Gel Filtration HPLC Research Articles

New Polyene Macrolide Compounds from Mangrove-Derived Strain Streptomyces hiroshimensis GXIMD 06359: Isolation, Antifungal Activity, and Mechanism against Talaromyces marneffei.

Mangrove-derived actinomycetes represent a rich source of novel bioactive natural products in drug discovery. In this study, four new polyene macrolide antibiotics antifungalmycin B-E (1-4), along with seven known analogs (5-11), were isolated from the fermentation broth of the mangrove strain Streptomyces hiroshimensis GXIMD 06359. All compounds from this strain were purified using semi-preparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity-guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds exhibited broad-spectrum antifungal activity against Talaromyces marneffei with minimum inhibitory concentration (MIC) values being in the range of 2-128 μg/mL except compound 2. This is the first report of polyene derivatives produced by S. hiroshimensis as bioactive compounds against T. marneffei. In vitro studies showed that compound 1 exerted a significantly stronger antifungal activity against T. marneffei than other new compounds, and the antifungal mechanism of compound 1 may be related to the disrupted cell membrane, which causes mitochondrial dysfunction, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for compound 1 preventing and controlling talaromycosis.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice.

Aujeszky's disease virus (ADV), also known as Suid alphaherpesvirus 1, which mainly infects swine, causes life-threatening neurological disorders. This disease is a serious global risk factor for economic losses in the swine industry. The development of new anti-ADV drugs is highly anticipated and required. Natto, a traditional Japanese fermented food made from soybeans, is a well-known health food. In our previous study, we confirmed that natto has the potential to inhibit viral infections by severe acute respiratory syndrome coronavirus 2 and bovine alphaherpesvirus 1 through their putative serine protease(s). In this study, we found that an agent(s) in natto functionally impaired ADV infection in cell culture assays. In addition, ADV treated with natto extract lost viral infectivity in the mice. We conducted an HPLC gel-filtration analysis of natto extract and molecular weight markers and confirmed that Fraction No. 10 had ADV-inactivating ability. Furthermore, the antiviral activity of Fraction No. 10 was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF). These results also suggest that Fraction No. 10, adjacent to the 12.5 kDa peak of the marker in natto extract, may inactivate ADV by proteolysis. Our findings provide new avenues of research for the prevention of Aujeszky's disease.

1760-P: Mining Animal Venoms as a Source of Insulinotropic Peptides for Potential Diabetes Therapy

T2D development and progression is widely known to be caused by pancreatic beta cell dysfunction, whether in the presence or absence of insulin resistance. There are however limited diabetes drugs that directly target the beta cell. Hence, there is a need to discover yet untapped pathways to increase or rescue beta cell function. Animal venoms provide a rich resource of bioactive compounds, many of which are highly potent and selective. Venom peptides have been harnessed for therapeutic drug development, such as the blockbuster diabetes drug, exenatide, a GLP-1 receptor agonist, being a synthetic form of exendin-4 that was first isolated from the saliva of the Gila monster. We hypothesize that we can further discover and develop new therapeutic agents that promote pancreatic beta cell function based on venom-derived compounds. We studied up to seven animal venoms from snakes, bees and scorpions. Pancreatic beta cells (including EndoC-βH1 cells, human islets, and MIN6 cells) were treated with crude animal venoms from each species and evaluated for insulin secretion capacity. We used a range of doses that elicit insulin secretion without causing cellular damage. Venoms from Apis mellifera (honey bee) and Naja naja (Indian cobra) were shown to robustly stimulate insulin secretion in a dose-dependent manner without affecting cell viability. Treatment with these venoms were also able to rescue diabetic cell models. We prioritized these two venoms for fractionation via gel filtration and reverse phase HPLC. After subsequent screening of the venom sub-fractions in insulin secretion assays, we pinpointed specific sub-fractions responsible for stimulating insulin release. Ongoing work is in progress to identify and validate selected peptides for insulinotropic activity, safety and stability. The insulinotropic peptides (and any modifications) may represent new therapeutic strategies for tackling T2D in future. Disclosure N.Ng: Stock/Shareholder; BetaLife. C.Koh: None. C.Ching: None. P.Gopalakrishnakone: None. A.Teo: None. Funding A*STAR Career Development Fund (202D800020)

Distinctive features of lipoprotein profiles in stroke patients.

