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- ItemPurification and characterization of fluorescent probes from blue green algae(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Luckana Laousoontornsiri; Dhirayos Wititsuwannakul; Pichit TosukhowongPhycocyanin which is a protein component of phycobilisomes has been purified from Spirulina platensis. It is an effective fluorescent protein with high quantum yield. Phycocyanin is gaining wide acceptance for use as fluorescent probe in various different analytical techniques. The cyanobacteria Spirulina platensis is a type of blue-green algae found as natural bloom in water environment. It has ability for CO(,2) fixation using the light harvesting phycobili some proteins in photosynthesis process. Phycocyanin was found as major components upon disruption of spirulina platensis by repeated freeze-thawing of the cell mass. The blue-green algae is abundant in natural water source and serves as a suitable staring terial. The deep blue supernate after removal of cell debris contained large amount of phycocyanin. A large scale preparation and purification procedure for phycocyanin was developed in this study. The protein was purified from DEAE cellulose collum chromatography by linear salt gradient elution. It has characteristics of specific absorbance at A620 which was used for monitoring the phycocyanin. The protein was eluted from the column at 0.15 M NaCl as almost pure phycocyanin peak. The absorbance ratio A620/A280 of fractions greater than 3.4 was found to be homogeneous phycocyanin and used as criteria for purity of the protein. Batch binding of phycocyanin with DEAE cellulose followed by step gradient salt elution was empioyed in large scale purification as modified and adapted from the collum method. The fluorescent properties of phycocyanin was characterized with maximum emission at 640 nm. It was sensitive to temperature above 50 C and pH8. The optimum ranges for fluorescence were 20C 40C and pH 4-8. Phycocyanin was also found sensitive to concentration quenching which is a typical nature of fluorophores. Subunit molecular weight was 17.5 kd and the native phycocyanin had molecular weight 208 kd, indicating the phycocyanin was multimeric protein containing 12 subunits in the active from. Avidin was also prepared and purified from egg-white in this investigation for conjugation study with phycocyanin. Large scale purification of avidin from diluted egg-white solution was developed by modification of the previously reported methods. The purification procedure was divided into two steps. The first step was batch binding of avidin with CM-cellulose followed by extensive washing and step wise elution with ammonium carbonate. Detection of avidin was monitored by specific dye binding assay using 4-hydroxyazobenzene 2-carboxylic acid (HABA) which displayed absorbance of A500. the second step employed 2-iminobiotin agarose as affinity column for purification of avidin eluted from CM-cellulose. Combination of two steps resulted in 74% recovery of purified avidin with a specific activity increased from 4.1 (ug biotion/g protein) ad CM-cellulose step to 15.7 in the final affinity step. The molecular weight of purified avidin was 18.5 kd as reported in this study. Conjugation of phycocyanin and avidin was carried out using different chemicals modifying method. Glutaraldehyde and carbodiimide were found unsatisfactory and no phycocyanin-avidin conjugate was resulted in these methods. However, the heterobifunctional reagent, SPDP, was found satisfactory for the formation of phycocyanin-avidin conjugate. The conjugate had both A620 property of phycocyanin and A500 for HABA binding to avidin in the same compels. The conjugate with molecular weight 37 kd probably represented 1:1 ratio and 1:2 ratio for the 55 kd conjugate. This study demonstrated that it was possible to perform large scale preparation of phycocyanin and avidin from simple and abundant starting materials as well as the utilization of the two biomolecules for analytical and practical purposes.
