Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro.

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma.

Polysaccharides from Pleurotus eryngii: Selective extraction methodologies and their modulatory effects on THP-1 macrophages.

Polysaccharides from P. eryngii mushroom were selectively extracted using low-cost technologies (water at different conditions of temperature and pressure). Mannogalactan was the main polysaccharide in cold-water extracted fraction (CWEF), while a linear (1→6)-ß-d-glucan was the main polymer in hot-water extracted fraction (HWEF). Autoclave-extracted fraction (AEF) contained a mixture of at least four different α- and ß-glucans. The report of linear (1→6)-ß-glucan and linear (1→3)-ß-glucan is a new finding for P. eryngii fruiting bodies. The immunostimulatory properties of the fractions on THP-1 macrophages were studied. All fractions at 50, 250 and 500 µg/mL were not cytotoxic and produced different stimulus on NO, IL-1ß and IL-10 secretion by the cells. Thus, our results showed that it is possible to concentrate different P. eryngii polysaccharides in selected fractions using a simple and low-cost procedure. Since biological effects depends on the polysaccharide structure, this technique allows the obtainment of fractions with distinct immunomodulatory activities.

Agaricus bisporus Crude Extract: Characterization and Analytical Application.

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p-nitrophenol, o-nitrophenol, guaiacol, o-cresol, m-cresol, p-cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10-4-1.0 × 10-3 M l-tyrosine, 3.1 × 10-6-1.0 × 10-4 M phenol, 5.4 × 10-5-1.0 × 10-3 M catechol, 8.5 × 10-5-1.0 × 10-3 M caffeic acid, 1.5 × 10-4-7.5 × 10-4 M chlorogenic acid, 6.8 × 10-5-1.0 × 10-3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

Colletotrichalactones A-Ca, unusual 5/6/10-fused tricyclic polyketides produced by an endophytic fungus, Colletotrichum sp. JS-0361.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC50s of 35.06 and 25.20 µM, respectively.

Inhibitory effects of Lentinus edodes mycelia polysaccharide on α-glucosidase, glycation activity and high glucose-induced cell damage.

At present, diabetes and diabetic complications have become one of the serious diseases affecting human health. In this study, the inhibitory effects of Lentinus edodes mycelia polysaccharide (LMP) on α-glucosidase activity, the formation of advanced glycation end products (AGEs) and high glucose-induced human umbilical vein endothelial cells (HUVECs) damage were explored. The interaction between LMP and α-glucosidase and the inhibition against AGEs formation were investigated with spectroscopic techniques. The results revealed that LMP had a reversible inhibition on α-glucosidase activity in a mixed-type manner. When the concentration of LMP was 2.7 mM, the inhibition rate was 34.38 %. LMP quenched the fluorescence of α-glucosidase through the static quenching and formed the LMP-α-glucosidase complex. At 310 K, the number of binding sites (n) and binding constant (Kb) were 1.01 and 3.71 × 104 L mol-1, respectively. In addition, LMP could inhibit the formation of AGEs. Compared with 40 mM glucose treatment group, treatment with 0.05 mM LMP for 48 h increased the cell viability from 70.17% to 91.14% and decreased ROS production from 3.33-fold to 1.21-fold. LMP inhibited high glucose-induced activation of MAPK signaling pathways. These findings may promote the application of LMP in the functional food industry.

Trametes robiniophila Murr in the treatment of breast cancer.

Breast cancer is the leading cause of cancer death among women across the world. Trametes robiniophila Murr (Huaier), a traditional herbal medicine, has been used in China to protect human health for about 1600 years. Recent years, Huaier had been proven to be effective for multiple types of malignancies. This systematic review focused on breast cancer treatment, summarizing the curative function of Huaier aqueous extract and polysaccharides in preclinical researches. Huaier could markedly inhibit breast cancer progression with low toxicity, enhance immune response and increase the sensitivity to radiation and chemotherapy. The therapeutic effect of Huaier granule in clinical studies was also included. This review amalgamated the current studies and highlighted the promising role of Huaier and its polysaccharides as complementary alternative medicine in breast cancer treatment.

Antioxidant and Neuroprotector Influence of Endo-Polyphenol Extract from Magnesium Acetate Multi-Stage Addition in the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes).