Classes of lipoproteins solubilize lipids in the blood, and their profiles are important for preventing atherosclerotic diseases. These can be identified by gel filtration HPLC, which has been analyzed in a manner that yields the same values as the de facto standard method, i.e., ultracentrifugation; however, previous studies have found that ultracentrifugation and its simplified alternatives, enzymatic methods, yield incorrect values. Here HPLC data of stroke patients and the controls were compared using data-driven analyses, without consideration for ultracentrifugation. The data well-separated patients from controls. In many patients, the level of HDL1 (a cholesterol scavenger) was low. The TG/cholesterol ratio of chylomicrons was found to be low in patients and high in the healthy elderly; the lower level may indicate a larger intake of animal fats. High levels of free glycerol in the elderly were hazardous, suggesting more dependence on lipids as an energy source. Statins had minimal effect on these factors. LDL cholesterol, the commonly-used risk indicator, was not a risk factor actually. Enzymatic methods failed to separate the patients from the control; hence, the existing guidelines for screening methods and medical treatment need to be revised. As an immediate step, glycerol would be an adaptable indicator.

The Antifreeze and Cryoprotective Activities of a Novel Antifreeze Peptide from Ctenopharyngodon idella Scales.

The purpose of this study is to obtain new antifreeze peptides (AFPs) that are natural, safe, and high activity from Ctenopharyngodon idella scales. The optimal hydrolysis conditions were investigated, and chromatography-based isolation was conducted using thermal hysteresis activity (THA) as an index. Molecular dynamic simulation (MDs) was explored to reveal the antifreeze mechanism of the AFPs. The results showed that the optimal hydrolysis conditions were 4000 U/g papain enzyme for 60 °C at pH 5.0 and substrate concentration (1:10) for 3 h, as unveiled by single-factor experiment results. The AFPs documented a THA of 2.7 °C when the Th was 1.3 °C. Hydrophilic peptide, named GCFSC-AFPs, with a THA of 5.09 °C when the Th was 1.1 °C was obtained after a series isolation of gel filtration, ion exchange, and reversed-phase HPLC chromatography. The AFPs had a molecular weight of 1107.54~1554.72 Da with three main peptides in the amino acid sequence of VGPAGPSGPSGPQ, RGSPGERGESGPAGPSG, and VGPAGPSGPSGPQG, respectively. The survival rate of yeast with GCFSC-AFPs reached 84.4% following one week of exposure at −20 °C. MDs indicated that GCFSC-AFPs interfered with the ice-water interaction and thus inhibited the ice crystallization process. Our data suggested that the GCFSC-AFPs were a novel and potential antifreeze agent in the food industry.

Characterization of novel umami-active peptides from Stropharia rugoso-annulata mushroom and in silico study on action mechanism

Umami peptides can supplement and enhance the overall taste of food, making it more harmonious, soft, and full-bodied. Stropharia rugoso-annulata mushroom is famous for its umami and pleasant flavor, however, there were no report on its umami peptides. In this study, umami peptides from S. rugoso-annulata were isolated and characterized, as well as to analyze the action mechanism in silico molecular docking approach. By ultrafiltration, gel filtration chromatography, HPLC, and UHPLC-Q-Orbitrap-MS2, five peptides, EPLCNQ, SGCVNEL, PHEMQ, SEPSHF, and ESCAPQL were identified. According to the sensory evaluation and electronic tongue analysis, they had good umami activity and their umami threshold values were in the range of 0.167–0.390 mmol/L. EPLCNQ and SGCVNEL had higher umami intensity than others. Molecular docking of the identified peptides with the umami taste receptor T1R1/T1R3 indicated that the peptides can enter the binding pocket in the Venus flytrap domain of T1R3 cavity, wherein Glu45, Ser104, and His145 may play major roles in the binding interactions of peptides and receptor, forming more stable hydrogen bonds to producing strong umami taste. The study expands the source range of umami peptides and potential raw material of value-added umami-flavorings, and provides further insight into understanding the flavor mechanisms of mushrooms.

