Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology / 现代检验医学杂志

Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

Comprehensive evaluation of the quality of Amomum tsao -ko based on entropy weight TOPSIS method / 中国药房

OBJECTIVE To evaluate the quality of Amomum tsao -ko from different origins and harvesting periods comprehensively. METHODS The contents of total volatile oil in A. tsao -ko were determined by volatile oil measurement method A stated in 2020 edition of Chinese Pharmacopoeia （part Ⅳ）；the contents of total flavonoids and total polyphenols in A. tsao -ko were determined by aluminum nitrate-sodium nitrite colorimetry and folin-ciocalteu method. The contents of α-pinene，β-pinene， 1，8-cineole，α-terpineol，geraniol and trans-nerolidol in the volatile oil of A. tsao -ko were determined by gas chromatography ；the contents of protocatechuate and vanillic acid in A. tsao -ko were determined by ulta high performance liquid chromatography. The above 11 indicators were selected ，and entropy weight TOPSIS method was used to comprehensively evaluate the quality of 16 batches of A. tsao -ko. RESULTS The contents of total volatile oil ，total flavonoids ，total polyphenols ，α-pinene，β-pinene， 1，8-cineole，α-terpineol，geraniol，trans-nerolidol，protocatechuate and vanillic acid in 16 batches of A. tsao -ko were 15.833 3- 28.000 0 μL/g，29.100 5-78.199 6 mg/g，6.789 8-35.797 7 mg/g，0.088 7-0.401 3 mg/g，0.106 3-0.408 0 mg/g，3.709 6-8.533 1 mg/g，0.259 8-0.599 6 mg/g，0.314 8-1.324 1 mg/g，0.272 3-0.576 4 mg/g，9.301 2-19.818 5 μg/g，8.180 9-27.666 3 μg/g， respectively. Entropy weight TOPSIS results showed that the top three of relative closeness rankings were A. tsao -ko produced by Yunnan Baoshan in July ，Yunnan Honghe in October ，Yunnan Wenshan in September ；the last three of relative closeness rankings were A. tsao -ko produced by Yunnan Dehong in September ，Yunnan Dehong in November ，Yunnan Dehong in December. CONCLUSIONS A. tsao -ko produced by Yunnan Baoshan in July ，Yunnan Honghe in October and Yunnan Wenshan in September present better quality.

CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats

BACKGROUND Cancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150­190 g, 6­8 weeks) to evaluate the capability of these cells to control cancer progression and metastasis. RESULTS qPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis. CONCLUSIONS Although further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer.

Study on in vitro Antioxidant Activities of Different Polar Parts of Ethanol Extract from Amomum tsao-ko / 中国药房

OBJECTIVE：To evaluate in vitro antioxidant activities of 4 different polar parts of ethanol extract from Amomum tsao-ko，and to lay a foundation for the research and development of antioxidant chemical components in the plant. METHODS ： The dried fruits of A. tsao-ko were crushed ，then were hearted and reflux extracted with 95％ ethanol. The extraction fluid was concentrated by rotary evaporation and evaporated in water bath to obtain the ethanol extract. The extract was dispersed in water ， and then extracted with petroleum ether ，ethyl acetate and n-butanol organic solvents one by one. Each solvent extract was combined and the lower water phase were collected. Finally ，the petroleum ether part ，ethyl acetate part ，n-butanol part and water part were obtained，after rotary evaporation concentration and water bath evaporation. Through in vitro antioxidant activity tests ，using 2， 6-di-tert-butyl-4-methylphenol（BHT）as positive control ，DPPH radical ，superoxide anion radical scavenging ability and Fe 3+ reducing ability of different polar parts of ethanol extract from A. tsao-ko were investigated. RESULTS ：The scavenging rates of 4 polar parts of ethanol extract from A. tsao-ko on DPPH radical were all over 80％；the order of scavenging ability was ethyl acetate part＞BHT＞n-butanol part ＞petroleum ether part ＞water part. Those of the 4 polar parts to superoxide anion radical were between about 30％-40％ mostly；the order of scavenging ability was n-butanol part ＞petroleum ether part ＞water part ＞ethyl acetate part ＞ BHT；but those were weaker than their scavenging ability to DPPH r adical. The polar parts of ethanol extract also had a certain reduction ability to Fe 3+；the order of the reduction ability was n-butanol part ＞BHT＞ethyl acetate part ＞petroleum ether part ＞ water part on the whole ，but that of water part rose to the stron- gest when its concentration was 4.0 μg/mL. CONCLUSIONS： The different polar parts of ethanol extract from A. tsao-kom have certain in vitro antioxidant capacity ，but the order of antioxidant activity of different polar parts was not the same in different antioxidant activity tests ；ethyl acetate part has the 163.com strongest scave nging effect on DPPH radical ，n-butanol part has the strongest scavenging ability on superoxide anion radical and reducing ability on Fe 3+.