The objective of this study was to explore the effect of magnesium acetate (MA) addition on the endo-polyphenol yield by Phellinus baumii and establish a feasible additive strategy. The optimal three-point MA addition strategy (0.05 g/L concentration of MA added at 0 h and 6 h, 0.9 g/L concentration of MA added at 12 h) was employed to obtain maximum endo-polyphenol yield. The maximum endo-polyphenol production was reached at 1.22 g/L, which was 1.39-fold higher than that of the control. Additionally, the endo-polyphenol showed stronger antioxidant activity in vitro compared with the control, including DPPH· scavenging capacity (78.76%) and Trolox equivalent antioxidant capacity (TEAC) (32.28 µmol Trolox/g sample). HPLC analysis showed that the endo-polyphenol production of the crude ethanol extracts was significantly higher than that of the control. Hispidin was isolated and identified from the ethanol extract of the culture mycelia from Ph. baumii with the three-point MA addition strategy. Hispidin showed a strong ability to scavenge DPPH free radicals and TEAC, equivalent to positive (vitamin C) value of 89.41% and 75.98%, respectively. Furthermore, hispidin protected H2O2-induced PC12 cells injured by decreased oxidative stress level. These results indicated that the MA multi-stage addition strategy was dependable, and could be used to develop new natural antioxidants for foods or medicines.

Antitumor Effects of Extract of the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes), on Human Melanoma Cells A375 In Vitro and In Vivo.

Melanoma is among the most aggressive and treatment-resistant human cancers. Phellinus baumii, a famous medicinal mushroom, has been used to treat different diseases, including cancer, in China and other east Asian countries. The purpose of this research was to explore its anticancer effects against melanoma, and the mechanisms that might be involved. CCK-8 assay exhibited that extracts of Ph. baumii (EPB) strongly inhibited cell viability of A375 melanoma cancer cell. Typical morphological changes of cell apoptosis were observed in EPB-treated A375 cells in Hoechst staining assay. Flow cytometry analysis indicated that EPB significantly induced A375 cells apoptosis and the cell cycle was disrupted in S phase. EPB increased the expression of Bax, and decreased Bcl-2 in A375 cells. EPB remarkably caused mitochondrial membrane potential collapse and induced a mitochondrial-dependent apoptosis in A375 cells evidenced by caspase-3 activation, followed by PARP cleavage. More importantly, EPB has shown a strong inhibitory effect on the migration and aggression of the A375 cells through the healing of the wound and transwell assay. In vivo, EPB was also found to strongly inhibit the growth of tumors in BALB/c nude mice. Our results indicated that Ph. baumii might be a natural therapeutic product for aggressive melanoma because it could induce apoptosis and inhibit metastasis in A375 cells.

Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries.

A simple SFC/MS (Supercritical Fluid Chromatography/Mass Spectrometry) method set was developed to effectively screen separations of various crude synthetic peptide products of pharmaceutical interest. Additives to the modifier methanol which were successful for these separations were found to include TFA (trifluoroacetic acid) and ammonia mixed with TFA, each at 0.1 % (v/v) composition in methanol. A final screening column set consisted of 2-ethylpyride (2-EP), 4-ethylpyridine (4-EP) and cross-linked diol (Luna™ HILIC) stationary phases. Small, linear and macrocyclic peptides with fewer than ten residues could all be eluted with good performance under at least one of the method conditions comprising the final screening protocol. For larger peptides either the 4-EP and HILIC columns with the above additives provided a good initial screening method set without 2-EP. The gradient was slightly longer and shifted to higher polarity relative to the gradients for smaller peptides. This method was often successful to elute large, hydrophilic peptides up to a 41-mer with acceptable peak profiles although the largest peptides with most ionizable residues were most challenging. For these peptides generally HILIC column performance was better with TFA + ammonia in the modifier than with TFA, while 4-EP performance was usually improved with TFA relative to TFA + ammonia.

Activity of Antioxidant Defense Enzymes in Rats with Experimental Allergic Encephalomyelitis.

We studied activities of antioxidant system enzymes in tissues of rats with experimental allergic encephalomyelitis. It was shown that the development of pathology is accompanied by deformation of the neurons and axonal degeneration, intensification of free radical oxidation, exhaustion of the reduced glutathione pool, and multidirectional changes in activities of antioxidant enzymes in rat tissues. The observed imbalance in the antioxidant defense system can be associated with excessive glutathione utilization in the glutathione transferase reaction and different severity of the pathological process in the brain and spinal cord. The received data necessitate the search for compounds that can prevent inhibition of antioxidant system components in order to analyze the possibility of their use in the treatment of multiple sclerosis.

Optimal conditions for the storage of German cockroach extract.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease­rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18­26˚C, RT) or refrigerated (2­8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease­rich cockroach extract.

Assessment of the Gastroprotective Effect of the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Against the Gastric Mucosal Ulceration Induced by Ethanol in Experimental Rats.