Anti-hyperglycemic activity of HPLC-fractionated Momordica charantia seed extract enriched in a novel napin-like protein in experimental diabetic rats and its validation with recombinant napin-like protein

Momordica charantia , a tropical plant, is used in traditional medicine to treat multiple ailments, including diabetes. Several studies have identified compounds with anti-hyperglycemic activity in crude or partially purified M. charantia extracts. However, systematic identification and purification studies validating the bioactivity of active components are unavailable to date. The present study aimed to use an extensive bioactivity guided purification strategy to identify and characterize the proteinaceous active components of M. charantia . The purification scheme, involving acid–ethanol extraction of M. charantia seeds, fractionation by isoelectric precipitation, gel filtration, and reverse-phase HPLC, yielded an anti-hyperglycemic protein fraction consisting of two heterodimeric proteins with molecular masses of 11.70 kDa and 11.47 kDa. Further analyses confirmed that both proteins had characteristics of napin-like proteins. Administration of the purified protein fraction to diabetic animals significantly decreased blood glucose. Acute toxicity studies involving repeated administration of the active peak fraction to experimental animals did not indicate any hepatotoxicity or nephrotoxicity. Furthermore, we evaluated the anti-hyperglycemic activity of a recombinant napin-like protein of M. charantia in diabetic animals, thus confirming our findings that purified napin-like proteins have anti-hyperglycemic activity.

Peptidomic analysis of low molecular weight antioxidative peptides prepared by lotus (Nelumbo nucifera Gaertn.) seed protein hydrolysates

Lotus (Nelumbo nucifera Gaertn.) seeds are rich in proteins and are considered to be a beneficial source of plant proteins. In this study, lotus seed protein hydrolysate (LSPH) was prepared using Flavourzyme and antioxidant activities were evaluated. The LSPH possessed the highest DPPH-scavenging (EC50 2.9 mg/mL), H2O2-scavenging (EC50 16.1 mg/mL), and reducing power activity (8.0 mg/mL) at 180 min of hydrolysis. After gel filtration and reverse phase HPLC purification, the low molecular weight fraction was then analyzed by Nano-LC-MS/MS. Sixteen peptides with corresponding mass from 1 to 2 kDa were identified. Peptides with hydrophobic features and acidic amino acids, Asp and Glu, are considered to contribute to overall antioxidant activities. These results show Flavourzyme hydrolysis of lotus seed protein successfully generates low molecular fragments that make them a potential nutraceutical or health promoting supplement.

Purification and Chemical Characterization of a Potent Acaricide and a Closely Related Inactive Metabolite Produced by Eurotium rubrum C47.

Mites are arthropods and some of them infest dry meat cured products and produce allergic reactions. Some mites, such as Tyrolichus casei, Tyrophagus putrescentiae, or Tyrophagus longior feed on filamentous fungi that grow during the meat curing process. Removal of mite infestation of meat products is extremely difficult and there are no adequate miticidal compounds. The filamentous fungus Eurotium rubrum growing on the surface of ham is able to exert a biocontrol of the population of mites due to the production of miticidal compound(s). We have purified two compounds by silica gel chromatography, gel filtration, semipreparative and analytical HPLC and determined their miticidal activity against T. casei using a mite feeding assay. Mass spectrometry and NMR analysis showed that these two compounds are prenylated salicilyl aldehydes with a C-7 alkyl chain differing in a double bond in the C-7 alkyl chain. Structures correspond to those of flavoglaucin and aspergin. Pure flavoglaucin has a miticidal activity resulting in more than 90% mite mortality whereas aspergin does not affect the mites. Both compounds were formed simultaneously by E. rubrum C47 cultures in different media suggesting that they are synthesized by the same pathway. Production of both compounds was higher in solid culture media and the products were associated with abundant formation of cleistothecia. In liquid cultures both compounds remained mainly cell-associated and only about 10% of the total compounds was released to the culture broth. This miticidal compound may be used to combat efficiently mite infestation in different habitats. These results, will promote further advances on the utilization of flavoglaucin in food preservation and in human health since this compound has antitumor activity.

Hydroxyethylcellulose as a methotrexate carrier in anticancer therapy

Clinical and experimental cancer therapy is multifaceted; one such facet is the use of drug carriers. Drug carriers are various nano- and macromolecules, e.g., oligosaccharides, proteins, and liposomes. The present study aimed to verify the suitability of cellulose as a carrier for methotrexate (MTX). Hydroxyethylcellulose, with a molecular weight of 90 kDa and soluble in water, was used. Methotrexate was linked to cellulose by methyl ester bonds. A conjugate containing on average 9.5 molecules of MTX per molecule of cellulose was developed. Gel filtration HPLC analysis showed that the conjugate contained approximately 2% free drug. Dynamic light scattering analysis showed an increase in the polydispersity of the conjugate. The degradation of the conjugate in phosphate buffer and plasma followed first-order kinetics. The conjugate showed the lowest stability (half-life 154 h) in plasma. The conjugate showed 10-fold lower cytotoxicity to the 4 T1 mammary tumour cell line than the free drug. In the in vivo experiment to treat orthotopically implanted mammary tumours, the conjugate and the free drug, both applied intravenously, showed maximum inhibition of tumour growth of 48.4% and 11.2%, respectively. In conclusion, cellulose, which is a non-biodegradable chain glucose polymer, can be successfully used as a drug carrier, which opens up new research perspectives.

