Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation

Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.     

柞蚕抗菌肽D中精氨酸、赖氨酸和色氨酸等氨基酸残基的化学修饰

用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。     

水稻原生质体产生细胞团的冰冻保存和冻后再生植株形成

水稻(Oryza sativa L.)原生质体产生的细胞团加上10-20％的二甲亚枫(DMSO)和10-20％的蔗糖,置于液氮中保存。冻后细胞生存率达到对照的40-50％。存活的细胞在附加2×10~(-5)mol/l 2,4-D 的Linsmier-Skoog(Ls)固体培养基上再生长,然后将形成的愈伤组织块转到附加10~(-6)mol/l NAA,4×10~(-6)mol/l 激动素和10~(-6)mol/l 2 IP 及8％的蔗糖的 LS培养基上分化出芽并形成植株。     

Mammalian DNMTs in the male germ line DNA of Drosophila

It is controversial whether DNA methylation plays a functional role in Drosophila. We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific beta2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.     

ABORTED GAMETOPHYTE 1 is required for gametogenesis in Arabidopsis

In flowering plants, the male and female gametogenesis is a crucial step of sexual reproduction. Although many genes have been identi fied as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1(AOG1) that is newly identi fied as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no signi ficant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG1 caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a signi ficant reduction in male and female fertility.Furthermore, the molecular analyses showed that the aog1 mutant signi ficantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gameto genesis at the stage prior to pollen mitosis I(PMI)in Arabidopsis, possibly through collaboration with other genes.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Factors Associated with Knowledge of and Willingness for Adult Male Circumcision in Changsha,China

Background

Male circumcision (MC) has been shown to reduce the risk of male genital diseases. MC is not commonly practiced among Chinese males and little is known about the factors associated with their knowledge of and willingness for MC. This study was to explore the knowledge regarding the foreskin among Chinese males and to identify factors associated with their willingness to undergo circumcision.

Methods

A total of 237 patients with redundant prepuce/phimosis were interviewed through face-to-face interviews. The items on the questionnaire included: demographics, an objective scale assessing knowledge about the foreskin, willingness to have MC, the attitudes of sexual partners and doctors toward redundant prepuce/phimosis, and the approaches that patients used to acquire knowledge regarding the prepuce. Univariate analysis and multiple logistic regression analysis were performed to identify factors that are associated with willingness to be circumcised (WTC).

Results

A total of 212 patients completed the interview. Multivariable logistic regression showed that three factors were significantly associated with WTC: being married (OR = 0.43), perceiving redundant prepuce/phimosis as a disease (OR = 1.93), and if a patient’s partner supported MC (OR = 1.39). 58% (n = 122) had received information about the foreskin from another party: 18% (n = 37) from school, 8% (n = 17) from family, 17% (n = 36) from friends, 27% (n = 57) from health care providers. About 4% (n = 8) believed that their partners disliked their redundant prepuce/phimosis. 20% (n = 42) had received doctors’ advice to undergo circumcision.

Conclusion

Knowledge about the foreskin was low among Chinese males. Our study elucidates the factors associated with WTC and suggests that more education of the population about the foreskin can help improve the recognition of a correctible abnormality and help patients assess the potential role of MC in their health.     

Stromal miR-200s contribute to breast cancer cell invasion through CAF activation and ECM remodeling