- ItemExtrarenal potassium homeostasis after chronic potassium deficiency and after postassium repletion in rats(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Phunpen Napradit; Chumpol Pholpramool; Samisukh Sophasan; Pawinee PiyachaturawatIn order to study the effect of chronic potassium deficiency and subsequent to potassium repletion on extrarenal potassium homeostasis, adult male and female Sprauge-Dawley rats were divided into 2 groups. Potassium depletion was induced in one group by feeding with a potassium-deficient diet for up to 120 days. The other group was paired-fed with normal rat chow at the same amount consumed by the first group serving as controls. During potassium depletion, the rats had low food intake and their growths were retarded compared to the control period. The males appeared to gain less but lost more weight than the female counterparts, however, these differences were statistically insignificant. Polyuria, polydipsia, an impairment of urinary concentrating ability and a reduction in urinary K+ excretion were also observed. The potassium-depleted males developed more severe polyuria and polydipsia than the females in the late phase of potassium deficiency. Dietary potassium deprivation for 16-17 weeks resulted in a significant decreased in plasma K+ concentration to 1.5-2 mEq/l, total blood K+ concentration, red cell K+ content and plasma Cl- concentration whereas plasma Na+ concentration was virtually not changed. In addition, potassium deficiency caused a metabolic alkalosis, a reduction in K + but an accumulation in Na+ contents in muscle. The K+ contents in SOL and EDL were decreased by 46 and 39 % in the males and by 38 and 33 % in the females, respectively. The reduction in muscle K+ content was not fully compensated for by an increase in Na+ content. More reduction in growth rate and muscle K+ content as well as severe polyuria and polydipsia were observed in potassium-depleted males indicate indirectly that prolonged and severe potassium deficiency may affect the males more than the females. However, 2 weeks repletion of potassium by feeding normal rat chow resulted in marked increases in body weight, rapid restoration to control levels in water intake and urine output. Furthermore, electrolyte changes in plasma, total blood, red cell and skeletal muscles as well as the change in acid base status were also reversible. To investigate the extrarenal potassium homeostasis, functional nephrectomy and adrenalectomy were performed in the potassium-depleted, potassium-supplemented (in which normal rat chow was fed for 14 days to the previously potassium-depleted rats), diet-restricted and normal-untreated rats. Acute K-loading was challenged by intravenous infusion with KC1 at a dose of 2 mEq/kg/hr for I hr. This resulted in elevations of plasma K+ concentration and muscle K+ content in all groups. The increment in plasma K+ concentration was significantly lower in the potassium-depleted rats versus the normal rats (max ΔpK 1.9 ± 0.3 vs. 3.9 ± 0.1 mEq/l, P<0.001) whereas the increases in muscle K+ content was higher than those in the other groups. Furthermore, following KC1 infusion blood pH was significantly decreased (P<0.05) and plasma Na+ concentration was increased (P<0.05) in the potassium-depleted rats but those in the other groups were virtually not changed. These results were related to a greater K+ uptake in the potassium-depleted group indicating an alteration in the extrarenal potassium disposal after chronic potassium deficiency. It is concluded that following KCI infusion the K+ ion may be an appropriate stimulus that leads to a marked activation of the residual Na(+)-K(+)pump in the potassium-depleted rats sufficient to lower plasma K+ concentration than in the normal rats. This effect may be mediated by insulin as well as an accumulation of Na(+) in the muscle, an increase in extracellular HCO(,3)- and metabolic alkalosis developed after chronic potassium deficiency. However, the alteration in extrarenal potassium disposal is completely reversed after potassium repletion.
- ItemHorizontal and vertical jumping ability in male and female athletes(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Chalermporn Ongvarrasopone; Thyon Chentanez; Thirayudh Glinsukon; Punya KaimukThe present study was designed to investigate the characteristic of vertical and horizontal jump at 0 degree c, 45 degree c, 90 degree c, 135 degree c, 180 degree c, 225 degree c, 270 degree c and 315 degree c and the relationship between these two jump tests and leg volume, leg muscles strength, body segments length, Vo2 max and anaerobic power. Twenty six men and eighteen women (age 22-24 yrs) were divided into sedentary and amateur athletes groups (Ms, Ma, Fs, Fa). The general physical fitness were measured. Vertical jump and reach test was recorded by digital indication jump meter. The horizontal jump test at various directions were performed with barefeet either one or both feet takeoff. The results showed that the vertical and horizontal jump distance at various directions in males did not have significantly difference between sedentary and amateur athletes except the horizontal jump at 180 degree c. While in female, insignificantly difference was found only in the horizontal jump distance at 135 degree c. It was apparently found that the greatest horizontal jump distance was found at 90 degree c while the shortest was found at 270 degree c. The horizontal jump in forward directions (0 degree c,45 degree c, 90 degree c, 135 degree c, 180 degree c ) had greater distance than that in backward directions (225 degree c,270 degree c,315 degree c). However no significant difference between left and right direction (180 degree c - 0 degree . 134 degree c - 45 degree c , 225 degree c - 315 degree c ) in each group of subjects except 180 degree c - 0 degree c in male amateur athletes which horizontal jump distance at 180 was greater than that at 0 degree c . The positive relationship between vertical and horizontal jump performance in every directions were found in sedentary group of males and females. A highly positive correlation between both legs volume, lean both legs volume, knee extensors strength and vertical, horizontal jump performance were found in all groups of subjects. However knee flexors strength related to the horizontal jump performance in some directions. A slightly low correlation was found between body segments length and jumping distance. Only female sedentary group that had significantly positive correlations between absolute Vo(,2) max and jumping performance. While female amateur athletes group did not have significantly correlation between anaerobic power and jumping performance. Strong relationship between anaerobic capacity and both types of jumping performance were found in every groups. The horizontal jump performance in mem were superior than those in women in every directions.