Screening Snake Venoms for Toxicity to Tetrahymena Pyriformis Revealed Anti-Protozoan Activity of Cobra Cytotoxins

Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis. The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians’ morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis.

Synthesis and preliminary evaluation of neoglycopolymers carrying multivalent N-glycopeptide units

In the present study, for the discovery of uncharacterized glycan-binding receptors or lectin-like receptors in plants, we developed neoglycopolymers to which three types of N-glycopeptides are conjugated; the first with plant complex type N-glycan (M3FX), the second with high-mannose type N-glycan (M8), and the third with animal complex type N-glycan (NeuAc2Gal2GN2M3). Three types of Asn-oligosaccharide (Asn-M3FX, Asn-M8, or Asn-NeuAc2Gal2GN2M3) were prepared from storage glycoproteins of Ginkgo biloba seeds, Vigna angularis seeds, and egg yolk glycopeptides from actinase digests of each glycoproteins or glycopeptide. Neoglycopolymers were synthesized such that the α-amino groups of Asn-oligosaccharide were coupled to the carboxyl groups of poly-γ-L-glutamic acid (γ-L-PGA) with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM). The resulting neoglycopolymers were purified through a combination of gel-filtration and reverse-phase HPLC. The incorporation of N-glycans into γ-L-PGA (mol%) was estimated through amino acid composition analysis after acid hydrolysis. The incorporation rates of Asn-M3FX, Asn-M8, and Asn-NeuAc2Gal2GN2M3 into γ-L-PGA were 15.4%, 8.6%, and 11.1%, indicating that nearly 890, 500, and 640 molecules of N-glycans were conjugated with γ-L-PGA, respectively. Furthermore, we confirmed that the neoglycopolymer carrying the multivalent high-mannose type N-glycans is a useful tool for rapid purification of mannose-binding protein, Concanavalin A, from jack bean extract.

Purification and characterization of catechol oxidase from Tadela (Phoenix dactylifera L.) date fruit

Catechol oxidase (PPO) was extracted and purified from Tadela (Phoenix dactylifera L.) date fruit, by a procedure that included (NH4)2SO4 precipitation followed by dialysis, Q-Sepharose bb ion-exchange chromatography and HPLC gel filtration chromatography. Some of its biochemical characteristics were studied. The purification rate and the yield were 80% and 20%, respectively. The Tadela date fruit catechol oxidase exhibited a molecular weight of 90 kDa using SDS-PAGE. The catechol oxidase showed only o‑diphenolase and triphenolase activities while no monophenolase activity was detected. A better affinity was observed using catechol as substrate (Km = 35 mM) with thus, a higher Vmax/Km ratio (80 U/mM·mL). This enzyme is thermostable in the temperature range (30–60 °C) with optimum activity in acidic range of pH. Four inhibitors were used for the control of enzymatic browning, of which sodium metabisulfite was the most potent (IC50 = 0, 11 mM). The values of KI and mechanism of inhibition were also determined. No significant change on enzyme activity was noticed in the presence of metal ion and detergents. Therefore, thermal inactivation was studied in the temperature range between 60 and 80 °C using catechol as substrate. Their kinetic (K, D, t1/2, Zt, Ea) and thermodynamic (ΔH, ΔG and ΔS) parameters were also estimated.

The characterization of androstenone transport in boar plasma

The transport of steroids by plasma proteins influences the amount of steroid available for uptake by the target tissue. In the boar, androstenone is transported to the adipose tissue where it accumulates to cause an off-odour or off-flavour in pork, known as boar taint. The mechanism of the transport of androstenone in the boar remains unclear, and the plasma protein responsible for binding androstenone has yet to be identified. Therefore, the purpose of the present study was to characterize the binding of androstenone to plasma proteins in the boar. The binding specificity of androstenone to plasma proteins was first investigated using a HPLC gel filtration method. [3H]-androstenone was incubated with plasma in the presence or absence of unlabeled competitors and the displacement of androstenone from plasma proteins was measured. In the presence of excess unlabeled competitors, [3H]-androstenone was only partially displaced from plasma proteins, indicating it binds to a low affinity high capacity plasma protein. Binding kinetics studies were also conducted to characterize the binding of androstenone and dehydroepiandrosterone (DHEA) to plasma proteins. The Bmax of androstenone and DHEA was approximately the same (89.1% and 92.3%, respectively). However, the binding affinity (K) of androstenone was 6.5 fold greater than DHEA (0.39 nmol/ml and 0.06 nmol/ml, respectively). Affinity chromatography was used to remove albumin from the plasma proteins. Following incubations with androstenone and DHEA, the binding observed in the albumin free protein fraction was reduced 2.6 and 2.1 fold, respectively relative to the binding in the albumin protein fractions. These results provide direct evidence that androstenone is transported non-specifically by albumin in the plasma of the boar.

