BMP-2、-3、-6和-12在骨肉瘤细胞株中的表达

目的研究骨形态发生蛋白(bone morphogenetic protein,BMP)-2、-3、-6和-12在骨肉瘤细胞株中的表达,为下一步研究BMPs在骨肉瘤发生发展中的作用奠定基础。方法利用免疫细胞化学法和Western blot法检测BMP-2、-3、-6和-12在人骨肉瘤细胞株MG63、U2OS和大鼠骨肉瘤细胞株UMR106中的内源性表达。结果在MG63、U2OS和UMR106细胞中,BMP-2、-3和-6均呈不同程度的阳性表达;而BMP-12在这3株骨肉瘤细胞中则均为阴性,并且两种检测方法所得结果完全一致。结论BMP-2、-3和-6在人骨肉瘤细胞株MG63、U2OS和大鼠骨肉瘤细胞株UMR106中均有内源性表达;而这3株骨肉瘤细胞中均未检出BMP-12的表达。     

Design, synthesis, and biological evaluation of linear 1-(4-, 3- or 2-methylsulfonylphenyl)-2-phenylacetylenes: a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric 1-(methylsulfonylphenyl)-2-phenylacetylenes possessing a COX-2 SO(2)Me pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 phenyl or substituted-phenyl ring substituent (3-F, 3-OMe, 3-OH, 3-OAc, 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target linear 1,2-diarylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction followed by oxidation of the respective 1-(methylthiophenyl)-2-phenylacetylene intermediate. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) as a potent COX-2 inhibitor (IC(50) = 0.32 microM) with a high COX-2 selectivity index (SI > 320) comparable to the reference compound rofecoxib (COX-2 IC(50) = 0.50 microM; COX-2 SI > 200). A molecular modeling study where (12d) was docked in the binding site of COX-2 showed that the MeSO(2) COX-2 pharmacophore was positioned in the vicinity of the secondary COX-2 binding site near Val(523). The 1-(4-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (11f, COX-1 IC(50) = 1.00 microM; COX-2 IC(50) = 0.06 microM; COX-2 SI = 16.7) and 1-(3-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (12f, COX-1 IC(50) = 6.5 microM; COX-2 IC(50) = 0.05 microM; COX-2 SI = 130) regioisomers exhibited comparable COX-2 inhibition, and moderately lower selective COX-2 selectivity, relative to the reference drug celecoxib (COX-1 IC(50) = 33.1 microM; COX-2 IC(50) = 0.07 microM; COX-2 SI = 472). The most potent anti-inflammatory agent 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) exhibited moderate oral anti-inflammatory activity (ED(50)= 129 mg/kg) at 3 h postdrug administration relative to the reference drug celecoxib (ED(50) = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. The structure-activity data acquired indicate that the acetylene moiety constitutes a suitable scaffold (template) to design novel acyclic 1,2-diarylacetylenes with selective COX-2, or dual COX-1/COX-2, inhibitory activities.     

Synthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

Derivatization of (+/-)-5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline,an imidazoline binding sites ligand,with (+)-(R)-alpha-methylbenzyl isocyanate for drug monitoring purposes

The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.     

Functional analysis of 2-5A-dependent RNase and 2-5a using 2',5'-oligoadenylate-cellulose

2-5A is an intracellular effector that has been implicated in interferon action, hormonal regulation, and cell growth control. 2-5A action is mediated through its activation of 2-5A-dependent RNase (RNase L, RNase F). Affinity resins [2-5A-cellulose and core (2-5A)-cellulose] were chemically synthesized for purification and immobilization of 2-5A-dependent RNase from mouse L cells and rabbit reticulocyte lysates. The breakdown of poly(U)-[3'-32P]Cp to acid-soluble fragments was demonstrated using the 2-5A-dependent RNase:2-5A -cellulose complex; this activity was enhanced by adding (free) 2-5A. In contrast, RNase activity was measured from the 2-5A-dependent RNase:core (2-5A)-cellulose complex only after the addition of free 2-5A. The rabbit reticulocyte 2-5A-dependent RNase is activated only by tetramer or higher oligomers of 2-5A; therefore there was breakdown of poly(U)-[3'-32P]Cp using core (2-5A)-cellulose-bound reticulocyte 2-5A-dependent RNase after addition of tetramer 2-5A but there was no poly(U) degradation in the presence of trimer 2-5A. The absence of significant general nuclease in the assays was demonstrated by the resistance to breakdown of poly(C)-[3'-32P]Cp (not susceptible to 2-5A-dependent RNase). Moreover, core (2-5A)-cellulose was used to develop a sensitive (subnanomolar) assay for the detection of authentic 2-5A. 2-5A, or the material to be tested, was added to mouse L-cell 2-5A-dependent RNase:core (2-5A)-cellulose complex in the presence of poly(U)-[3'-32P]Cp. The concentration of 2-5A in the sample could be measured from the amount of poly(U) degradation. Several closely related analogs of 2-5A were tested and found to be completely inactive. The technology described herein may be applied to the study of the regulation of 2-5A-dependent RNase, the detection of 2-5A from cells and tissues, and other aspects of the 2-5A system.     

