PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制

通过PCR扩增获得了包含多聚半乳糖醛酸酶(PG)全部阅读框架的1.5kb cDNA,经限制酶酶谱和部分序列分析鉴定无误后,将其以反方向插入含两个增强子的35s启动子和Nos3＇端之间,构建成表达PG反义RNA的双元载体,经农杆菌途径转化番茄品种“丽春”,获得了60株抗卡那霉素再生植株,经PCR检测,证明有2／3的再生植株有外源PG基因导入,成熟果实的PG粗提液的SDS—PAGE分析表明：若干株系中PG蛋白量较对照有不同程度的下降。PG活性亦同步下降,其中一个株系3^＃,PG酶活下降了93%。这些结果表明外源PG基因的反方向导入有效地抑制了内源PG基因的表达。     

Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16

PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.     

Prostaglandin (PG) accumulation by mouse ovaries, oviducts, and uteri

Uteri, ovaries and oviducts from mice were collected at autopsy. Tissue slices were incubated with [³H]-PGE₂ in the presence or absence of a large excess (100 fold) of nonradioactive PGE₂ using 0.01M sodium phosphate buffer (pH 7.2). Bound and free PGs were separated by a filtration technique. PGE₂ accumulation by the uteri was evaluated as a function of incubation time, wet weight of tissues, and reproductive state. The tissue to medium ratio (T/M) was greater than 1.0 for uteri as the time of incubation increased. This suggests the presence of PGE₂ binding sites in mouse uterine tissue. Also, PGE₂ accumulation was not observed in oviducts or in ovaries.     

The Role of Polygalacturonase (PG) in the Ripening of Apple Fruits

Polygalacturonase (PG) activity and changes in respiratory intensity of apple fruits were investigated. The respiratory rate was decreased to a preclimacteric minimum from 30 Aug. to 20 Sept., Then increased to a climacteric peak (20–30 Sept.) and again drop down gradually with approaching the senescence stage. The PG activity was undetectable in a developing fruit until the onset climacteric phase. It rose rapidly after harvest, and reaching its highest level on 27 Oct. Just a month after the climacteric peak. The PG activity fell gradually. The amount of the fractions of pectic acid in fruits changed with the modifications of PG activity. With the ripening of fruits, the content of alcohol-soluble small molecules of pectic acids was increased from 12 to 13 5 mg/100 g of tissue, while the amount of alcohol- insoluble large molecules of pectic acids reduced from 530 to 280/100 g of tissue. PG activity would indicate the destruction of cell walls and the separation of cells. The onset of softening of fruits occurred 20 days after the rise of PG activity. It is supposed that the process of softening is directly controlled by PG activity.     

Prostaglandin(PG) E2 in regulation of immunity and inflammatory diseases

Prostaglandin(PG) E2 is an important metabolic product of arachidonic acid. PGE2 plays important roles in regulation of fever, inflammatory responses and blood pressure via four functionally antagonistic E-prostanoid (EP) receptors, which are designated as EP1, EP2, EP3 and EP4, respectively. Recently, there is increasing evidence that PGE2 also regulates the maturation of immune cells and immune response. This review aims to briefly summarize and discuss the recent findings regarding the role of PGE2 in regulation of immunity.     

前列腺素E2对免疫细胞及炎症相关疾病的调控作用

前列腺素E2(prostaglandin E2,PGE2)是一种极其重要的脂质代谢产物,在受到生理或病理的各种刺激,尤其是有害刺激时被释放,在发热、炎症和血压调节中均发挥着重要作用.PGE2通过4种功能相互拮抗的的受体(E-prostanoid receptors,EP1、EP2、EP3和EP4)而广泛参与机体及细胞代谢过程.一直以来,人们都认为前列腺素在免疫过程中发挥的作用相当有限,部分原因是因为抑制类前列腺素合成的非甾体类抗炎药在机体免疫过程中没有发挥明显作用.但近来的研究表明,PGE2很可能在免疫细胞的发育分化及免疫应答过程中也起重要作用.本文主要对前列腺素与免疫系统的关系进行分析和总结,并重点讨论相关研究的新近进展.     

Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 Å, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming ∼20 Å above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.To create antibodies capable of effectively neutralizing human immunodeficiency virus type 1 (HIV-1), the adaptive humoral response is driven to exceptional lengths (reviewed in reference 8). Indeed, the response often fails, and sera from individuals infected with HIV-1 typically display limited neutralization breadth (59). After several years of infection, however, antibodies capable of neutralizing diverse viral strains develop in 15 to 25% of infected individuals (3, 16, 32, 33, 49, 53). Details of the adaptive changes that allow for effective recognition are of direct vaccine relevance, and clues from rare neutralizing antibodies have been eagerly sought.Two broadly neutralizing antibodies, PG9 and PG16, were recently identified with single cell-sequencing techniques after direct microneutralization assessment of secreted antibody from individually plated, stimulated B cells (58). These antibodies are somatically related and appear to be derived from the same recombination of heavy and light chains. They both recognize a site on HIV-1 gp120 composed of elements from the second and third variable regions (V2 and V3). Despite the vaunted diversity of the HIV-1 gp120 envelope and the even higher sequence variability in the V2 and V3 regions (26), neutralization assays indicate that the recognized epitope is conserved in 70 to 80% of circulating viral isolates (58).To investigate the molecular features of PG9 and PG16 that account for their neutralization effectiveness, we prepared antigen-binding fragments (Fabs) of each antibody and screened for crystallization. We were able to obtain a number of crystals, and those of PG16 proved suitable for structural analysis. Determination of the PG16 structure visualized several unusual features, and structure-function analysis indicated that two features, extensive affinity maturation and an exceptionally long heavy chain-third complementarity-determining region (CDR H3), were critical to its neutralization effectiveness. Barriers to eliciting these two features provide a likely explanation for the rarity of antibodies like PG9 and PG16; understanding and overcoming such barriers may form the basis for an effective HIV-1 vaccine.     