The chaga medicinal mushroom (Inonotus obliquus) was traditionally used to treat various ailments. To establish the pharmacological properties of I. obliquus, studies were performed to show the antiulcer activity of the ethanolic extract. The ethanolic extract of I. obliquus was prepared. The antiulcer activity of I. obliquus was determined using gastric ulcerated rats (ulceration induced by ethanol). The ethanolic extract of I. obliquus (200 mg/kg) did not cause any sign of toxicity or sensitivity to rats when the extracts were administered by oral feed. Oral administration of ethanolic extract of I. obliquus exhibited antiulcer activity in all models used. The ethanolic extract of I. obliquus showed an effective antiulcer activity, which could be due to the presence of various biologically active compounds. This confirmed the traditional uses of I. obliquus in the treatment of ailments.

Antiproliferative, Immunostimulatory, and Anti-Inflammatory Activities of Extracts Derived from Mushrooms Collected in Haida Gwaii, British Columbia (Canada).

Wild mushrooms, while largely explored for their ecological significance, have not been systematically studied for their medicinal properties. This is the first report of biological activities of mushrooms from Haida Gwaii, British Columbia, Canada. The 17 mushroom species in this study were collected from multiple locations on Haida Gwaii and were screened for antiproliferative, immunostimulatory, and anti-inflammatory activities. Prior to screening, mushrooms were genetically identified and then sequentially fractionated into four crude extracts using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. We report here the strong antiproliferative and antiinflammatory activities of Amanita augusta, Phellodon atratus, Guepinia helvelloides, Chroogomphus tomentosus, Laetiporus conifericola, and Inocybe sp. In addition, A. augusta, G. helvelloides, and Inocybe sp. showed potent immunostimulatory activity. Two other species (Ganoderma tsugae and Pleurotus ostreatus) displayed strong immunostimulatory activity consistent with previous reports by others, suggesting that similar constituents are present in the same species from Haida Gwaii. For nine species (Russula paludosa, Hygrophoropsis aurantiaca, Tricholomopsis rutilans, Tyromyces chioneus, Hydnum repandum, Hypholoma fasciculare, Clavulina cinerea, P. ostreatus and Ramaria cystidiophora), we describe antiproliferative, immunostimulatory, and/or anti-inflammatory activities that have never been reported before. The new findings serve as a platform for future investigations into the potentially novel bioactive constituents of these mushrooms, as well as an incentive to further study a wider array of wild mushrooms for medicinal properties.

Nattokinase Crude Extract Inhibits Hepatocellular Carcinoma Growth in Mice.

Nattokinase (NK, E.C. 3.4.21.62) is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of hepatocellular carcinoma (HCC), a principal malignancy of the liver that causes morbidity and mortality worldwide. Crude extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE-treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and vimentin in the NCE groups. Taken together, these data demonstrate that NCE from Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.

Utilization of enzyme extract self-encapsulated within polypyrrole in sensitive detection of catechol.

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1-60 µM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test.

Spirulina consumption effectively reduces anti-inflammatory and pain related infectious diseases.

BACKGROUND: Inflammation and pain triggers several pathological illnesses. Synthetic drugs used for the controlling of inflammatory conditions convey significant toxic effects. Global scientific community continually attempt to improve effective, economic and harmless naturally derived remedies against inflammation and pain. The present study aimed to quantify the phytochemical constituents of the freshly cultivated Spirulina and targeted to examining the anti-inflammatory and analgesic activity of Spirulina extract (SE) derived from Arthrospira platensis. METHODS: The anti-inflammatory effect of SE was evaluated in animal models including carrageenan-induced rat hind paw oedema, and cotton pellet-induced granuloma formation. Analgesic effects of SE were evaluated by acetic acid induced writhing response and hot plate test. RESULTS: Phytochemical quantification guided to identify seven carbohydrates, thirteen amino acids, eleven fatty acids and polyphenolic compounds respectively. The results indicated that SE significantly attenuated carrageenan-induced hind paw oedema, and cotton pellet-induced granuloma. Preliminary molecular mechanistic studies established that SE decreased the productions of TNF-α, IL-1ß, IL-6, PGE2 and NO, and suppressed the activities of COX-2 and iNOS. CONCLUSION: These results provide a strong scientific foundation for the anti-inflammatory and analgesic activities of SE against different studies in animal models.

Aqueous extracts of se-enriched Auricularia auricular attenuates D-galactose-induced cognitive deficits, oxidative stress and neuroinflammation via suppressing RAGE/MAPK/NF-κB pathway.

