Scriptaid is a prospective agent for improving human asthenozoospermic sample quality and fertilization rate in vitro.

ABSTRACT: Male infertility is a global issue caused by poor sperm quality, particularly motility. Enhancement of the sperm quality may improve the fertilization rate in assisted reproductive technology (ART) treatment. Scriptaid, with a novel human sperm motility-stimulating activity, has been investigated as a prospective agent for improving sperm quality and fertilization rate in ART. We evaluated the effects of Scriptaid on asthenozoospermic (AZS) semen, including its impact on motility stimulation and protective effects on cryopreservation and duration of motility, by computer-aided sperm analysis (CASA). Sperm quality improvement by Scriptaid was characterized by increased hyaluronan-binding activity, tyrosine phosphorylation, adenosine triphosphate (ATP) concentration, mitochondrial membrane potential, and an ameliorated AZS fertilization rate in clinical intracytoplasmic sperm injection (ICSI) experiments. Furthermore, our identification of active Scriptaid analogs and different metabolites induced by Scriptaid in spermatozoa lays a solid foundation for the future biomechanical exploration of sperm function. In summary, Scriptaid is a potential candidate for the treatment of male infertility in vitro as it improves sperm quality, prolongs sperm viability, and increases the fertilization rate.

Phosphodiesterase 10A inhibitor PF-2545920 as a prospective agent for the clinical promotion of sperm motility.

Phosphodiesterase (PDE) inhibitors can improve sperm motility in patients with asthenozoospermia. However, the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity. We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil. After seminal plasma was discarded, several semen samples were subjected to four treatments (control, PF-2545920, pentoxifylline, and sildenafil) to evaluate their ability to affect motility, viability, and spontaneous acrosome reactions. Intracellular calcium and adenosine triphosphate (ATP), mitochondrial membrane potential, and penetration through viscous medium were assessed by flow cytometry, luciferase, and hyaluronic acid after treatment with PF-2545920. Statistical analyses were performed using the analysis of variance statistical test. PF-2545920 elevated the percentage of motile spermatozoa compared to the control, pentoxifylline, and sildenafil groups at 10 µmol l -1 ( P < 0.01). It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions ( P < 0.05). PF-2545920 also increased mitochondrial membrane potential ( P < 0.001) and altered intracellular calcium ( P < 0.05) in a dose-dependent manner, including increasing sperm hyaluronic acid penetrating ability ( P < 0.05). Therefore, PF-2545920 might be an excellent choice for stimulating the sperm motility.

Soluble expression and purification of human ß-defensin DEFB136 in Escherichia coli and identification of its bioactivity.

Human ß-defensins are an important family of innate host defense peptides with pleiotropic activities. Human ß-defensin 36 (DEFB136) is a novel member of the ß-defensin family which have not been characterized so far. In the present research, the DEFB136 peptide was expressed successfully and purified using the IMPACT-TWIN 1 expression system. The purified DEFB136 peptide was identified by MALDI-TOF mass spectrometry and circular dichroism spectroscopy. While the recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans strains, but had low cytotoxicity to human erythrocytes. In addition, the result of the octet assay showed that the DEFB136 had a high lipopolysaccharide (LPS)-binding affinity, suggesting the DEFB136 may be involved in immunoregulation through its LPS neutralization. These results may help lay the groundwork to understand better the complex interaction between innate host defense and the diversity of the defensin family.

The truncated human beta-defensin 118 can modulate lipopolysaccharide mediated inflammatory response in RAW264.7 macrophages.

The family of human ß-defensins consists of small cysteine-rich peptides, which are receiving significant attention due to their antimicrobial activity. The N-terminal cysteine motif of ß-defensin is considered to contribute to its biological activity. Human ß-defensin 118 (DEFB 118) is a particular anion ß-defensin expressed predominantly in the male reproductive tract, but its physiological activity has not yet been revealed. In order to verify the potential role of the N-terminal domain of DEFB118 peptide in the regulation of infection, the truncated ß-defensin core region of DEFB118 peptide was expressed with IMPACT-pTWIN1 system in Escherichia coli. Herein, the purified homogeneous DEFB118 peptide was identified by mass spectrometry and circular dichroism spectroscopy. The in vitro experiments revealed that DEFB118 peptide exhibited prominent LPS-binding potency (KD: 2.94 nM). Moreover, the DEFB118 core peptide significantly inhibited the mRNA level of LPS-induced inflammatory cytokines including IL-α, IL-1ß, IL-6 and TNF-α in RAW264.7 cells, and correspondingly decreased secretion of IL-6 and TNF-α. We concluded that strong binding of DEFB118 to LPS might prevent LPS from binding to its receptor, and hence inhibited cytokines secretion. The results of this study may be a benefit to elucidate the immune protection of DEFB118 in the male reproductive tract.