Characterization of bone PG II cDNA and its relationship to PG II mRNA from other connective tissues.

Two cDNA clones encoding the small proteoglycan II (PG II) of bone were isolated from a lambda gt11 expression library. These clones expressed recombinant protein which was cross-reactive with polyclonal and monoclonal antisera to PG II molecules from several connective tissues. The longest clone, lambda Pg 20 was studied in detail. The clone was shown to encode PG II by hybrid selected translation and immunoprecipitation. Northern analysis showed two species of the PG II message of approximately 1.4 and 1.8 kb. Substantial amounts of PG II message were found in bone, tendon, articular cartilage, skin, smooth muscle and cornea. Trace amounts of message were also detected in liver and brain. Radiolabeled bovine PG II cDNA hybridized to RNA from several other species including the human, rat and chicken. The level of PG II mRNA in chick embryonic fibroblasts was sensitive to transformation by Rous sarcoma virus.     

Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16

The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1^−/−) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.It is argued that an effective HIV vaccine should include a component that induces a broadly neutralizing antibody response (2, 3, 21, 25, 32, 37, 39, 54). The key target for broadly neutralizing HIV antibodies is the envelope spike, which consists of a compact, metastable heterodimeric trimer of the glycoproteins gp120 and gp41 (43, 62).gp120 is one of the most heavily glycosylated proteins known, with up to 50% of its mass arising from carbohydrates attached to roughly 25 N-linked glycosylation sites (31) determined by the NXT/S consensus sequence (where X can be any amino acid except Pro) (1). Glycosylation significantly impacts the folding and conformation of envelope spikes, thus affecting antigenicity and immunogenicity (30, 35). Carbohydrates are generally poorly immunogenic, and the dense covering of glycans is often referred to as the “silent face” or “glycan shield” (58). The glycans have also been suggested to have an important role in viral transmission through interaction with lectins, in particular the C-type lectin DC-SIGN, which is found on the surfaces of dendritic cells and is thought to aid the transport of virus to anatomical sites rich in CD4⁺ T cells, such as lymph nodes (8, 16).Although the positioning of N-linked protein glycosylation is encoded by the protein sequence (1), the type of glycan displayed (high mannose, hybrid, or complex) is not under direct genetic control but is determined by the three-dimensional structure of a protein and its interaction with the biosynthetic cellular environment, including accessibility to glycan-processing enzymes (50). For example, highly clustered glycans prevent access of the processing enzymes, leading to high-mannose-type glycans being displayed (6, 23). Therefore, the glycosylation of recombinant HIV envelope proteins can vary significantly depending on the protein sequence, structure, and the cell in which they are expressed (50). Although the positions of many glycans are relatively conserved between isolates and clades (60), there can be variation in the occupancy and precise nature of the glycans displayed at these positions on recombinant envelope (7, 17-19, 61). However, we have recently observed major differences between the glycosylation of recombinant envelope proteins and envelope expressed on the virion surface, with the latter being dominated by Man_5-9GlcNAc₂ oligomannose glycans (9). Nevertheless, significant glycan heterogeneity remains on the virion surface.Recently, two new neutralizing antibodies, PG9 and PG16, were isolated from an African clade A-infected donor and shown to be both broad and potent (56). From a panel of 162 viruses, PG9 neutralized 127 and PG16 neutralized 119 viruses at a median potency that exceeded that of the broadly neutralizing antibodies—2G12, b12, 2F5, and 4E10—by about an order of magnitude. In a TZM-bl neutralization assay, PG9 has been shown to neutralize 87% of a panel of 82 viruses (M. Seaman, unpublished data). Both PG9 and PG16 show preferential trimer binding and interact with an epitope formed from conserved regions of the V1/V2 and V3 variable loops. Mutation of N160, an N-linked glycosylation site in the V2 loop, completely abolishes PG9 and PG16 neutralization, suggesting the N160 glycan is important in forming the PG9 and PG16 epitope. Further, PG9 shows significant binding to monomeric gp120 DU422 and treatment of the glycoprotein with Endo H (removing high-mannose glycans) results in significant reduction in antibody binding. Occasionally, neutralization of some pseudoviruses by PG16 in particular has revealed an unusual neutralization profile with a shallow slope and plateaus at <100%. We hypothesized that this unusual neutralization profile may be related to antibody sensitivity to glycosylation and, more specifically, could be due to glycan profile or partial glycosylation at critical sites.We show here that loss of any one of several glycosylation sites in the V1, V2, and V3 loops has significant effects on the sensitivity of pseudovirus to neutralization by PG9 and PG16. Generating pseudovirus in the presence of various glycosidase inhibitors also has notable effects on antibody neutralization. We show that some of these effects are isolate dependent and others are universal and can be used to help identify the presence of PG9 and PG16-like antibodies in the serum of HIV-1-infected patients (57). For some isolates displaying aberrant neutralization profiles as described above, we found that changing the glycan profile on the HIV-1 trimer using glycosidase inhibitors or a mutant cell line resulted in higher neutralization plateaus and neutralization profiles with the more usual sigmoidal shape. Changes in sensitivity to neutralization were also observed for some but not all isolates. The antibody-gp120 interaction was not inhibited by sugar monosaccharides found in glycans on the HIV envelope. The results suggest PG9 and PG16 recognize a conformational epitope that is dependent on the glycosylation at specific variable loop N-linked glycosylation sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.     

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.