The activation of cancer-associated fibroblasts (CAFs) is a key event in tumor progression, and alternative extracellular matrix (ECM) proteins derived from CAFs induce ECM remodeling and cancer cell invasion. Here we found that miR-200 s, which are generally downregulated in activated CAFs in breast cancer tissues and in normal fibroblasts (NFs) activated by breast cancer cells, are direct mediators of NF reprogramming into CAFs and of ECM remodeling. NFs with downregulated miR-200 s displayed the traits of activated CAFs, including accelerated migration and invasion. Ectopic expression of miR-200 s in CAFs at least partially restored the phenotypes of NFs. CAF activation may be governed by the targets of miR-200 s, Fli-1 and TCF12, which are responsible for cell development and differentiation; Fli-1 and TCF12 were obviously elevated in CAFs. Furthermore, miR-200 s and their targets influenced collagen contraction by CAFs. The upregulation of fibronectin and lysyl oxidase directly by miR-200 or indirectly through Fli-1 or TCF12 contributed to ECM remodeling, triggering the invasion and metastasis of breast cancer cells both in vitro and vivo. Thus, these data provide important and novel insights into breast CAF activation and ECM remodeling, which trigger tumor cell invasion.It has been well established that a reactive microenvironment induces cancer cells to proliferate, migrate and become invasive. Cancer-associated fibroblasts (CAFs) are thought to be the main players among the cohabitating stromal cell types, and they favor tumor progression. The cancer-promoting ability of CAFs is dependent on their activation; however, this process has not been fully explored.The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans and glycoproteins that exerts biochemical and mechanical effects on cells. An increasing body of evidence suggests that ECM remodeling has an important role in cell morphogenesis,¹ survival,² migration and invasion.³ CAFs can deposit certain ECM components and facilitate the directional migration and invasion of carcinoma cells through mechanotransduction-triggered architectural remodeling of the microenvironment.^{4, 5} However, the mechanism by which activated CAFs stimulate the dysregulation of ECM proteins, thus influencing cancer cell invasion, is not well understood.Previously, our team identified a set of dysregulated miRNAs in breast CAFs using a miRNA microarray, and it was found that the levels of miR-200 family members were noticeably suppressed,⁶ indicating their importance in CAF function. Whether these downregulated miR-200 s in the stroma drive the activated phenotype of CAFs as well as aberrant ECM protein expression to promote cancer cell invasion is an intriguing question.The miR-200 s family can be functionally divided into cluster 1 (miR-200a and miR-141) and cluster 2 (miR-200b and miR-200c) according to their ''seed'' region for binding to mRNA. The effects of the miR-200 s on fibrosis, epithelial cell characteristics, cell differentiation and tumor progression have been discussed. For example, miR-200b is essential for the regulation of renal fibrogenesis⁷ and the intestinal fibrosis of Crohn''s disease.⁸ In aggressive carcinoma cells, the maintenance of EMT,⁹ tumor growth,¹⁰ migration,¹¹ invasion,⁹ anoikis resistance¹² and poor response to chemotherapy¹³ are enhanced by the reduced expression of miR-200 s. Furthermore, miR-200 s are upregulated during mammary differentiation¹⁴ but are downregulated in breast cancer stem cells,¹⁵ and these molecules support the maintenance of pluripotent stem cells.¹⁶ These previous reports indicate that miR-200 s may have a significant role in CAF activation.In the current work, we first determined that miR-200 s were commonly downregulated in breast CAFs, and this result was also demonstrated in normal fibroblasts (NFs) co-cultured with breast cancer cells. miR-200 s induced the conversion of NFs into CAFs by targeting Fli-1 and TCF12. Re-expression of miR-200 s in CAFs attenuated the activation-associated CAF phenotype. In particular, miR-200 s and their targets all contributed to CAF-associated ECM remodeling through two key ECM remodeling proteins, fibronectin (FN) and lysyl oxidase, further fueling cancer cell invasion and metastasis. Therefore, our data provide new information regarding the role of CAF activation and function in the promotion of cancer cell invasion through ECM remodeling and provide a considerable amount of information that will be useful for the development of stromal therapeutic targets.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Forest characteristics and population structure of Glyptostrobus pensilis,a globally endangered relict species of southeastern China

The Chinese water pine Glyptostrobus pensilis is the sole surviving species of the genus Glyptostrobus. It is endemic to southern China, central Vietnam, and eastern Laos, and today it is nearly extinct in the wild. Forest community characteristics and population structure of G. pensilis in China have remained un-known up to now. We investigated six swamp forest stands and analyzed their forest community characteristics (i.e. vertical stratification, species composition, and diversity) and population structure, including the frequency distribution of DBH (diameter at breast height) and age-classes as found in Fujian Province, southeastern China. The vertical stratifications of all the forest stands were rather simple. The remaining wild specimens ranged from roughly 15 to some 357 years for an average of ca. 85 years, with only a few individuals less than 20 years old. Compared with the stands and populations of G. pensilis in Vietnam, the taxonomic compositions of the stands in the two regions were different, except for the dominant species-G. pensilis. The Shannon-Wiener index showed the overstory of each stand had much lower diversity (0.26 on average) in Fujian Province than that (1.97 on average) in Vietnam, whereas the diversity indices were about the same (around 2.41) for the understories in the two regions. Furthermore, we discovered 18 G. pensilis seedlings at the study sites in Fujian Province. This discovery demonstrates that G. pensilis regeneration is extremely poor and its populations are declining, although these populations are rela-tively healthier than those in Vietnam.