Study on the Optimization of Purification Technology of Total Flavonoids from Amomum tsao-ko by Macroporous Adsorption Resin / 中国药房

OBJECTIVE：To establish a method for the content determination of to tal flavonoids from Amomum tsao-ko ，and to optimize the purification technology by macroporous resin. METHODS ：The content of total flavonoids was measured by HPLC. The determination was performed on Eclipse Plus C 18 column with mobile phase consisted of acetonitrile- 1％ acetic acid solution （15∶85，V/V）at the flow rate of 0.8 mL/min. The column temperature was 40 ℃，and the detection wavelength was set at 256 nm. The sample size was 10 μL. Taking the adsorption and desorption performance as indexes，6 kinds of macroporous resins were screened out by static adsorption and desorption tests ；adsorption and desorption time were investigated by static adsorption and desorption kinetics tests. Using the content of total flavonoids （calculated by rutin ）as index ，with sample concentration ，sample pH，ethanol volume fraction and elution amount as factors ，based on single factor test ，orthogonal design was used to optimize the purification technology of total flavonoids from A. tsao-ko ，and validation test was performed. RESULTS ：The linear range of rutin were 0.028-0.281 mg/mL（r＝0.999 9）. The limit of quantification was 437.5 ng/mL and the limit of detection was 109.4 ng/mL. RSDs of precision ，stability and reproducibility tests were all lower than 2％；the recoveries were 96.24％-99.75％（RSD＜2％，n＝ 6）. The comprehensive capacity of adsorption and desorption of HPD 450 macroporous resin was the most suitable ，and the best static adsorption and desorption time both were 12 h. The optimal purification technology was 1.854 4 mg/mL ； ethanol elution was 8 times of the column volume. Vertificationtests show that after optimized ，the content of total flavonoids from A. tsao-ko increased from 22.556 7 mg/g to 57.728 2 mg/g. The purity of was 2.56 and stable for the content determination. Optimal purification technology is stable and feasible ，which is suitable for purifieation of total flavonoids from A. tsao-ko .

Nitric Oxide Inhibitory Constituents from the Fruits of Amomum tsao-ko

Bioactivity-guided fractionation of MeOH extract of the dried fruits of Amomum tsao-ko led to isolation of nine compounds (1 – 9). Their structures were elucidated by spectroscopic methods including extensive 1D and 2D-NMR, as alpinetin (1), naringenin-5-O-methyl ether (2), naringenin (3), hesperetin (4), 2′,4′,6′-trihydroxy-4-methoxy chalcone (5), tsaokoin (6), boesenbergin B (7), 4-hydroxyboesenbergin B (8), and tsaokoarylone (9). Of these, compound 8 was isolated from a natural source for the first time, which was previously reported as a synthetic product. The isolated compounds (1 – 9) were tested for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages. Among them, three chalcone derivatives (compounds 5, 7, and 8) and a diarylheptanoid (compound 9) exhibited significant inhibitory activity on the NO production with IC₅₀ values ranging from 10.9 to 22.5 µM.