Protective Effects of Oridonin on Acute Liver Injury via Impeding Posttranslational Modifications of Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) in the Toll-Like Receptor 4 (TLR4) Signaling Pathway.

OBJECTIVE: Recent researches have demonstrated that inflammation-related diseases are effectively regulated by posttranslational modifications (PTMs) including phosphorylation and acetylation. Our previous study found a new acetyltransferase inhibitor, oridonin, which had a protective effect on acute liver injury (ALI). In the present study, we further investigated its protective mechanism against D-galactosamine (D-Gal) combined with lipopolysaccharide- (LPS-) induced ALI in mice. METHODS: Intraperitoneal injections of LPS (40 µg/mouse)/D-Gal (5 mg/mouse) were given to the mice, and the experimental group was pretreated with intraperitoneal injection of oridonin (0.2 mg/mouse). To elucidate the protective mechanism of oridonin, we collected liver specimens and used RNA-sequencing (RNA-Seq) analysis. We focused on the genes that were upregulated by LPS/D-Gal and downregulated after pretreatment with oridonin. The downregulated genes examined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were further verified by real-time polymerase chain reaction (PCR) and western blot. RESULTS: GO analysis showed that genes that were downregulated after pretreatment with oridonin were extremely concentrated in immune response, chemotaxis, and inflammatory response. Real-time PCR confirmed that the expression of these genes was upregulated by LPS/D-Gal induction and reduced after treatment with oridonin, which was consistent with RNA-Seq results. KEGG pathway analysis showed a significantly enriched downregulated gene that was present in the Toll-like receptor (TLR) 4 signaling cascade. Our results manifested that phosphorylation levels of upstream signaling molecules in the TLR4 signaling cascade, including extracellular signal-regulated kinase (ERK), P38, and IκB, were significantly inhibited by oridonin. Furthermore, LPS/D-Gal stimulation triggered posttranslational modifications of related gene loci in the TLR4 signaling pathway, including phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4 T345/S346) and acetylation of IRAK4 (K34). However, after treatment with oridonin, the modification pattern of IRAK4 expression stimulated by LPS/D-Gal was suggestively attenuated. CONCLUSION: Our study revealed that the protective effects of oridonin on LPS/D-Gal-induced ALI mediated by inhibition of the PTMs of IRAK4, including phosphorylation of T345/S346 and acetylation of K34.

Tideglusib, a prospective alternative to nonoxynol-9 contraceptive.

OBJECTIVES: We aimed to evaluate the antifertility activity and vaginal irritation effects of tideglusib in vivo using rabbit models and to evaluate the cytotoxical effects of tideglusib to sperm, vaginal cells and vaginal bacteria (L. acidophilus) in vitro. STUDY DESIGN: We treated female rabbits with vaginal tideglusib 1 mM, nonoxynol-9 (N-9) or vehicle control (Poloxamer 407). In experiment 1, we sacrificed females (n = 6 each) after 10 days of daily administration and assessed vaginal histological changes using Eckstein irritation score. In experiment 2, females (n = 9 each) received estradiol benzoate to induce ovulation 24 h prior to vaginal treatment followed by introduction of a fertile male. These females underwent necropsy at the 21st day to assess pregnancy status. In experiment 3, we used an HTM-TOX IVOS sperm motility analyzer and scanning electron microscopy (SEM) to evaluate the effect of tideglusib on human sperm samples. In experiment 4, we evaluated the effect of tideglusib on lactobacillus and vaginal cell growth in vitro. RESULTS: The total irritation score of tideglusib vs. N-9 was 3.4 ±â€¯2.07 vs. 7.8 ±â€¯3.82, p <.05. The pregnancy rate of tideglusib, N-9 and control group was 11.1%, 0% and 88.9%, respectively. Tideglusib exhibited a dose-dependent spermostatic/spermicidal activity, and the minimum effective concentrations of tideglusib and N-9 were 8.724 ±â€¯3.047 µM and 219.75 ±â€¯41.78 µM, respectively. SEM and transmission electron microscopy revealed acrosomal membrane impairments caused by tideglusib. Tideglusib was much less toxic to vaginal cells and L. acidophilus than N-9 in vitro. CONCLUSIONS: Evaluation using rabbit models indicated that tideglusib is a prospective spermicidal contraceptive with low vaginal irritation effects. IMPLICATIONS: Tideglusib or tideglusib analogues may be a contraceptive with perspective to replace N-9. It is possible for a spermicide to balance spermicidal activity and vaginal/cervical irritation effects very well.