Characterization of the Candidate Reference Material of Timothy Pollen Allergen Extract in Terms of Protein Profile and Specific Allergenic Components

Medicinal products based on pollen allergen extracts are widely used for diagnosis and therapy of pollinosis. According to the requirements of the State Pharmacopoeia of the Russian Federation, 13th ed., the allergenic potency of such products is assessed against certified reference materials whose biological activity was confirmed in vivo in subjects sensitized to particular tested allergens. The finished dosage form of a candidate In-House Reference Material (IHRM) of timothy pollen allergen extract and the corresponding allergen pollen extract (intermediate) were characterized in terms of protein profile and allergenic composition using SDS-PAGE, gel- filtration HPLC, and western blotting. The test samples were compared to finished pharmaceutical products (FPP) and pollen allergen extracts produced at different dates and made from pollen harvested at different dates, as well as to the WHO International Standard (IS) of Timothy Pollen Extract (NIBSC code: 82/520). SDS- PAGE and gelfiltration HPLC showed that the candidate IHRM protein profile was comparable in terms of major protein fractions to those of all the FPP batches and pollen allergen extracts produced at different dates. HPLC confirmed the comparability of the protein profiles of the candidate IHRM and the IS (NIBSC code: 82/520), but showed minor variations in the ratio of the main protein fractions. Western blotting confirmed the presence of the main allergenic components with relative molecular masses ranging from 50 to 60 kDa and from 27 to 35 kDa. The composition of specific allergenic components of timothy pollen allergen products manufactured by JSC «SIC «Microgen» was identical with that of the WHO IS of Timothy Pollen Extract in terms of the main protein and allergenic components.

A sodium binding system alleviates acute salt stress during seawater acclimation in eels

BackgroundTeleosts transiting from freshwater (FW) to seawater (SW) environments face an immediate osmotic stress from ion influxes and water loss, but some euryhaline species such as eels can maintain a stable plasma osmolality during early SW exposure. The time course changes in the gene expression, protein abundance, and localization of key ion transporters suggested that the reversal of the ion transport systems was gradual, and we investigate how eels utilize a Na-binding strategy to slow down the ion invasion and complement the transporter-mediated osmoregulation.ResultsUsing an electron probe micro-analyzer, we localized bound Na in various eel tissues in response to SW transfer, suggesting that the Na-binding molecules were produced to sequester excess ionic Na+ to negate its osmotic potential, thus preventing acute cellular dehydration. Mucus cells were acutely activated in digestive tract, gill, and skin after SW transfer, producing Na-binding molecule-containing mucus layers that fence off high osmolality of SW. Using gel filtration HPLC, some molecules at 18 kDa were found to bind Na in the luminal secretion of esophagus and intestine, and higher binding was associated with SW transfer. Transcriptome and protein interaction results indicated that downregulation of Notch and β-catenin pathways, and dynamic changes in TGFβ pathways in intestine were involved during early SW transition, supporting the observed histological changes on epithelial desquamation and increased mucus production.ConclusionsThe timing for the activation of the Na-binding mechanism to alleviate the adverse osmotic gradient was temporally complementary to the subsequent remodeling of branchial ionocytes and transporting epithelia of the digestive tract. The strategy to manipulate the osmotic potential of Na+ by specific binding molecules is similar to the osmotically inactive Na described in human skin and muscle. The Na-binding molecules provide a buffer to tolerate the salinity changes, which is advantageous to the estuary and migrating fishes. Our data pave the way to identify this unknown class of molecules and open a new area of vertebrate osmoregulation research.

Angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory activities of transglutaminase treated sodium caseinate hydrolysates

Angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities of cross-linked and non-cross-linked sodium caseinate (NaCN) hydrolysates were studied. Three different samples were generated: NaCN hydrolysed with Prolyve 1000™ (Prolyve), NaCN cross-linked with transglutaminase (TGase) pre-Prolyve hydrolysis and NaCN cross-linked post-Prolyve hydrolysis. Gel filtration and reverse phase HPLC analysis of the resulting samples indicated that the hydrolysates had similar peptide profiles. Hydrolysates showed higher (p < 0.05) ACE and DPP-IV inhibitory activity compared with intact protein, but no significant (p > 0.05) differences in activity were found between cross-linked and non-cross-linked hydrolysate samples. Hydrolysate IC50 values for ACE and DPP-IV inhibition ranged from 0.10 to 0.17 mg mL−1 and 0.85–1.18 mg mL−1, respectively. Simulated gastrointestinal digestion had no significant (p > 0.05) effect on the bioactivities of the hydrolysates. The results demonstrated that incubation with TGase before or after NaCN hydrolysis with Prolyve had no effect on ACE or DPP-IV inhibitory activities.

A novel function of vitellogenin subdomain, vWF type D, as a toxin-binding protein in the pufferfish Takifugu pardalis ovary

Marine pufferfish of the Tetraodontidae family contain high levels of tetrodotoxin (TTX) in the liver and ovary. TTX is suggested to transfer from the liver to the ovary in female pufferfish during maturation. TTX in pufferfish eggs may act as a repellent against predators and as a sexual pheromone to attract male pufferfish. The toxification mechanism of the pufferfish ovary is poorly understood. Here we evaluated the chemical form of TTX and its related substances in the ovary of the panther pufferfish Takifugu pardalis by LC-ESI/MS. TTX and its analogs 4-epi-TTX, 4, 9-anhydroTTX, deoxyTTX, dideoxyTTX, and trideoxyTTX were detected in a low molecular weight fraction by Sephacryl S-400 column chromatography. The finding of an unknown TTX-related substance in a high molecular weight fraction from the Sephacryl S-400 column suggested the occurrence of toxin-binding protein in the ovary. The toxin-binding protein in the ovary was purified by ion-exchange HPLC, gel filtration HPLC, and SDS-PAGE. Amino acid sequencing and cDNA cloning revealed that the toxin-binding protein, TPOBP-10 (Takifugu pardalis ovary toxin-binding protein with a molecular mass of 10 kDa) was homologous with the predicted vitellogenin-1-like protein [Takifugu rubripes] subdomain, a von Willebrand factor type D domain. TPOBP-10 mRNA was highly expressed in the ovary and liver and less in other organs of female individuals based on RT-PCR. These findings reveal a novel function of the vitellogenin subdomain as binding with TTX-related substances, and its involvement in the toxification of the pufferfish ovary.

A Chromatographic Method for Assay of Sodium Caprylate in the Formulation “Albumin, Solution for Infusions”

A method for assaying sodium caprylate content using gel filtration HPLC and recording of the refractometric detector signal is described. Validation studies demonstrated the suitability of the method. Inclusion of a spectrophotometer in the chromatography system provided for monitoring two parameters (“Sodium caprylate” and “Polymers and aggregates”) in a single test.

Discussion and Analysis of &lt;em&gt;Flammulina velutipes Polysaccharides&lt;/em&gt; Compositions, Molecular Weight and Monosaccharide Composition

The aim of the present work was to explore the composition, molecular weight and monosaccharide composition of ,Flammulina velutipes polysaccharides by DEAE Cellulose-52, High Performance Gel Filtration Chromatography (HPGFC) and HPLC with PMP derivative with the FVP isolated and purified from Flammulina velutipes. The results showed that Flammulina velutipes polysaccharides were composed of three kinds of polysaccharides; the molecular weight of FVP was composed of 4191338, 372779, 19002; the molecular weight of 372779, 19002, one of the biggest average molecular weight polysaccharides is neutral polysaccharide, the rest of the two kinds of polysaccharide composition of less average molecular weight is acidic polysaccharides and the proportion of two kind of acidic polysaccharides significantly greater than neutral polysaccharide, 44.31 and 37.76% respectively, only 17.93% of neutral polysaccharide. FVP was composed of Glc, Man, Gal, Xyl, Fuc; the highest content of Glc, followed by Gal and Man, Xyl and Fuc content is relatively low. The molar ratio was relatively 13.05:2.75:3.16:1.48:1.00. In conclusion, FVP contained the more Glc, the less Fuc and a small amount of galacturonic acid and glucuronic acid, which showed FVP may contain acidic polysaccharides.