Together with cytochrome P450 (CYP), flavin-containing monooxygenase (FMO) present in liver microsomes oxidizes various endogenous and exogenous chemicals. In an effort to determine the human FMO activity, we have developed two non-invasive urine analysis methods using caffeine (CA) and ranitidine (RA) as the probe compounds. As the production of theobromine (TB) and ranitidine N-oxide (RANO) from CA and RA is catalyzed primarily by the hepatic FMO, we have assigned the urinary molar ratios of TB/CA and RA/RANO as the in vivo FMO activity. In 200 age-matched Korean volunteers, the obtained TB/CA ratio ranged from 0.4 to 15.2 (38-fold difference) and the RA/RANO ratio from 5.7 to 27.2 (4.8-fold). The FMO activity of 20's, determined by caffeine metabolism, was the highest (2.5$\pm$l.9) and those of 30's, 40's, 50's, 60's and 70's were 40%, 50%, 24%, 39% and 36% of the 20's, respectively. Intake of grapefruit juice, known to contain flavonoids, inhibited the in vivo FMO (TB/CA) activity by 79%. Addition of the flavonoids like naringin, quercitrin and kaempferol, present in grapefruit juice, to the in vitro microso-mal FMO assay, thiobenzamide S-oxidation, produced 75%, 70% and 60% inhibition, respectively. Obtained Ki values of quercitrin, kaempferol and naringin on the in vitro FMO activity were 6.2, 12.0 and 13.9 $\mu\textrm{M}$, respectively. This suggested that the dose of drug should need to be adjusted to suit the individual FMO activities when the drugs metabolized by FMO are given to patients. As the intake of grapefruit juice has been identified to inhibit the FMO as well as CYP3A4 and lA2 activities, patients taking drugs metabolized by these enzymes should not drink grapefruit juice as the carrier.
Recently, we reported that GS 389 has vasodilating action without cardiac inotropic action (Chang et al., Can. J. Physiol. Pharmacol. 72, 327-334, 1994). However the mechanism of action of GS 389 has not been thoroughly evaluated. In the present study, we performed functional study of GS 389 in rat trachealis, thoracic aorta, pig coronary artery by isometric tension and in human platelets by aggregation experiments. We also tested if GS 389 influences on $Ca^{2+}$movement and inositol phosphate metabolism. In rat trachealis, GS 389 concentration-dependently relaxed carbachol (0.1 $\mu$M)- and high $K^{+}$(65.4 mM)-induced contraction with p$IC_{50}$/ of 4.43$\pm$ 0.19 and 4.11$\pm$0.12, respectively. In $Ca^{2+}$-free media, GS 389 inhibited carbachol-induced phasic contraction. In rat thoracic aorta, GS 389 inhibited $^{45}$ Ca uptake due to norepinephrine and high $K^{+}$, indicating that GS 389 has direct inhibitory action of $Ca^{2+}$movement. Furthermore, GS 389 competitively inhibited U46619-induced contraction in rat thoracic aorta and pig coronary artery with K, values of 5.23$\pm$0.12 and 5.56$\pm$0.14, respectively, and inhibited U 46619-induced phosphatidylinositide (PI) turnover in rat aorta. GS 389 also concentration-dependently inhibited the human platelet aggregation against U 46619 with p$IC_{50}$/ 5.66$\pm$0.02. These results indicate that GS 389 has thromboxane $A_2$ antagonistic action in vascular and platelets as well as direct action on $Ca^{2+}$ movement, which may account, at least in part, for relaxing action of rat trachealis. trachealis.