DNA barcoding sequence analysis of Amomum tsao-ko germplasm resources in Yunnan Province / 中草药

Objective: To screen and evaluate DNA barcoding of Amomun tsao-ko populations in Yunnan. Methods: ITS, psbA-trnH, matK, rbcL, and ycf1 sequences were screened and evaluated using A. tsao-ko as samples. The samples of A. tsao-ko population were amplified and sequenced. The sequences were spliced with Genestar, and then processed with Mega for data processing. And A. tsao-ko diversity and identification were analyzed and discussed. Results: The length of the amplified fragments of primers ITS5 and ITS4 was approximately 520 bp; The length of the amplified fragments of the primers rbcLa-F and rbcLa-R was approximately 498 bp; The length of the amplified fragments of the primers ycf1-bF and ycf1-bR was approximately 800 bp; The length of the amplified fragments of the primers psbA-trnH-1F and psbA-trnH-1R was approximately 400 bp; The length of the amplified fragments of the primers matK-2F and matK-2R was approximately 470 bp. The success rate of amplification and sequencing was high, and most of the results were available. By analyzing the amplification results of ITS, psbA-trnH, matK and ycf1 sequences of A. tsao-ko, A. tsao-ko and other Amomum genus plants can be clearly distinguished; All samples of the ITS sequence were divided into MG5 white flower A. tsao-ko population and other populations; All samples of the psbA-trnH sequence were divided into MG5 white flower A. tsao-ko population, MG6 yellow flower A. tsao-ko population and other populations; All samples of the matK sequence were divided into MG6 A. tsao-ko population and other populations. The MG5 white flower A. tsao-ko sample failed to be amplified; All samples of the ycf1 sequence were divided into the MG6 yellow flower A. tsao-ko population and other populations, and the MG5 white flower A. tsao-ko population was clustered with the other 22 A. tsao-ko populations; The amplification of rbcL sequence was consistent for all samples. Conclusion: The ITS, matK, psbA-trnH and ycf1 sequences can accurately distinguish A. tsao-ko from other plants of Amomum genus; The sequence site variations were found in matK, psbA-trnH and ycf1 sequences of MG6. This research has contributed to the selection and breeding of A. tsao-ko varieties. ITS and psbA-trnHsequences can distinguish yellow flower and white flower of A. tsao-ko; There is no variation in the rbcL sequence of all samples of white and yellow flowers of A. tsao-ko, and Amomum tsao-ko and other plants of Amomum genus cannot be identified with the rbcL sequence, which can be discarded.

In Vivo Validation Model of a Novel Anti-Inflammatory Scaffold in Interleukin-10 Knockout Mouse

BACKGROUND: We fabricated anti-inflammatory scaffold using Mg(OH)2-incorporated polylactic acid-polyglycolic acid copolymer (MH-PLGA). To demonstrate the anti-inflammatory effects of the MH-PLGA scaffold, an animal model should be sensitive to inflammatory responses. The interleukin-10 knockout (IL-10 KO) mouse is a widely used bowel disease model for evaluating inflammatory responses, however, few studies have evaluated this mouse for the anti-inflammatory scaffold. METHODS: To compare the sensitivity of the inflammatory reaction, the PLGA scaffold was implanted into IL-10 KO and C57BL/6 mouse kidneys. Morphology, histology, immunohistochemistry, and gene expression analyses were carried out at weeks 1, 4, 8, and 12. The anti-inflammatory effect and renal regeneration potency of the MH-PLGA scaffold was also compared to those of PLGA in IL-10 KO mice. RESULTS: The PLGA scaffold-implanted IL-10 KO mice showed kidneys relatively shrunken by fibrosis, significantly increased inflammatory cell infiltration, high levels of acidic debris residue, more frequent CD8-, C-reactive protein-, and ectodysplasin A-positive cells, and higher expression of pro-inflammatory and fibrotic factors compared to the control group. The MH-PLGA scaffold group showed lower expression of pro-inflammatory and fibrotic factors, low immune cell infiltration, and significantly higher expression of anti-inflammatory factors and renal differentiation related genes compared to the PLGA scaffold group. CONCLUSION: These results indicate that the MH-PLGA scaffold had anti-inflammatory effects and high renal regeneration potency. Therefore, IL-10 KO mice are a suitable animal model for in vivo validation of novel anti-inflammatory scaffolds.