- ItemEffects of cadmium, chromium, and their combinations on puntius gonionotus bleeker(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Urasa Sriboonlue; Suchart Upatham; Rojana Keawjam; Siriwan JantataemeThe fingerling stage of the common silver barb, Puntius gonionotus Bleeker, was exposed to cadmium and chromium as individual metals and as mixtures for 96 hours by using the static bioassay experiment in order to determine the LC50 values. The results showed that the 96-h LC50 values of cadmium and chromium were 2.77 and 69.03 mg/l, respectively, whereas in the Cd-Cr mixtures at toxicity ratios 1:1, 1:2, and 2:1 were 0.14, 0.16, and 0.13 toxic units, respectively. Furthermore, the mixtures appeared to interact more than additively in all ratios. In the accumulation experiments, fish were exposed to cadmium and chromium in single and in mixture (1:1) with the concentration of 5% of 96-h LC50 value, but 1% of 96-h LC50 value was also done in cadmium. The whole body contents of metals were determined every week until 8 weeks. The results indicated that the whole body metal contents increased with increasing exposure time, and the accumulation of one metal was influenced by the presence of the other.
- ItemEffects of zinc and pH on Puntius gonionotus bleeker(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Sompong Boonklomjitr; Suchart Upatham; Rojana Keawjam; Siriwan JantataemeThe toxicity of zinc sulphate to Puntius gonionotus Bleeker was investigated with an exposure period of 96 hours using static bioassay. The results of 24, 48, 72, and 96-h LC50 values of fish exposed to zinc were 7.06, 5.98, 5.83 and 5.91 mg/l, respectively. The fish also were exposed in zinc sulphate solutions at pH 4.5, 5.0, 6.0, 7.0, 8.0, and 9.0, and their 96-h LC50 values at pH 4.5, 5.0, 6.0, 7.0, 8.0, and 9.0 were 10.154, 15.203, 7.212, 6.417, 4.081, and 7.328 mg/l, respectively. The toxiciy of zinc increased at pH 5-8 and increased at pH below 5 also whereas it decreased at pH 8-9. Accumulation of zinc in the whole bodies of fish showed an increasing trend from week 1 to week 8, using Atomic absortion spectrophotometer.