2-(p-Nitrophenyl)-2''-deoxyadenosine, a new type of mutagenic nucleoside.

A crude preparation of 2-phenyladenosine was found to be mutagenic in the Ames Salmonella assay. In the purification of this preparation, it was revealed that 2-phenyladenosine itself was nonmutagenic but that 2-(m- and p-nitrophenyl)-adenosines (5m,p) contaminating the sample were the mutagenic principles. A structure-activity relationship study was carried out, and it was found that 5p, 2-(p-nitrophenyl)-adenine (7p), and 2-(p-nitrophenyl)-2'-deoxyadenosine (15p) were strongly mutagenic toward S. typhimurium TA98 and TA100 without metabolic activation, the potency being in the order 15p greater than 7p greater than 5p. The potency of 15p in TA98 was one order of magnitude greater than that of 4-nitroquinoline N-oxide. 15p also showed mutagenicity in the mouse cell line FM3A in culture.     

Temporal and spatial expression of MMP-2,-9,-14 and their inhibitors TIMP-1,-2,-3 in the corpus luteum of the cycling rhesus monkey

A CL develops by extensive cellular reorganization and neovascularization of the remnants of the evacu-ated follicle following ovulation. In both rodent and primate, the development of CL is a rapid process with very high cellular turnover[1,2]. A CL is u…     

Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

Temporal and spatial expression of MMP-2,-9,-14 and their inhibitors TIMP-1,-2,-3 in the corpus luteum of the cycling rhesus monkey

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. If implantation is unsuccessful, luteolysis is initiated. Extensive tissue remodeling occurs during CL formation and luteolysis. In this study, we have studied the possible involvement of MMP-2,-9,-14, and their inhibitors, TIMP-1,-2,-3 in the CL of cycling rhesus monkey at various stages by in situ hybridization, immunohistochemistry and microscopic assessment. The results showed that the MMP-2 mRNA and protein were mainly expressed in the endothelial cells at the early and middle stages of the CL development, while their expressions were observed in the luteal cells at the late stage during luteal regression. MMP-9 protein was detected in the CL at the early and middle stages, and obviously increased at the late stage. The expressions of MMP-14 and TIMP-1 mRNA were high at the early and late stages, and low at the middle stage. TIMP-2 mRNA was high throughout all the stages, the highest level could be observed at the late stage. The TIMP-3 production was detected throughout all the stages, but obviously declined during CL regression. MMP-9,-14 and TIMP-1,-2,-3 were mainly localized in the cytoplasm of the steroidogenic cells. The results suggest that the MMP/TIMP system is involved in regulation of CL development in the primate, and the coordinated expression of MMP-2,-14 and TIMP-1,-3 may have a potential role in the CL formation and the functional maintaining, while the interaction of MMP-2,-9,-14 and TIMP-1,-2,-3 might also play a role in CL regression at the late stage of CL development in the primate.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Chiral synthesis of (2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol

Resolution of (2RS,3RS)-2-[alpha-(2-methoxymethoxyphenoxy)phenylmethyl]morpholine, 11, with (+) mandelic acid led to the formation of (+)-(2S,3S)-2-[alpha-(2-methoxymethoxyphenoxy)phenyl methyl] morpholine (11a). Compound 11 was synthesized in seven steps from (2RS,3RS)-cinnamyl alcohol-2,3-epoxide (4), with an overall yield of 17%. Cleavage of the methoxymethyl group of the Fmoc derivative 12 with catalytic amounts of p-toluenesulfonic acid in methanol afforded (+)-(2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2. The synthetic utility as well as the configuration of compound 2 has been demonstrated by converting (S,S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2 to (2S,3S)-2-[alpha-(2-ethoxyphenoxy)phenylmethyl]morpholine (1) and (2S,3S)-2-(2-methoxyphenoxy) benzyl)morpholine (16), two potential norepinephrine reuptake inhibitors under clinical evaluation.     