Analysis of genetic diversity and genetic relationship of Amomum tsao-ko germplasm resources in Yunnan by SSR markers / 中草药

Objective To evaluate the genetic diversity and phylogenetic relationships of Amomun tsao-ko populations in Yunnan. Methods Seven pairs of microsatellite (SSR) primers were used to analyze 24 A. tsao-ko populations; First, GenALEx was used to calculate genetic diversity parameters, PCoA and AMOVA analysis was carried out; NTsys software was then used to draw population clusters map; And finally, the Structure software was used to calculate the best K value. Results The average of Shannon’s diversity index (H) of the 24 A. tsao-ko populations was 0.49, the average of heterozygosity (He) was 0.32, the genetic differentiation coefficient (Fst) was 0.090, and the gene flow (Nm) was 2.930. Eighty-one percent of the genetic differentiation among the 24 populations of A. tsao-ko existed within the population, and only 19% existed among the populations. The genetic identity (I) of the 23 A. tsao-ko populations of yellow flowers was 0.631 8-0.982 4. The genetic distance (D) was in the range of 0.017 7- 0.459 2, while the consistency degree of the A. tsao-ko population of white flower (MG5) and 23 other yellow flowers was 0.369 7-0.609 0. However, cluster analysis showed that A. tsao-ko population of the white flowers and yellow flowers were clearly separated at the genetic distance of 0.49. Structure clustering showed 209 A. tsao-ko resources can be divided into four groups when K value was 4. Conclusion The genetic diversity of A. tsao-ko populations of yellow flowers of Yunnan is higher on average, and the genetic variation is mainly found in population rather than among populations. According to the genotypes, A. tsao-ko of yellow flower and white flower are clearly divided into two categories, and the genetic distance is very far; and the yellow flower of A. tsao-ko is roughly divided into four groups.

Chemical constituents from fruits of Amomum paratsao-ko / 中草药

Objective: To investigate the chemical constituents of Amomum paratsao-ko. Methods: The chemical constituents were separated and purified consecutively by silica gel, Sephadex LH-20 column chromatography, recrystallization as well as preparative HPLC. Their structures were determined by physicochemical properties and spectral analyses. Results: Fourteen compounds were isolated and identified as 2(E)-2-decene-1,10-di-yl-acetate (1), 2(E)-2-decene-1,10-diol (2), β-sitosterol (3), 3,5-dihydroxy- 7,4’-dimethoxyflavone (4), rhamnocitrin (5), kaempferol (6), rhamnetin (7), kaempferol-3,7,4’-O-trimethylether (8), ombuin (9), quercetin (10), kumatakenin (11), octyl ferulate (12), (E)-decyl-3-(4-hydroxy-3-methoxyphenyl) propenoate (13), and trans-p-hydroxycinnamic acid (14). Conclusion: Compound 1 is a new compound named paratsaokoster, and compounds 2-14 are isolated from this plant for the first time.

Chemical constituents from mangrove plant Sonneratia paracaseolaris / 中草药

Objective: To study the chemical constituents of the methanol extract from the aerial parts of the mangrove plant Sonneratia paracaseolaris. Methods: The methanol extract was isolated and purified with various chromatographic methods, including silica gel, ODS, Sephadex LH-20 columns, TLC, and HPLC. The compounds were identified by their physical chemical properties and 1H-NMR and 13C-NMR data. Results: Seventeen compounds were obtained from the methanol extract of Sonneratia paracaseolaris and identified as phytol (1), stigmasta-4-ene-3,6-dione (2), stigmata-4,22-diene-3,6-dione (3), cholesterol (4), (22E)-cholesterol-5,22-diene-3β-alcohol (5), diosmetin (6), tricin (7), 5,3′,5′-trihydroxy-7,4′-dimethoxyflavone (8), 5-hydroxy- 7,4′-dimethoxyflavone (9), 5-hydroxyl-7,3′,4′-trimethoxydihydroflavone (10), vanillin (11), p-hydroxy benzaldehyde (12), salicylic acid (13), trans-p-hydroxyl ethyl cinnamate (14), 4-hydroxy-2,6-dimethoxy-benzaldehyde (15), 3,4,5-trimethoxybenzoic acid (16), and 3,3′,4-trimethoxyellagic acid (17). Conclusion: All the compounds except 4, 11, 15, and 17 are obtained from genus Sonneratia. All compounds are isolated from S. paracaseolaris for the first time.