- ItemProduction of oxidase enzymes for clinical assyas(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Sumalai Phongying; Pichit Tosukhowong; Bhinyo Panijpan; Kachorn CharoensiriThe oxidase enzymes have a high potential in biotechnological applications. Screen for a good source of a particular oxidase has been actively carried out in various laboratories by using plating technique and liquid culture technique. In this investigation, nutrient selective medium technique was used to screen for microorganisms which produced choline oxidase, glucose oxidase, L-α-glycerophosphate oxidase, sarcosine oxidase and urate oxidase. Sarcosine oxidase and urate oxidase producing micro-organims were successfully screened from 50 soil samples whereas microorganism producing choline oxidase, glucose oxidase and L-α-glycerophosphate oxidae were not found. Sarcosine oxidase and urate oxidase producing microorganisms were identified as a genus Arthrobacter based on Bergeys manual and arbitarily name Arthrobacter sp S-1 and Arthrobacter sp U-1, respectively. Arthrobacter sp S-1 produced only intracellular sarcosine oxidase with specific activity about 0.06 unit/mg protein. Crude sarcosine oxidase was purified 20 folds, 72% recovery with a specific activity of 1.2 unit/mg protein. Molecular weight of this sarcosine oxidase was about 190,000 daltons estimated by gel filtration. Optimal pH and optimal temperature were about 7.7 and 45 degree C, respectively. This enzyme was stable upto 30 degree C in phosphate buffer pH 7.5 and it was stable at 4 degree C in the presence of NaN(,3). K(,m) was about 7.0 mM and it was specific to sarcosine. Effects of various activators and inhibitors were studied. Arthrobacter sp U-1 produced both intracellular and extracel-lular urate oxidase in the ratio of 20:1 and about 0.34 unit/mg protein of crude intracellular urate oxidase was obtained. This crude enzyme was purified 15 folds, 70% recovery with a specific activity of 5 unit/mg protein. The molecular weight of this urate oxidase was about 120,000 daltons estimated by gel filtration. Optimal pH was about 9.0. Optimal temperature was about 35 degree C in phosphate buffer pH 7.5 and about 50 degree C in the presence of sodium borate. This enzyme was stable upto 35 degree C in phosphate buffer pH 7.5 and 45 degree C in the presence of sodium borate. It was stable at 4 degree C in the presence of NaN(,3). K(,m) was about 7.0 x 10(,-5)M and it was specific to uric acid. Effects of various activators and inhibitors were studied. It was concluded that both enzymes from the two microorganisms are similar to the commercially available enzyme.
- ItemMichael reaction of [alpha]-chloro sulfonyl carbanion(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Chongkon Sumpanwatanakun; Vichai Reutrakul; Manat PohmakotrThe purpose of this research is to study the reaction of α-chloaro sulfonyl carbanion with various α,β-unsaturated compounds of monoesters, diesters, mononitrile, dinitrile, aldehydes, ketones, 1,3-ketorster, and 1,3-diketone. The two method for generating the α-chloro sulfonyl carbanion were investigates i.e. : 1. Lithium diisopropylamide (LDA) in tetrahydrofuran (THF) with hexamethylphosphoramide (HMPA) as a cobolvent (LDA method) 2. K(,2)Co(,3)/ benzyltriethylammonium chloride (TEBA) / aprotic solvent (PTC method) The results from the investigations could be summarized as follows : 1. Reaction of lithio chlormethyl phenyl sulfone anion with alpha, beta-unsaturated monoesters, diesters, mono and dinitrile gave the corresponding cyclopanes. 2. Reaction of alpha-chloro sulfonyl carbaion with methyl acrylate and crotonitrile yielded the cyclopentyl products in low yields. These products derived from a 1+2+2 MIMI-RC reaction. The reaction of methyl acrylate with alpha-chloro sulfonyl carbanion gave cyclohexyl derivative. This product could derived from a Michael-Michael-Dieckmann reaction. 3. The products from the reaction of α-chloro sulfonyl carbanion with α,β-unsaturated ketones depended no the tpye of ketones. The results obtained as follows.a. Methyl vinyl ketone, the major products are the corresponding cyclopropane and cyclohexyl compounds derived from Michael-Michael-Aldol reaction.b. Chalcone gave cyclopropanes and cyclopropyl epoxide.c. Dibenzalacetone, only cyclopropyl products were isolated.d. Cyclopentenone and cyclohexennone yielded cyclopropyl derivatives and the tricyclic compounds. These reactions provide a quick entry to polycyclic compounds. 4. The addition of lithio chloromethyl phenyl sulfone anion to α,β-unsaturated aldehydes gave 1,2-addition product exclusively. 5. The reaction of α,β-unsaturated 1,3-ketoester with lithio chloromethyl phenyl sulfone anion gave the expected cyclopropane in good yield. For α,β-unsaturated 1,3-diketone, dihydrofuran derivative was isolated in high yield.
