Adjuvanticity of Processed Aloe vera gel for Influenza Vaccination in Mice.

The effectiveness of current influenza vaccines is considered suboptimal, and 1 way to improve the vaccines is using adjuvants. However, the current pool of adjuvants used in influenza vaccination is limited due to safety concerns. Aloe vera, or aloe, has been shown to have immunomodulatory functions and to be safe for oral intake. In this study, we explored the potential of orally administered processed Aloe vera gel (PAG) as an adjuvant for influenza vaccines in C57BL/6 mice. We first evaluated its adjuvanticity with a split-type pandemic H1N1 (pH1N1) Ag by subjecting the mice to lethal homologous influenza challenge. Oral PAG administration with the pH1N1 Ag increased survival rates in mice to levels similar to those of alum and MF59, which are currently used as adjuvants in influenza vaccine formulations. Similarly, oral PAG administration improved the survival of mice immunized with a commercial trivalent influenza vaccine against lethal homologous and heterologous virus challenge. PAG also increased hemagglutination inhibition and virus neutralization Ab titers against homologous and heterologous influenza strains following immunization with the split-type pH1N1 Ag or the commercial trivalent vaccine. Therefore, this study demonstrates that PAG may potentially be used as an adjuvant for influenza vaccines.

Plausible Minimal Substrate for Erm Protein.

Erm proteins methylate a specific adenine residue (A2058, Escherichia coli coordinates) conferring macrolide-lincosamide-streptogramin B (MLSB) antibiotic resistance on a variety of microorganisms, ranging from antibiotic producers to pathogens. To identify the minimal motif required to be recognized and methylated by the Erm protein, various RNA substrates from 23S rRNA were constructed, and the substrate activity of these constructs was studied using three Erm proteins, namely, ErmB from Firmicutes and ErmE and ErmS from Actinobacteria The shortest motif of 15 nucleotides (nt) could be recognized and methylated by ErmS, consisting of A2051 to the methylatable adenine (A2058) and its base-pairing counterpart strand, presumably assuming a quite similar structure to that in 23S rRNA, an unpaired target adenine immediately followed by an irregular double-stranded RNA region. This observation confirms the ultimate end of each side in helix 73 for methylation, determined by the approaches described above, and could reveal the mechanism behind the binding, recognition, induced fit, methylation, and conformational change for product release in the minimal context of substrate, presumably with the help of structural determination of the protein-RNA complex. In the course of determining the minimal portion of substrate from domain V, protein-specific features could be observed among the Erm proteins in terms of the methylation of RNA substrate and cooperativity and/or allostery between the region in helix 73 furthest away from the target adenine and the large portion of domain V above the methylatable adenine.

Development of functional composts using spent coffee grounds, poultry manure and biochar through microbial bioaugmentation.

Spent coffee grounds (SCG), poultry manure, and agricultural waste-derived biochar were used to manufacture functional composts through microbial bioaugmentation. The highest yield of tomato stalk-based biochar (40.7%) was obtained at 450°C with a surface area of 2.35 m2 g-1. Four pilot-scale composting reactors were established to perform composting for 45 days. The ratios of NH4+-N/NO3--N, which served as an indicator of compost maturity, indicate rapid, and successful composting via microbial bioaugmentation and biochar amendment. Moreover, germination indices for radish also increased by 14-34% through augmentation and biochar amendment. Microbial diversity was also enhanced in the augmented and biochar-amended composts by 7.1-8.9%, where two species of Sphingobacteriaceae were dominant (29-43%). The scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) were enhanced by 14.1% and 8.6% in the fruits of pepper plants grown in the presence of the TR-2 (augmentation applied only) and TR-3 (both augmentation and biochar amendment applied) composts, respectively. Total phenolic content was also enhanced by 68% in the fruits of the crops grown in TR-3. Moreover, the other compost, TR-L (augmentation applied only), boosted DPPH scavenging activity by 111% in leeks compared with commercial organic fertilizer, while TR-3 increased the phenolic content by 44.8%. Composting facilitated by microbial augmentation and biochar amendment shortened the composting time and enhanced the quality of the functional compost. These results indicate that functional compost has great potential to compete with commercially available organic fertilizers and that the novel composting technology could significantly contribute to the eco-friendly recycling of organic wastes such as spent coffee grounds, poultry manure, and agricultural wastes.