Analysis and comparison of RNA expression profiles in the heart and liver of Fkbp51 knockout mice / 中国比较医学杂志

Objective To study the function of Fkbp51 in the heart and liver by analyzing the differential RNA expression profiles in the wild-type mice (WT) and Fkbp51 knockout (KO) mice, and to elucidate the role of Fkbp51 gene in metabolic pathways in the heart and liver.Methods Using the second generation of high-throughput gene sequencing technology, the mRNA expression profiles of heart and liver were sequenced in WT and Fkbp51 KO mice.The data of sequencing of heart tissues were analyzed by DEGseq, and the results of sequencing of liver tissues were analyzed by BRB-Array Tools.The differential genes of the heart and liver in the mice were screened respectively.Gene ontology (GO) analysis and KEGG pathway analysis were performed to analyze the differentially expressed genes using the online tool DAVID.In addition, the differential genes of the two organ tissues were analyzed by Venn diagram.The interaction network of proteins was analyzed using the STRING database.Results (1) The absence of Fkbp51 led to changes in mRNA expressions of heart-related signal pathways such as vascular smooth muscle contraction, chemokine, retinol, and MAPK signaling pathways.(2) The lack of Fkbp51 mostly induced changes in cholesterol synthesis and metabolism, lipid metabolism, redox and other related genes and pathways in the liver.(3) In the heart and liver, Fkbp51 deletionresult ed in four co-differential genes, among them, down-regulation of Rnaset2b, Hmga1 and Fkbp51, while Cyp2b10 was down-regulated in the heart but up-regulated in the liver.All these proteins may interact with HSP90 protein and participat in the metabolism of heart and liver tissues.Conclusions Fkbp51 is involved in different metabolic and gene expression regulation pathways of heart and liver, and the roles are both independent and interrelated.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

No expression of porcine endogenous retrovirus after pig to monkey xenotransplantation / 한국실험동물학회지

This study was performed to investigate the expression of two porcine endogenous retrovirus (PERV) elements, PERV gag and full-length conserved PERV, in blood cells collected periodically from organ-recipient monkeys that underwent pig to non-human primate xenotransplantation. The heart and kidney-respectively acquired from alpha-1,3-galactosyltransferase knockout (GT-KO) pigs that survived for24 and 25 days-were xenografted into cynomolgus monkeys. The two PERV elements expressed in the xenografted GT-KO pig organs were not present in the blood cells of the recipient monkeys. In the present study, we deduced that PERVs are not transmitted during GT-KO pig to monkey xenotransplantation.

<b>Kampo Medicine and Immune Systems as Revealed </b><b>in the Ko-tei-nai-kei </b> / 日本東洋医学雑誌

From around 1990, it has been suggested that our internal defense system is composed of two distinct elements ; local innate immunity principally arranged on surface areas to establish barriers against various pathogens, and systemic acquired immunity mainly seen in systemic compartments to survey and control internal damage and disorders. The former innate surface barrier is chiefly regulated via species-restricted CD 1 antigen-presenting molecules, through lipid/glycolipid antigens presented mainly by dendritic cells (DCs) and lacking antigen-specific memory through gene-rearrangements, while the latter acquired barrier is controlled by individually restricted MHC molecules and gains antigen-specific memory through gene-rearrangements. Surprisingly, it had been revealed more than 2,000 years before in the ancient Chinese medicine textbook, <i>Ko-tei-nai-kei</i>, that our defense system is also classified into two categories, named “defense-qi” and “nutritional-qi”, and shown that the former “defense-qi” is arranged at the surface of skin to control our sweat and interact with “muddy” substances, while the latter “nutritional-qi” is situated on and within blood vessels and produces purified nutrients from food, drink and other exogenous substances. In this review, based on our recent understanding of immunological progress and the modern concepts of immunity, the possible relationship between “defense-qi” and innate immunity as well as “nutritional-qi” and acquired immunity are discussed.

