in the embryos. 7. The content compositions of seed 1) Fat contents become gradually increased after the early May, at the adult stage it occupies 65~85% more fat than walnut and palm. Embryo includes 78.8% fat, and 57.0% fat in endosperm. 2) Sugar content after pollination becomes greatly increased as in the case of reducing sugar, while non-reducing sugar becomes increased in the early June. 3) Crude protein content becomes gradually increased after the early May, and at the adult stage it becomes 48.8%. Endosperm is made up with more protein than embryo. 8. The test of germination The collected optimum period of Pinus koraiensis seeds at an adequate maturity was collected in the early September, and used for the germination test of reduction-method and embryo culture. Seeds were taken at the interval of 7 days from the middle of July to the middle of September for the germination test at germination apparatus.