Overview of Glucagon-Like Peptide-1 Receptor Agonists for the Treatment of Patients with Type 2 Diabetes.

BACKGROUND: It is estimated that 29.1 million people or 9.3% of the US population have diabetes, which contributes to considerable medical and financial burden. Type 2 diabetes mellitus is characterized by insulin resistance and insulin secretion impairment leading to hyperglycemia. The presence of insulin resistance is strongly correlated with obesity. OBJECTIVE: This article reviews the available glucagon-like peptide-1 (GLP-1) receptor agonists and their role in the management of patients with diabetes, to help guide the selection of the most suitable agent for the individualized treatment of patients with type 2 diabetes. DISCUSSION: This article reviews the evidence from phase 3 clinical trials for each of the 5 GLP-1 receptor agonists by comparing them against one another and with other existing therapies, including metformin, dipeptidyl peptidase-4 (DPP-4) inhibitors, and sulfonylureas. Incretin-based therapies have emerged as attractive agents for the treatment of type 2 diabetes. They target the GLP-1 hormone, which is partly responsible for insulin release and for attenuating hyperglycemia during meals (ie, the incretin effect). The 2 classes of incretin-based therapy currently available are GLP-1 receptor agonists and DPP-4 inhibitors, which prevent the breakdown of GLP-1. Both classes are attractive options, given their glucose-lowering effects without the adverse effects of hypoglycemia and weight gain. The different mechanisms of action of these therapies result in generally greater efficacy with GLP-1 receptor agonists, albeit at the expense of slightly increased gastrointestinal symptoms. These agents exert their effects by improving glucose-dependent insulin release, suppressing glucagon release, suppressing hepatic glucose output, and decreasing the rate of gastric emptying, thereby reducing appetite. Currently, 5 GLP-1 receptor agonists are available, including exenatide, liraglutide, albiglutide, dulaglutide, and lixisenatide; semaglutide may soon become available as the newest agent. With the exception of the investigational oral semaglutide, which has shown promising results, the other 5 agents are administered as subcutaneous injections, at different dosing intervals. CONCLUSION: Currently, 5 GLP-1 receptor agonists are available for use in the United States. Although they are all in the same drug class, some significant differences exist among the various GLP-1 receptor agonists. The choice of a specific GLP-1 receptor agonist will depend on the patient preferences, potential adverse effects, and cost.

Prevention of azoxymethane/dextran sodium sulfate-induced mouse colon carcinogenesis by processed Aloe vera gel.

The preventive effect of a processed Aloe vera gel (PAG) on colon carcinogenesis was examined using an azoxymethane (AOM)-initiated and dextran sodium sulfate (DSS)-promoted mouse colon carcinogenesis model. Oral administration of PAG (200, or 400mg/kg/day) significantly reduced the multiplicity of colonic adenomas and adenocarcinomas compared with the AOM/DSS only-treated mice. In the mice treated with 400mg/kg of PAG, adenoma and adenocarcinoma development was reduced to 80% and 60%, respectively, compared to 100% in the PAG-untreated AOM/DSS-treated mice. Western blot analysis using colon extracts showed that PAG reduced the activation of nuclear factor kappa B (NF-κB), resulting in the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression. PAG appeared to inhibit the NF-κB activation through the activation of peroxisome proliferator-activated receptor gamma. PAG also inhibited the expression and phosphorylation of signal transducer and activator of transcription 3, which is known to connect inflammation and cancer. In addition, PAG inhibited cell cycle progression-inducing cellular factors, such as extracellular signal-regulated kinases 1/2, cyclin-dependent kinase 4, and cyclin D1. On the other hand, PAG increased the expression of Caudal-related homeobox transcription factor 2, which is known to be a tumor suppressor in colorectal cancer. These findings show that PAG suppresses colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon.