Immunohistochemical Study on Early Odontogenesis in Hsp70 Knock-out Mice Fetuses Exposed by Maternal Hyperthermia / 대한해부학회지

To investigate the effects of maternal hyperthermia on early odontogenesis,pregnant Hsp70 knock-out and wild type mice at embryonic day (ED)8.5 were immersed in a 43 degrees C water bath until their core body temperature reached that temperature,and then given a further 5 min of hyperthermia.Untreated Hsp70 WT mice fetuses were used as the control group.Fetuses were collected at EDs 13.5,15.5 and 17.5.Developing teeth in the mandible were processed for histological and immunohistochemical studies.Tissue sections were immunostained for FGF-8 and FGF -4 and observed using light microscopy.In the controls, FGF-8 immunolocalization was observed in cells within the dental lamina and in apically located dental epithelium at ED 13.5.However,a few cells were immunopositive in the heat shocked (HS)group.At EDs 15.5 and 17.5 of the control group,the basal lamina adjacent to the dental pulp showed positive immunostaining.In contrast,most of the dental epithelium was immunopositive at ED 15.5 in the HS group and inner and outer dental epithelial cells were continuously immunopositive by ED 17.5.FGF-4 immunolocalization was found in apical dental epithelium at ED 13.3 in the control group,but no such positive reaction was observed in the HS group.At ED 15.5 in the controls,basal lamina and dental epithelium near the cervical loop were immunopositive.In contrast,early cap-stage teeth had cells near the mouth of the dental bud and cervical loop that were immunopositive to FGF-4 in the HS group.In controls at ED 17.5,cells near the future secondary enamel knot were immunopositive,whereas most of the dental epithelium except for cells in the mouth of the dental lamina was negative in the HS group.Thus,maternal hyperthermia may inhibit normal odontogenesis through sustained production of FGF-8 and downregulation of FGF-4.

Effects of Commercially Available Mineral Waters on Decoction of Kampo Medicines / 日本東洋医学雑誌

Kampo medicines containing Bupleuri Radix (Sho-saiko-to and Sho-saiko-to plus Fossilia ossis mastodi and Ostreae testa) were decocted with four kinds of mineral waters and tap water, and the extracts were analyzed for saikosaponin contents by HPLC. The results indicated that the yield of the extracted materials was the largest when Kampo medicines were decocted with the hard water compared with other mineral water extracts. However, the same extract contained the smallest amount of saikosaponin b₂ of those tested. Extractions made with the mineral waters having a weakly acidic or weakly alkaline nature gave similar yields of the extracted materials and saikosaponin b₂ contents.<br>Present results suggest a possibility that decoction using a hard water significantly affects extraction of certain ingredients in Kampo medicine.

A Case of Contact Dermatitis due to Shiun-ko and Taitsu-ko / 日本東洋医学雑誌

A 50-year-old female developed severe dermatitis on her face during topical therapy with Shiun-ko and Taitsu-ko. The skin lesions almost disappeared in two weeks with oral steroid therapy and did not recur. Patch testing showed positive reactions to Shiun-ko, Taitsu-ko and Lithospermi radix, and beeswax, which were constituents of Shiun-ko and Taitsu-ko. The result suggests that the patient was sensitized with these topical medicaments and developed allergic contact dermatitis.

Experiences in the Use of Shiun-ko for Verruca Vulgaris / 日本東洋医学雑誌

Twenty-seven cases with verruca vulgaris were examined in which the treatment consisted of oral administration of biscoclaurin alkaloids and Kampo medicine (Jumi-haidoku-to) with Siun-ko ointment. Seven of these cases were cured within one month-12 cases were cured within three months; and 8 cases required more than 3 months for a cure to be effected. However, there were no regressions or ineffective cases. Histopathological features of 5 cases examined after two or four weeks usage of Siun-ko ointment showed swelling of keratinocytes with formation of lacunae or fissures. This combination is thought to be an effective and useful treatment.