The effect of the level of casein phosphopeptide (CPP) on mineral (Ca and P) bioavailabilties and bone biomarker of aged ovariectomized (OVX) Sprague-Dawley rats were studied as a model for postmenopausal bone loss. Forty five Spargue dawley rats, 220-230 g of body weight were fed a control diet (AIN 93M) or containing different level of CPP diet for 7 weeks: $0\%$ (sham control; SC, OVX control; OC), $1\%$ (OVX low CPP diet: OL), $2\%$ (OVX medium CPP diet; OM), $3\%$ (OVX high CPP diet; OH) Ca absorption was unaffected by increasing CPP content from 0 to $3\%$. Urinary Ca excretion was increased by OVX, and decreased by CPP significantly (p < 0.05) with no evident doserelationship. The urinary P excretion was increased by CPP intake in OVX rats. The fecal excretion of P given CPP decreased in OVX with dose dependent manner. Ca and P contents of femur significantly increased by adding 2 or $3\%$ of CPP when compared with OC group and OL group (p < 0.05). There were no significant differences in serum alkaline phosphatase activity and c-terminal telopeptide excretion in experimental groups. Although ovariectomy induced the increase in urinary c-terminal telopeptide excretion, 2 or $3\%$ of CPP in the diet decreased urinary c-terminal telopetide excretion significantly. These finding suggest the usefulness of CPP in the prevention of postmenopausal bone loss by decreasing urinary Ca excretion and bone resorption. Over 2 percent of CPP in the diet was effective to prevent postmenopausal bone loss.
Ultraviolet (UV) radiation-induced loss of dermal extracellular matrix is associated with skin photoaging. Recent studies demonstrated that keratinocyte-releasable stratifin (SFN) plays a critical role in skin collagen metabolism by inducing matrix metalloproteinase 1 (MMP1) expression in target fibroblasts. In the present study, we examined whether SFN released from UVB-irradiated epidermal keratinocytes increases MMP1 release from dermal fibroblasts, and whether these events are affected by p-coumaric acid (p-CA), a natural phenolic compound with UVB-shielding and antioxidant properties. HaCaT cells were exposed to UVB in the absence and presence of p-CA, and the conditioned medium was used to stimulate fibroblasts in medium transfer experiments. The cells and media were analyzed to determine the expressions/releases of SFN and MMP1. UVB exposure increased SFN release from keratinocytes into the medium. The conditioned medium of UVB-irradiated keratinocytes increased MMP1 release from fibroblasts. The depletion of SFN using a siRNA rendered the conditioned medium of UVB-irradiated keratinocytes ineffective at stimulating fibroblasts to release MMP1. p-CA mitigated UVB-induced SFN expression in keratinocytes, and attenuated the MMP1 release by fibroblasts in medium transfer experiments. In conclusion, the present study demonstrated that the use of UV absorbers such as p-CA would reduce UV-induced SFN-centered signaling events involved in skin photoaging.
Jung, Ahjin;Yun, Ji-Sook;Kim, Shinae;Kim, Sang Ryong;Shin, Minsang;Cho, Dong Hyung;Choi, Kwang Shik;Chang, Jeong Ho
Molecules and Cells
/
제42권1호
/
pp.56-66
/
2019
Histidine triad nucleotide-binding protein (HINT) is a member of the histidine triad (HIT) superfamily, which has hydrolase activity owing to a histidine triad motif. The HIT superfamily can be divided to five classes with functions in galactose metabolism, DNA repair, and tumor suppression. HINTs are highly conserved from archaea to humans and function as tumor suppressors, translation regulators, and neuropathy inhibitors. Although the structures of HINT proteins from various species have been reported, limited structural information is available for fungal species. Here, to elucidate the structural features and functional diversity of HINTs, we determined the crystal structure of HINT from the pathogenic fungus Candida albicans (CaHINT) in complex with zinc ions at a resolution of $2.5{\AA}$. Based on structural comparisons, the monomer of CaHINT overlaid best with HINT protein from the protozoal species Leishmania major. Additionally, structural comparisons with human HINT revealed an additional helix at the C-terminus of CaHINT. Interestingly, the extended C-terminal helix interacted with the N-terminal loop (${\alpha}1-{\beta}1$) and with the ${\alpha}3$ helix, which appeared to stabilize the dimerization of CaHINT. In the C-terminal region, structural and sequence comparisons showed strong relationships among 19 diverse species from archea to humans, suggesting early separation in the course of evolution. Further studies are required to address the functional significance of variations in the C-terminal region. This structural analysis of CaHINT provided important insights into the molecular aspects of evolution within the HIT superfamily.