From the Cover: Ethylmercury-Induced Oxidative and Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death: Involvement of Autophagosome-Lysosome Fusion Arrest.

Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Ca2+ levels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

Induction of mast cell degranulation by triterpenoidal saponins obtained from Cimicifugae rhizoma.

Cimicifugae rhizoma has been widely used as a traditional herbal medicine to treat inflammation and menopausal symptoms. In this study, we found that some of the triterpenoidal saponins purified from the ethanol extract of Cimicifugae rhizoma dramatically induced histamine release. The structure-related induction of mast cell degranulation by them and the mechanism of action were determined. ß-Hexosaminidase release in HMC-1 cells was increased in a concentration-dependent manner, with maximal 6.5- and 8.5-fold increases, by 200 µg/mL 24-epi-7,8-didehydrocimigenol-3-O-xyloside (comp 1) and cimigenol 3-O-beta-d-xyloside (comp 4) compared with those treated with phorbol 12-myristate 13-acetate and A23187 (PMACI), respectively. However, ß-hexosaminidase release was not changed by 7,8-dihydrocimigenol (comp 3), or 23-OAc-shengmanol-3-O-xyloside (comp 7). These triterpenoidal saponins changed neither the intracellular Ca(2+ )level nor the activation of PKC, both of which play essential roles in histamine release. However, cromolyn and ketotifen, membrane stabilizers, effectively inhibited the ß-hexosaminidase release induced by comp 1 or comp 4 by 39 and 45%, respectively. Collectively, xylose on the cimigenol-related backbone among triterpene glycosides isolated from Cimicifugae rhizoma may play an important role in activating mast cells and induction of degranulation partly via membrane destabilization of mast cells.

Physical and Antibacterial Properties of Gold-Poly(ethyl methacrylate) Nanocomposites Prepared by In-Situ Polymerization.

In this paper, the physical and antimicrobial properties of gold-poly(ethyl methacrylate) nanocomposites (Au-PEMA) are evaluated. Characterization of gold nanoparticles was carried out based on UV-Vis spectroscopy and transmission electron microscopy (TEM). The specimens were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and field emission scanning electron microscope (FE-SEM). We identified the thermal stability of Au-PEMA nanocomposites and the inhibitory effect of live bacterial attachment of Au-PEMA nanocomposites against S. mutans was also evaluated.

Diagnosis and treatment of patients with thyroid cancer.

BACKGROUND: Thyroid cancer is the most common malignancy of the endocrine system, representing 3.8% of all new cancer cases in the United States and is the ninth most common cancer overall. The American Cancer Society estimates that 62,450 people in the United States will be diagnosed with thyroid cancer in 2015, and 1950 deaths will result from the disease. OBJECTIVE: To review the current approach to the diagnosis and treatment of patients with thyroid cancer. DISCUSSION: Over the past 3 decades, there has been a dramatic increase in the number of people diagnosed with thyroid cancer, which may be attributable to the wide use of imaging studies, including ultrasounds, computed tomography, magnetic resonance imaging, and positron emission tomography scans that incidentally detect thyroid nodules. Thyroid cancer is divided into several main types, with papillary thyroid cancer being the most common. The treatment options for patients with thyroid cancer include the surgical removal of the entire thyroid gland (total thyroidectomy), radioactive iodine therapy, and molecular-targeted therapies with tyrosine kinase inhibitors. This article summarizes the diagnosis and treatment of thyroid cancer, with recommendations from the American Thyroid Association regarding thyroid nodules and differentiated thyroid cancer. Recently approved drugs and treatment trends are also explored. CONCLUSION: The prognosis and treatment of thyroid cancer depend on the tumor type and its stage at the time of diagnosis. Many thyroid cancers remain stable, microscopic, and indolent. The increasing treatment options for patients with thyroid cancer, including therapies that were recently approved by the US Food and Drug Administration, have kept the mortality rate from this malignancy low, despite the increase in its incidence. Early diagnosis and appropriate treatment can improve prognosis and reduce mortality.