B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB.

B-lymphocyte development requires the basic helix-loop-helix proteins encoded by the E2A gene. In this study, the control mechanism of E2A was further explored by disruption of the E2A-related genes, E2-2 and HEB. In contrast to E2A, E2-2 and HEB are not essential for the establishment of the B-cell lineage. However, both E2-2 and HEB are required for the generation of the normal numbers of pro-B cells in mouse embryos. Breeding tests among mice carrying different mutations revealed that E2-2 and HEB interact with E2A in many developmental processes including generation of B cells. Specifically, mice transheterozygous for any two mutations of these three genes produced fewer pro-B cells than the singly heterozygous littermates. This study indicates that B-cell development is dependent not only on an essential function provided by the E2A gene but also on a combined dosage set by E2A, E2-2, and HEB.     

Adiponectin stimulates release of CCL2, -3, -4 and -5 while the surface abundance of CCR2 and -5 is simultaneously reduced in primary human monocytes

The adipokine adiponectin is well known to affect the function of immune cells and upregulation of CCL2 by adiponectin in monocytes/macrophages has already been reported. In the current study the effect of adiponectin on CCL2, -3, -4, and -5 and their corresponding receptors CCR1, CCR2, and CCR5 has been analyzed. Adiponectin elevates mRNA and protein of the CC chemokines in primary human monocytes. Simultaneously the surface abundance of CCR2 and CCR5 is reduced while CCR1 is not affected. Downregulation of CCR2 by adiponectin is blocked by a CCR2 antagonist although expression of the CCL2 regulated genes CCR2 and TGF-beta 1 is not altered in the adiponectin-incubated monocytes. CCL2, -3, and -5 concentrations measured in supernatants of monocytes of normal-weight (NW), overweight (OW), and type 2 diabetic (T2D) patients positively correlate with BMI and are increased in obesity and T2D. In contrast CCL4 is similarly abundant in the supernatants of all of these monocytes. The degree of adiponectin-mediated induction of the chemokines CCL3, -4, and -5 negatively correlates with their basal levels and upregulation of CCL3 and CCL5 is significantly impaired in OW and T2D cells. Serum concentrations of these chemokines are almost equal in the three groups and do not correlate with the levels in monocyte supernatants. In conclusion these data demonstrate that adiponectin stimulates release of CCL2 to CCL5 in primary human monocytes, and induction in cells of overweight probands is partly impaired. Adiponectin also lowers surface abundance of CCR2 and CCR5 and downregulation of CCR2 seems to depend on autocrine/paracrine effects of CCL2.     

Synthesis and antibacterial activity of N-[2-[5-(methylthio)thiophen-2-yl]-2-oxoethyl] and N-[2-[5-(methylthio)thiophen-2-yl]-2-(oxyimino)ethyl]piperazinylquinolone derivatives

A number of N-substituted piperazinylquinolone derivatives were synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative bacteria. Preliminary results indicated that most compounds tested in this study demonstrated comparable or better activity against Staphylococcus aureus and Staphylococcus epidermidis than their parent piperazinylquinolones as reference drugs. Among these derivatives, ciprofloxacin derivative 5a, containing N-[2-[5-(methylthio)thiophen-2-yl]-2-oxoethyl] residue, showed significant improvement of potency against staphylococci, maintaining Gram-negative coverage.     

Syntheses of 2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and Its (2S,3R)-Isomer

2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S,3R)-isomer were respectively synthesized from allyl 2-[(2R,3S)-3-(benzyloxycarbonyloxy)-2-fluorotetradecanamido]-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside and its corresponding (2S,3R)-isomer. Both target compounds did not activate macrophage, but the (2S,3R)-analogue strongly inhibited the binding of LPS to macrophage.     

Synthesis and preliminary biological evaluation of (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines, two novel conformationally constrained l-AP4 analogues

Two novel constrained l-AP4 analogues, (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines (7) and (8), were synthesized and evaluated as mGluR ligands. Compound 7 showed to be a group III mGluRs agonist with micromolar activity.