Phosphorus (P) is a macro mineral needed for bone mineralization and cell membrane structure and P is also involved in several fundamental pathways of metabolism in the body. Because of the low concentration and digestibility of P in plant ingredients that are the main components of diets for poultry and pigs, feed phosphates are usually included in diets in addition to the P contributed by plant ingredients. The most widely used feed phosphates in poultry and swine diets are dicalcium phosphate (DCP) and monocalcium phosphate (MCP), but tricalcium phosphate (TCP), monosodium phosphate (MSP), and magnesium phosphate (MgP) may be used as well. Because feed phosphates are mostly produced from rock phosphate, feed phosphates have impurities that contain minerals other than P. Concentrations of P in feed phosphates range from 14.8% (MgP) to 25.7% (MSP). The standardized total tract digestibility (STTD) of P in pigs ranges from 71% (TCP) to 95% (MSP). The STTD of Ca and the standardized ileal digestibility (SID) of P and Ca in feed phosphates fed to pigs and poultry have been determined only in a few experiments. Available data indicate that the STTD of Ca and SID of P in MCP are greater than in DCP in both poultry and pigs, but the SID of Ca is similar between DCP and MCP fed to broilers. Information on mineral concentrations and digestibility values in feed phosphates is needed in diet formulation for pigs and poultry, but if diets are formulated to contain equal concentrations of digestible P and Ca, it is unlikely that animal performance will be impacted by the source of feed phosphates used in the diet.
This study explored the effect of calcium levels and/or ovariectomy on bone metabolism using female Sprague-Dawley weanling rats as a model . Rats received a low (0.1%) calcium diet for 8 weeks. The rats were then divided into three subgroups that were fed 0.1% ,0.5% and 1.5% calcium diets for 8 weeks after operation. The results of this experiment indicate that body weight gin was higher in ovariectomy groups than in sham groups regardless of calcium level and food intake. Serum Ca and P concentrations were of normal level regardless of calcium level and ovariectomy. Estrogen concentration was low in the ovariectomized group. Serum alkaline phophatase activity and urinary hydroxyproline have been used as markers of bone formation and resorption. These values were increased in ovariectomized groups. The weight, length and breaking force of femur were not significantly different between the groups. Ash, Ca, P and total lipid contents in femur and lumbar were decreased in the groups fed low calcium . Mg content was decreased in the ovariectomy and total protein content was not affected by calcium level and ovariectomy. The effect groups of ovarectomy on calcium contents of bone was more prominent in lumbar than in femur. In conclusion, though low calcium intakes during growth period may retard the attainment of peak bone mass, calcium supplementation after this period increased bone growth and mineral contents, but not significant effect in three calcium levels. Furthermore, calcium intake was shown to have a greater influence on the mineral contents of femur than of lumbar, and removal of endogenous estrogen production by ovariectomy was shown to be more deleterious on the ash and calcium contents of the lumbar than of femur.
We have recently reported that $Mg^{++}-deficiency$ showed endothelium dependent relaxation in isolated canine coronary arteries precontracted with $PGF_{2{\alpha}}$. To differentiate the release of EDRF or $PGI_2$ from the endothelium cells as the cause of vasorelaxation by $Mg^{++}-deficiency$, effects of several inhibitors of arachidonic acid metabolism on the relaxation by $Mg^{++}-deficiency$ were evaluated and also compared with that of acetylcholine. Ibuprofen and tranylcypromine ($10{\mu}M$), an inhibitor of cyclo-oxygenase and $PGI_2$ synthetase, respectively, did not effect on $Mg^{++}-free$ induced vasorelaxation. Pretreatment of quinacrine ($10{\mu}M$), an inhibitor of phospholipase $A_2$ and also $Ca^{++}$ uptake, blocked vasorelaxation by $Mg^{++}-free$. But trifluoperazine ($10{\mu}M$), which is about as potent as quinacrine in the inhibition of $Ca^{++}$ uptake, did not effect on $Mg^{++}-deficiency$ induced vasorelaxation. NDGA ($10{\mu}M$), an inhibitor of lipoxygenase, completely restored $Mg^{++}-free$ induced vasorelaxation, even though pretreatment of that was not blocked which might be due to the characteristics of vasorelaxation of NDGA itself. Pretreatment of methylene blue ($10{\mu}M$), which is known as a inhibitor of EDRF through the blocking effect of guanylate cyclase, completely blocked vasorelaxation by $Mg^{++}-free$ as well as acetylcholine ($0.1{\mu}M$). Acetylcholine-induced dose response curve was also antagonized by pretreatment of quinacrine ($10{\mu}M$), but not by ibuprofen, tranylcypromine and NDGA. These results appear to suggest that $Mg^{++}-free$ induced vasorelaxation was mediated by the release of EDRF through the activation of phospholipase $A_2$ and noncyclo-oxygenase on arachidonate metabolism.