Estrogenic endocrine-disrupting chemicals modulate the production of inflammatory mediators and cell viability of lipopolysaccharide-stimulated macrophages.

Estrogenic endocrine-disrupting chemicals (EDCs) are exogenous substances that act as competitive inhibitors of estrogen in the endocrine system. By disrupting the endocrine system, EDCs can cause severe disabilities and diseases, including cancers and altered sexual development. Although the influence of these molecules in the endocrine system is evident, the effects of EDCs on the immune system as well as their cytotoxicity have been poorly examined. Therefore, we selected 21 EDCs that are commonly found in Korean ecosystems, such as surface waters and effluents, and studied their immunologic effects by comparing nitric oxide (NO) production and cytotoxicity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (RAW cells), a macrophage cell line. Among the EDCs tested, fenitrothion (FTH) inhibited the messenger RNA (mRNA) expression of inducible NO synthase (iNOS), resulting in reduced NO production, while treatment with andostenedione (AD), diethyl phthalate, di-n-butyl phthalate (DBP), estriol, or molinate decreased production of NO in an iNOS-independent fashion. In contrast, benzo(a)pyrene (B(a)P) increased the production of NO in RAW cells. In addition, AD, DBP, or FTH inhibited the mRNA expression of tumor necrosis factor alpha or interleukin-1 beta. Treatment with 17-α-ethynylestradiol, 17-ß-estradiol, 4-n-butyl phenol, or alachlor induced apoptosis of RAW cells, while dicyclohexyl phthalate and B(a)P caused cell death in an apoptosis-independent manner. These data suggest that EDCs can influence the immune response to pathogens by modulating the functions of macrophages.

Processed Aloe vera gel ameliorates cyclophosphamide-induced immunotoxicity.

The effects of processed Aloe vera gel (PAG) on cyclophosphamide (CP)-induced immunotoxicity were examined in mice. Intraperitoneal injection of CP significantly reduced the total number of lymphocytes and erythrocytes in the blood. Oral administration of PAG quickly restored CP-induced lymphopenia and erythropenia in a dose-dependent manner. The reversal of CP-induced hematotoxicity by PAG was mediated by the functional preservation of Peyer's patch cells. Peyer's patch cells isolated from CP-treated mice, which were administered PAG, produced higher levels of T helper 1 cytokines and colony-stimulating factors (CSF) in response to concanavalin A stimulation as compared with those isolated from CP-treated control mice. PAG-derived polysaccharides directly activated Peyer's patch cells isolated from normal mice to produce cytokines including interleukin (IL)-6, IL-12, interferon-γ, granulocyte-CSF, and granulocyte-macrophage-CSF. The cytokines produced by polysaccharide-stimulated Peyer's patch cells had potent proliferation-inducing activity on mouse bone marrow cells. In addition, oral administration of PAG restored IgA secretion in the intestine after CP treatment. These results indicated that PAG could be an effective immunomodulator and that it could prevent CP-induced immunotoxic side effects.

Effects of a new sustained-release microsphere formulation of exenatide, DA-3091, on obese and non-alcoholic fatty liver disease mice.

The aim of this study was to examine the effects of a new sustained-release (SR) microsphere formulation of exenatide, DA-3091, on body weight gain and hepatic injury in high fat diet (HFD)-induced obese mice and high sucrose diet (HSD)-induced non-alcoholic fatty liver disease (NAFLD) mice. Then, we determined whether DA-3091 has the potency as a drug for the treatment of metabolic disease. In obese mice, after 8-week treatment, the body weight gain was significantly more suppressed by both 1 mg/kg and 2 mg/kg of DA-3091, monthly subcutaneous administered, than by 10 mg/kg/day of sibutramin, a drug against obesity. In NAFLD mice, a significant reduction in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, representative markers of hepatic injury, was observed after biweekly subcutaneous administration of 1 mg/kg and 2 mg/kg of DA-3091 for 8 weeks. A significant reduction in hepatic lipid accumulation was observed in DA-3091 treated groups as well. Based on these results, it is demonstrated that DA-3091 has the potency as a drug for the treatment of metabolic disease.