Oridonin ameliorates lipopolysaccharide/D-galactosamine-induced acute liver injury in mice via inhibition of apoptosis.

We investigated the protective effects exerted by oridonin, the main active constituent of the Chinese medicinal herb Rabdosiarubescens, against lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury (ALI). An ALI model was induced in mice using LPS (40 µg/0.5 ml) and D-Gal (5 mg/0.5 ml). The mice were randomly divided into the following five groups of six mice each: one control group (a), one ALI group (b), two oridonin treatment groups (c and d), and one oridonin control group (e). Oridonin (0.2 mg/0.5 ml) was administered once 1 h prior to the LPS/D-Gal challenge in group c and a total of three times over a period of four days, with the last dose given at 1 h before the LPS/D-Gal challenge, in group d. Pretreatment with oridonin improved the survival rate, alleviated histopathological abnormalities, and suppressed plasma aminotransferases in the LPS/D-Gal-challenged mice. Importantly, oridonin attenuated LPS/D-Gal-induced apoptosis in hepatocytes by reducing pro-apoptotic signals (P<0.05), such as tumor necrosis factor-α (TNF-α) and c-Jun N-terminal kinases (JNK). Furthermore, JNK-associated mitochondrial pro-apoptotic proteins were also suppressed by pretreatment with oridonin. Taken together, these data show that oridonin exerts protective effects against LPS/D-Gal-induced ALI in mice via a mechanism that may involve the suppression of the pro-apoptotic cytokine TNF-α and JNK-associated pro-apoptotic signaling.

DDX11-AS1 as potential therapy targets for human hepatocellular carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most fatal cancers, whose incidence and death rates are still rising. Here, we report the identification of long non-coding RNAs (IncRNAs) that associated with HCC progression and metabolism based on the systematically analysis of large scale RNA-seq data from HCC patients. We identified seven lncRNAs with high confidence which were highly related with prognostic of HCC. Of note, three of them had quite different expression patterns between the control samples and the patients, and their critical roles in cancer progression were validated. We proposed that DDX11-AS1 play important role during HCC oncogenesis and may serve as potential therapy target for HCC.

Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis.

Several enzymes involved in central carbon metabolism such as isocitrate lyase and phosphoenolpyruvate carboxykinase are key determinants of pathogenesis of Mycobacterium tuberculosis (M. tb). In this study, we found that lysine acetylation plays an important role in the modulation of central carbon metabolism in M. tb. Mutant of M. tb defective in sirtuin deacetylase exhibited improved growth in fatty acid-containing media. Global analysis of lysine acetylome of M. tb identified three acetylated lysine residues (K322, K331, and K392) of isocitrate lyase (ICL1). Using a genetically encoding system, we demonstrated that acetylation of K392 increased the enzyme activity of ICL1, whereas acetylation of K322 decreased its activity. Antibodies that specifically recognized acetyllysine at 392 and 322 of ICL1 were used to monitor the levels of ICL1 acetylation in M. tb cultures. The physiological significance of ICL1 acetylation was demonstrated by the observation that M. tb altered the levels of acetylated K392 in response to changes of carbon sources, and that acetylation of K392 affected the abundance of ICL1 protein. Our study has uncovered another regulatory mechanism of ICL1.

Histone deacetylase inhibitor induces cell apoptosis and cycle arrest in lung cancer cells via mitochondrial injury and p53 up-acetylation.