In order to study the effect of aqua-acupuncture with Cervi Cornu (C.C.) extract solution manufactured by water-alcohol method on the lipid metabolism and osteoporosis, C.C. aqua-acupuncture was carried out every two days for 60 days on the corresponding bilateral loci of Shinsu (BL23), Taejo (BL11), and Hyonjong (GB39) from the 10th day after the ovariectomy for induced osteoporosis and thereafter the level of serum triglyceride, LDL, Ca, phosphorus and osteocalcin, as well as urine hydroxyproline were measured in the ovariectomized rats. The rats being studied were divided into a normal group (sham operation group), a control group (ovariectomized group), and a C.C. aqua-acupuncture group (group with C.C. aqua-acupunctured to the points of Shinsu, Taejo, and Hyonjong every two days for 60 days from the 10th day after the ovariectomy). The following results were obtained: 1. The serum triglyceride indicated an inhibited increase with statistical significance in the C.C. aqua-acupuncture group compared with the control group. 2. The serum LDL indicated an inhibited increase without any statistical significance in the C.C. aqua-acupuncture group compared with the control group. 3. The serum Ca showed an inhibited decrease without any statistical significance in the C.C. aqua-acupuncture group compared with the control group. 4. The serum phosphorus showed an inhibited decrease with statistical significance in the C.C. aqua-acupuncture group compared with the control group. 5. The serum osteocalcin indicated an inhibited decrease without any statistical significance in the C.C. aqua-acupuncture group compared with the control group. 6. The urine hydroxyproline showed an inhibited increase with statistical significance in the C.C. aqua-acupuncture group compared with the control group.
This study investigated the influence of anthropometric data and nutrient intake on bone mineral density(BMD) and biochemical markers of bone metabolism The mean age of 21 premenopausal women were 47.0 years and that of 41 postmenopausal women whose menopausal age was 49.46 years were 60.56 years. The waist and WHR of postmenopausal women were significantly higher than those of premenopausal ones. The animal protein intake of premenopausal and postmenopausal women were 38.5 and 21.03 g which comprised 54.35 and $31.84\%$ of total protein intake, respectively. The calcium intake of premenopausal and postmenopausal women were 446.45 and 546.97mg which was 63.78 and $78.14\%$ of Korean RDA, respectively. The ALP(Alkaline phosphatase) of premenopausal women was 65.81 U/L, which was significantly lower than that(90.24 U/L) of postmenopausal women (p<0.01). BMD of lumbar spine of premenopausal women was correlated significantly with body weight(r=0.690, p<0.01), waist(r=0.682, p<0.01), WHR(r=0.672, p<0.01), BMI(r=0.559, p<0.01), and body fat(r=0.457, p<0.01). Urinary Ca/creatinine ratio of the premenopausal women was negatively correlated with plant protein(r=-0.529, p<0.05) and plant calcium(r=-0.579, p<0.05). BMD of lumbar spine of postmenopausal women showed positive correlation with lean body mass(r=0.469, p<0.01) and body weight(r=0.383, p<0.05). Urinary Ca/creatinine ratio for the postmenopausal women was positively correlated with ALP(r=0.404, p<0.01) and urinary Na/creatinine ratio(r=0.389, p<0.05). In conclusion, it is necessary to maintain adequate body weight and to increase calcium intake for the premenopausal women. It is also important to increase muscle mass and reduce salt intake for the postmenopausal women.
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