2,3,6-Trisubstituted quinoxaline derivative, a small molecule inhibitor of the Wnt/beta-catenin signaling pathway, suppresses cell proliferation and enhances radiosensitivity in A549/Wnt2 cells.

GDK-100017, a 2,3,6-trisubstituted quinoxaline derivative, reduced ß-catenin-T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity and inhibited cell proliferation in a dose-dependent manner with an IC50 value of about 10 µM in A549/Wnt2 cells. GDK-100017 down-regulated the expression of Wnt/ß-catenin pathway target genes such as cyclin D1 and Dkk1 but not c-myc or survivin. GDK-100017 inhibited cell proliferation by arresting the cell cycle in the G1 phase not only in A549/wnt2 cells but also in SW480 colon cancer cells. In addition to its wnt signaling inhibitory properties, GDK-100017 also enhanced the radiosensitivity of the A549 human NSCLC line. These results suggest that GDK-100017 possesses potential anti-cancer activity by inhibiting the Wnt/ß-catenin signal pathway, blocking the ß-catenin-TCF/LEF interaction, and enhancing radiosensitivity.

A tobacco CBL-interacting protein kinase homolog is involved in phosphorylation of the N-terminal domain of the cucumber mosaic virus polymerase 2a protein.

The replication and transcription of cucumber mosaic virus (CMV) are catalyzed by multi-protein complex RNA-dependent RNA polymerase (RdRp), which is composed of the viral-encoded 1a and 2a proteins with host factors. We have reported that the N-terminal region of the polymerase 2a protein, composed of 126 amino acids, is required for interaction with the helicase 1a protein, and that the phosphorylation of the region abrogated interaction with the 1a protein, suggesting a mechanism of resistance in host plants against viral infection. Here, we found that three protein 2a kinases, of 60, 55, and 38 kDa, co-purified with the tobacco membrane fraction in an in-gel kinase assay. By yeast two-hybrid library screening using the N-terminal 126 amino acids of 2a as a bait, we identified CBL-interacting protein kinase 12 (NtCIPK12) corresponding to 55 kDa protein 2a kinase. The bacterially expressed protein kinase showed protein 2a kinase (t2aK) activity in vitro. We found that NtCIPK12 stabilized upon CMV infection at the post-translational level, and accumulated more heavily to the membrane than in the cytosol.

Comparability studies of new 3rd generation recombinant human factor VIII GreenGene F after improvement of formulation and viral inactivation/removal process.

A new 3rd generation recombinant factor VIII (rFVIII), GreenGene F (WHO INN: beroctocog alfa), which is a highly homogenous B-domain deleted FVIII protein comprising of two peptides as heavy chain (A1 and A2 domain) and light chain (A3, C1, and C2 domain) at 80 and 90 kDa, was developed from its predecessor product GreenGene (2nd generation product previously approved by Korea FDA after clinical studies in South Korea) by process improvements of i) addition of Solvent/Detergent treatment for virus inactivation, ii) nanofiltration (20 nm pore size) for viral removal and iii) alterations to an albumin-free formulation to minimize the risk of viral contamination. An assessment of comparability between the two products was made to see if process improvements for safer product manufacturing affected the rFVIII structural and functional characteristics. Physicochemical and physiological characteristics were observed, in vivo efficacy following a single intravenous administration to FVIII knock-out mice and toxicity by various GLP in vivo tests were evaluated. All results showed equivalence, proving that no changes in protein characteristics of rFVIII occurred from process changes in formulation, viral inactivation, and viral removal which minimize the risk of pathogen transmission to enhance safety.

Mixture of three amino acids as stabilizers replacing albumin in lyophilization of new third generation recombinant factor VIII GreenGene F.

A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood-derived albumin. L-Arginine (hydrophilic amino acid, positively charged side chain), L-glutamic acid (hydrophilic amino acid, negatively charged side chain), and L-isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient.

Dietary Aloe QDM Complex Reduces Obesity-Induced Insulin Resistance and Adipogenesis in Obese Mice Fed a High-Fat Diet.