- ItemStudies of inosine on the metabolic status of thalassemic red blood cell in vitro(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Pathama Leewanich; Udom Chantharaksri; Suthat Fucharoen; Pipat CherdrungsiThalassemic red blood cells (RBCs) are known to have shorter survivals with accompanying numbers of metabolic defects and virtually all kinds of morphologic abnormalities are described. They are under continuous oxidative stress with exhausted antioxidative components. It is anticipated that the excessive uses or insufficient supplies of high energy phosphate (ATP) in thalassemic RBCs may be a presumptive factor that normally requires for maintaining the well-being of RBCs. This study was designed to determine the metabolic status of thalassemic RBCs and assess in vitro if the effect of inosine, a known metabolic activator shows any phar-macologic benefit. Heparinized blood of normal volunteers, non-splenectomized and splenectomized thalassemias (THAL) were subjected to various conditions in vitro. Levels of ATP and FDP (furctose 1, 6-diphosphate, an intracellular glycolytic regulator) were respectively determined by HPLC and enzymatic methods. It was found that the levels of ATP in thalassemic RBCs of both non-splenectomized (NS) and splenectomized (SP) were lower than those of volunteers (NORMAL). The elevated FDP levels in the thalasssemic RBCs suggested that they were under continuous stress of having to compensate for the ATP-depleted RBCs. Inosine in the dose of 10 mM was found effectively in elevating both ATP and FDP in the RBCs, especially in ATP-depleted RBCs, of NORMAL and THAL. However, the restoring ability of inosine on the levels of intraerythrocytic ATP in THAL was not as marked as that in the NORMAL though the levels of FDP were higher elevated in THAL than that of NORMAL. These findings demonstrate that inosine is effective in maintaining the metabolic status of human RBCs of both NORMAL and THAL in vitro, and suggest that specific biochemical defect(s) may have occurred to the glycolytic regulatory path-way in the thalassemic RBCs.
- ItemThe risk factors for gastric cancer in Thai people(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Siriluk Apivanich; Siriluk Apivanich; Jayanton Patumanond; Natchaporn Pichainarong; Dusit Sujirarat; Suthus Sriphojanart
- ItemStudies on seed germination and plant growth development of Thai wild orchid, aerides houlletiana and acampe rigida(Mahidol University. Mahidol University Library and Knowledge Center, 2024) Karnchana Rungruchkanont; Sarinee Chaicharoen; Maleeya Kruatrachue; Suchart UpathamSeeds of Aerides houlletiana and Acampe rigida were cultured on the combination of three modified media; modified Vacin and Went plus iron of Murashige and Skoog (VN), modified Vacin and Went plus minor element, vitamin and iron of Murashige and Skoog (VM) and modified Shenk and Hildebrandt (SH), and four different concentrations of saccharose, 0, 10, 20 and 30 g/l in the light and dark conditions. Seeds of the two orchid species germinated in the light and dark conditions but the light condition gave slightly early germination than the dark condition. Seeds of A. houlletiana germinated and had good protocorm development on VM medium without saccharose (VMO), whereas seeds of A. rigida germinated and had good protocorm development on VN and VM media with 10 g/l saccharose (VN10 and VM10). The study on the effect of three additive substances, namely, banana, potato and charcoal on protocorm growth of A. houlletiana and A. rigida showed that the addition of banana, potato and charcoal in the culture medium enhanced growth and development of protocorms. For protocorm proliferation, VM medium plus 10 g/l saccharose without peptone (VSl0P0) was the suitable medium for both orchid species. During the stage of plant development, modified Knudson medium (KS) was the best medium for A. houlletiana, whereas modified Vacin and Went medium (VW) was the most suitable for A. rigida. In the histological study, differences among A. houlletiana and A. rigida in each stage of development from seeds were observed. The proembryo of A. houlletiana contained several meristematic loci among parenchyma tissues. On the other hand, the seed of A. rigida contained a single embryo. Inactive callus was found only in A. rigida, while active callus was found in A. rigida and A. houlletiana. The shoot apex of A. houlletiana contained well differentiated meristematic cells with very dense cytoplasm and large nuclei. However, the meristematic cells in the shoot apex of A. rigida appeared to be more diffused. In cytological observation, both A. houlletiana and A. rigida have the same chromosome number, 2n = 38.