The reversibility of non-genotoxic phenotypic changes has been explored in order to develop novel preventive and therapeutic approaches for cancer. Quisinostat (JNJ-26481585), a novel second-generation histone deacetylase inhibitor (HDACi), has efficient therapeutic actions on non-small cell lung cancer (NSCLC) cell. The present study aims at investigating underlying molecular mechanisms involved in the therapeutic activity of quisinostat on NSCLC cells. We found that quisinostat significantly inhibited A549 cell proliferation in dose- and time-dependent manners. Up-acetylation of histones H3 and H4 and non-histone protein α-tubulin was induced by quisinostat treatment in a nanomolar concentration. We also demonstrated that quisinostat increased reactive oxygen species (ROS) production and destroyed mitochondrial membrane potential (ΔΨm), inducing mitochondria-mediated cell apoptosis. Furthermore, exposure of A549 cells to quisinostat significantly suppressed cell migration by inhibiting epithelial-mesenchymal transition (EMT) process. Bioinformatics analysis indicated that effects of quisinostat on NSCLC cells were associated with activated p53 signaling pathway. We found that quisinostat increased p53 acetylation at K382/K373 sites, upregulated the expression of p21(Waf1/Cip1), and resulted in G1 phase arrest. Thus, our results suggest that the histone deacetylase can be a therapeutic target of NSCLC to discover and develop a new category of therapy for lung cancer.

Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms.

Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms.

Characterization of ß-defensin 42 expressed in principal cells at the initial segment of the rat epididymis.

ß-defensins, preferentially expressed in male reproductive tracts, particularly in the testes and epididymis with region-specific patterns, play an important role in both innate immunity and sperm fertility. Expressed in the caput region of epididymis, ß-defensins have been known to contribute to innate immunity, sperm motility initiation, and maintenance. However, ß-defensins of the initial region remain to be uncharacterized. In this study, rat ß-defensin 42 (Defb42) was revealed to be exclusively located in the principal cells at the initial segment of the rat epididymis and its sperm's acrosome. Furthermore, the expression of Defb42 was dependent on luminal testicular factors and developmental phases. The recombinant Defb42 was predominantly antimicrobial not against Candida albicans, but against Escherichia coli and Staphylococcus aureus. Based on these findings, Defb42 was suggested to play a dual role in sperm fertility and host defense in rat epididymis.

Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions.

Production and characterization of recombinant human beta-defensin DEFB120.

Public health of human beings is threatened by superbugs. Novel human beta-defensins, which contribute to host defense against pathogen invasion and innate immune protection, might be a potent natural candidate pool for new antibiotic lead screening. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli and identified the purified homogeneous proteins using MALDI-TOF mass spectrometry. Then, we performed the fundamental studies on the structure and biological functions for the DEFB120 peptide. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response. These results may pave a way for exploiting the essential physiological functions of DEFB120 and also for the development of natural antibiotic pools.

Soluble fusion expression, characterization and localization of human ß-defensin 6.

Human ß­defensin 6 (DEFB106) is an antimicrobial peptide expressed in the epididymis, testis and lung, which indicates that DEFB106 may be involved in innate immunity and fertility. However, as a ß­defensin, this protein has not been well characterized. Using an intein­mediated fusion expression system, the recombinant DEFB106 was expressed and purified (yield, 3­5 mg/l) under optimized conditions. The purified protein was characterized using mass spectrometry and circular dichroism spectroscopy. The measured molecular weight was consistent with its theoretical value and the predominant secondary structure was ß­sheet, the common structure of ß­defensin family members. The purified DEFB106 showed antimicrobial activity against not only Escherichia coli (E. coli) and Candida albicans (C. albicans) SC5314, but also Staphylococcus aureus (S. aureus) CMCC26003. Furthermore, it exhibited a high affinity for heparin and lipopolysaccharide. In addition, it was determined that native DEFB106 was located in the epididymis, bone marrow and skin. These observations may aid in the determination of the physiological and pathological functions of DEFB106.

[Expression of recombinant human ZP3 protein using the baculovirus expression system].

OBJECTIVE: To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3. METHODS: The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored. RESULTS: The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm. CONCLUSION: It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.

The novel human ß-defensin 114 regulates lipopolysaccharide (LPS)-mediated inflammation and protects sperm from motility loss.