Obesity-induced disorders contribute to the development of metabolic diseases such as insulin resistance, fatty liver diseases, and type 2 diabetes (T2D). In this study, we evaluated whether the Aloe QDM complex could improve metabolic disorders related to blood glucose levels and insulin resistance. Male C57BL/6 obese mice fed a high-fat diet for 54 days received a supplement of Aloe QDM complex or pioglitazone (PGZ) or metformin (Met) and were compared with unsupplemented controls (high-fat diet; HFD) or mice fed a regular diet (RD). RT-PCR and western blot analysis were used to quantify the expression of obesity-induced inflammation. Dietary Aloe QDM complex lowered body weight, fasting blood glucose, plasma insulin, and leptin levels, and markedly reduced the impairment of glucose tolerance in obese mice. Also, Aloe QDM complex significantly enhanced plasma adiponectin levels and insulin sensitivity via AMPK activity in muscles. At the same time, Aloe QDM decreased the mRNA and protein of PPARγ/LXRα and scavenger receptors in white adipose tissue (WAT). Dietary Aloe QDM complex reduces obesity-induced glucose tolerance not only by suppressing PPARγ/LXRα but also by enhancing AMPK activity in the WAT and muscles, both of which are important peripheral tissues affecting insulin resistance. The Aloe QDM complex could be used as a nutritional intervention against T2D.

Estrogenic/antiestrogenic activities of a Epimedium koreanum extract and its major components: in vitro and in vivo studies.

The estrogenic and antiestrogenic activities of Epimedii Herba, which is a traditional medicinal herb used in Korea and China were investigated in this study. The in vitro estrogen receptor (ER) mediated estrogenic/antiestrogenic activities of an Epimedii Herba extract (Epi ext) and its major components were determined using an estrogen responsive element driven reporter gene assay in MCF-7/ERE and HEK293T cells. The Epi ext exhibited ERα- and ERß-mediated estrogenic activity with an EC(50) of 5.0 and 17.8 µM in HEK293T cells, respectively. Prenylflavonoid glycosides such as icariin (ICA), epimedin A, B, and C did not show any in vitro estrogenic or antiestrogenic activities. Icaritin (ICT) and quercetin exhibited in vitro ER mediated estrogenic activity with a more potent interaction with ERß. In vivo estrogenic activities of the Epi ext, ICA and ICT were compared using an uterotrophic assay. Although the potency of in vitro estrogenic activity was in the order of ICT>Epi ext>ICA, ICA had the strongest estrogenic activity and next ICT in ovariectomized rats. These results collectively suggest that phytoestrogens possess both estrogenic and antiestrogenic activity, and that the differential expression of these two compounds with opposing activities is dependent on the physiological environment in terms of estrogen level, which may be the case in humans.

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group.

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, Met(N-terminal), and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, Met(N-terminal)-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, Met(N-terminal), and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.

Peripubertal administration of icariin and icaritin advances pubertal development in female rats.

Epimedii Herba is a traditional medicinal herb used in Korea and China and exerts estrogenic activity. In this study, we investigated the effect of peripubertal administration of Epimedii Herba on pubertal development in female rats using a modified protocol of the rodent 20-day pubertal female assay. Female Sprague-Dawley rats (21 days old after weaning, 10 rats per group) were divided into five groups: saline (Con), ethinyl estradiol (E2), Epimedii Herba ext (Ext), icariin (ICI), and icaritin (ICT), which were administered by oral gavage (E2 by subcutaneous injection) from postnatal day (PND) 21 through PND40. The time to vaginal opening (VO) was shorter for the Epimedii groups, particularly for the ICT group (p<0.05). Treatment with ICI and ICT significantly increased the duration of the estrus cycle (ICI, 2.78 days; ICT, 4.0 days; control, 1.78 days). Ovary weight was reduced by E2 treatment and increased by the Ext, ICI, and ICT treatments while the weight of the uterus and pituitary glands increased significantly only in the E2 and ICT groups. Although Epimedii Herba displayed relatively weak estrogenic activity, its repeated administration could affect pubertal development in female rats.