Aging is a natural process that accompanied with progressive cognitive deficits and functional decline in organisms. Selenium (Se), an essential trace element, exhibits antioxidative and anti-inflammatory abilities. Here, our study aimed to investigate the protective effects of aqueous extracts of Se-enriched Auricularia auricular (AESAA) on aging mice induced by d-galactose (D-gal) and explore its potential mechanism. d-gal was administered (100 mg/kg) subcutaneously for 12 weeks to establish an aging mouse model. Morris water maze (MWM) test was conducted to assess the cognitive deficits of mice. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and malondialdehyde (MDA) level in hippocampus were measured to evaluate oxidative stress. The contents of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) in hippocampus were determined by ELISA method. Further, hippocampal levels of RAGE, p-Erk, p-JNK, p-P38 and p-NF-κB were detected by western blot and the RAGE expression was confirmed by immunohistochemistry. We found that AESAA supplementation significantly decreased d-gal-induced cognitive deficits, as evidenced by better performance in the MWM test. Furthermore, AESAA treatment attenuated oxidative stress and decreased the contents of pro-inflammatory cytokines in hippocampus. Importantly, AESAA inhibited the up-regulation of RAGE, p-Erk, p-JNK, p-P38 in the hippocampus of d-gal treated mice. Moreover, the results also indicated that AESAA inhibited p-NF-κB and p-IκBα expression. In conclusion, our findings suggest that AESAA effectively decreases cognitive impairment, alleviates oxidative damage and neuroinflammation in mice through s RAGE/MAPK/NF-κB signaling pathway, which provides a potential therapy for delaying the aging process.

Integrating Molecular Network and Culture Media Variation to Explore the Production of Bioactive Metabolites by Vibrio diabolicus A1SM3.

Vibrio diabolicus A1SM3 strain was isolated from a sediment sample from Manaure Solar Saltern in La Guajira and the produced crude extracts have shown antibacterial activity against methicillin-resistant Staphylococcus aureus and cytotoxic activity against human lung cell line. Thus, the aim of this research was to identify the main compound responsible for the biological activity observed and to systematically study how each carbon and nitrogen source in the growth media, and variation of the salinity, affect its production. For the characterization of the bioactive metabolites, 15 fractions obtained from Vibrio diabolicus A1SM3 crude extract were analyzed by HPLC-MS/MS and their activity was established. The bioactive fractions were dereplicated with Antibase and Marinlit databases, which combined with nuclear magnetic resonance (NMR) spectra and fragmentation by MS/MS, led to the identification of 2,2-di(3-indolyl)-3-indolone (isotrisindoline), an indole-derivative antibiotic, previously isolated from marine organisms. The influence of the variations of the culture media in isotrisindoline production was established by molecular network and MZmine showing that the media containing starch and peptone at 7% NaCl was the best culture media to produce it. Also, polyhydroxybutyrates (PHB) identification was established by MS/MS mainly in casamino acids media, contributing to the first report on PHB production by this strain.

Effect of betaine and selenium on the growth and photosynthetic pigment production in Dunaliella salina as biostimulants.

The aim of our study was to establish the effect of selenium and betaine on the growth of D. salina, accumulation of photosynthetic pigments and antioxidant activity of the hydrophobic fraction. This approach was an attempt to demonstrate 'microalgae biostimulant' effects, similar to 'plant biostimulant' effects, i.e. increased tolerance to abiotic stress and enhanced accumulation of bioactive compounds. A high-throughput assay was done in 24-well microplates, at 15% NaCl and different concentrations of sodium selenite (0, 0.5, 2 and 8 µM) or betaine (0, 5, 50 and 500 µM). Both selenium and betaine induced a slight delay in algae growth during the actively growing stage but the final density reached similar values to the control. Betaine significantly enhanced (50%-100%) carotenoids and chlorophyll a accumulation, in a concentration depending manner. Antioxidant activity increased almost 3-fold in extracts of algae treated with 50 µM betaine. Selenium had a much more discrete effect than betaine on pigments biosynthesis. The antioxidant activity of the extracts increased 2-fold in the presence of Se compared to the control. Our work proves that it is possible to enhance production and activity of bioactive compounds from microalgae by using ingredients, which already proved to act as plant biostimulants.

Chromosome Cohesion and Condensation in Xenopus Egg Extracts.

Chromosome structure in both interphase and M-phase cells is strongly influenced by the action of the cohesin and condensin protein complexes. The cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication and promotes normal interphase chromosome structure and gene expression. In contrast, condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system is uniquely suited to analyze the functions of both complexes. Egg extracts, in which the cell cycle state can be manipulated, contain stockpiles of nuclear proteins (including condensin and cohesin) sufficient for the assembly of thousands of nuclei per microliter. Extract prepared from unfertilized eggs is arrested by the presence of cytostatic factor (CSF) in a state with high levels of M-phase kinase activity, but can be stimulated to enter interphase, in which DNA replication occurs spontaneously. For cohesion assays, demembranated sperm nuclei are incubated in interphase extract, where they undergo rapid and synchronous DNA replication and cohesion establishment through the recruitment of proteins and other factors (e.g., nucleotides) from the extract. Sister chromatid cohesion is assessed by then driving the extract into M phase by the addition of fresh CSF-arrested extract. In contrast, because chromosome condensation occurs spontaneously in M-phase extracts, sperm nuclei are added directly to CSF extracts to assay condensation.