Lipopolysaccharide (LPS) is an important pathological factor involved in serious inflammatory diseases and male reproductive impairments. Emerging evidence demonstrates that antimicrobial peptides possess protective activity in response to LPS-induced inflammation. However, the LPS-binding and/or immunosuppressive activity of ß-defensins (DEFBs) has been underestimated. In the present work, we characterized a novel human defensin, DEFB114, which was expressed predominantly in the epididymis and gingival cells at the RNA level. Homogenous recombinant DEFB114 peptides were prepared and characterized using mass spectrometry. DEFB114 protein exhibited a broad spectrum of antimicrobial activity with salt sensitivity against typical pathogenic microbes (i.e. Escherichia coli, Staphylococcus aureus, and Candida albicans). Interestingly, DEFB114 demonstrated novel LPS-binding activity in vitro and inhibited TNF-α release in RAW264.7 cultures through the inhibition of MAPK p42/44 when challenged with LPS. Moreover, DEFB114 could also rescue the LPS-induced reduction of human sperm motility in vitro and protect d-galactosamine-sensitized C57BL/6 mice from LPS-induced lethality in vivo. The protective activity of DEFB114 on RAW264.7, human sperm, and the d-galactosamine-sensitized mice was disulfide bond-dependent because alkylated DEFB114 lost its activity. The low cytotoxicity of the DEFB114 peptide toward human erythrocytes is indicative of its potential therapeutic use in the treatment of LPS-induced inflammation, LPS contamination, and potentially septic shock.

Spink13, an epididymis-specific gene of the Kazal-type serine protease inhibitor (SPINK) family, is essential for the acrosomal integrity and male fertility.

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.

Human beta-defensin DEFB126 is capable of inhibiting LPS-mediated inflammation.

ß-Defensins are cationic, antimicrobial peptides that participate in antimicrobial defense as well as the regulation of innate and adaptive immunity. Human ß-defensin 126 (DEFB126) is a multifunctional glycoprotein consisting of a conserved ß-defensin core and a unique long glycosylated peptide tail. The long glycosylated peptide tail has been proven to be critical for efficient transport of sperm in the female reproductive tract, preventing their immune recognition, and efficient delivery of capacitated sperm to the site of fertilization. However, the functions of the conserved ß-defensin core remain to be fully elucidated. In the present work, the conserved ß-defensin core of the DEFB126 was expressed to explore its potential antimicrobial and anti-inflammatory activities. The DEFB126 core peptide exhibited both high potency for binding and neutralizing lipopolysaccharide (LPS) in vitro, and potent anti-inflammatory ability by down-regulating the mRNA expression of pro-inflammatory cytokines including IL-α, IL-1ß, IL-6 and TNF-α in a murine macrophage cell line RAW264.7. The treatment with the DEFB126 core peptide also led to correspondingly decreased secretion of IL-6 and TNF-α. The blockade of LPS-induced p42/44 and p38 MAPK signal pathway might contribute to the anti-inflammation effects of the DEFB126 core peptide. Furthermore, fluorescence-labeled DEFB126 could enter RAW 264.7 cells and reduce the production of LPS-stimulated inflammatory factors, implying that DEFB126 might also participate in intracellular regulation beyond its direct LPS neutralization. In summary, our results demonstrate that the DEFB 126 core peptide has critical functions in parallel to its C-terminal tail by showing LPS-binding activity, anti-inflammatory effects and intracellular regulatory function.

Soluble fusion expression and characterization of human beta-defensin 3 using a novel approach.

Human ß-defensin 3 (DEFB103) is a recently identified small cysteine-rich cationic peptide expressed ubiquitously upon local microbial invasion. A number of accumulating evidences indicate that this peptide is involved in many biological processes, including microbicidal activities, chemoattraction, and immunomodulation. In this article, we describe a novel approach through which we performed the expression and purification of the recombinant DEFB103 peptide in Escherichia coli (E. coli) based on the pTWIN1 expression system. This approach does not introduce any extra residues to the peptide product, and also eliminates the requirement of removing the fusion tag by exogenous proteases. A high yield of 112 mg of soluble fusion DEFB103 was obtained in 1 liter of Luria-Bertani (LB) medium. By one-step affinity chromatography and on-column, auto-cleavage of the fusion tag, the mature DEFB103 peptide was produced with a yield of 30 mg/L LB. The purified DEFB103 peptide demonstrated strong antimicrobial activities against E. coli, S. aureus and C. albicans, which were representatives of Gram-negative and Gram-positive bacteria and fungi, respectively. Using this novel approach, we have successfully expressed and purified several human defensins with significant bioactivities. Our work may be helpful for structural and functional studies of other human defensins, and also for the production of human defensins.