Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

The effect of GnRH,eCG and progestin type on estrous synchronization following laparoscopic AI in ewes

The aim of the experiment was to evaluate the effects of GnRH and/or eCG and progestin type (implant versus CIDR) on the induction of estrus and pregnancy rate following laparoscopic AI (LAI) with frozen semen. In the first trial, ewes (n = 129) were treated with norgestomet implants for 14 days. At implant removal ewes received eCG (400 IU) and/or GnRH (25 μg) 36 h after removal, resulting in control, eCG, GnRH, and eCG/GnRH groups (n = 30–34/group). In trial 2, ewes (n = 36) were treated with intravaginal fluorogestone acetate sponges (FGA) or CIDR for 12 days. After withdrawal, half of the ewes from each progestin group received eCG (400 IU), resulting in sponge, sponge/eCG, CIDR and CIDR/eCG groups (n = 8–10/group). In both trials, estrous activity was assessed using a vasectomized ram from the time of progestin removal to laparoscopic AI with frozen semen 58–60 h (trial 1) or 54–56 h (trial 2) following cessation of treatment. In trial 1, GnRH decreased (P < 0.05) the percentage of ewes in estrus (GnRH, 75.8% versus control, 93.8% versus eCG/GnRH, 94.1%), however pregnancy rates were similar in all groups (control, 53.1%; eCG, 70.0%; GnRH, 51.5%; eCG/GnRH, 55.9%, respectively). In trial 2, neither the type of progestin nor eCG treatment effected the percentage of ewes in estrus (sponge, 75.0%; sponge/eCG, 100.0%; CIDR, 100.0%; CIDR/eCG, 90.0%). However, pregnancy rates following LAI were higher (P < 0.05) when ewes were treated with eCG (progestin + eCG, 73.7% versus progestin alone, 41.2%). Results demonstrate that the source of progestin does not influence the expression of estrus or the proportion of ewes pregnant following LAI. When progestin treatment protocols are used in combination with eCG, pregnancy rates can be increased. A dose of GnRH near the end of progestin treatment may decrease the estrous response, by inducing ovulation before normal expression of estrus.     

Inulinase biosynthesis using immobilized mycelium of Aspergillus niger

Conidia of Aspergillus niger 20 Osm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 °C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant.     

Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-³H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K_d of 1.30 ± 0.56 nM and 1.54 ± 0.35 nM, respectively. Binding of [24-³H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC₅₀ of 92 ± 8.4 nM and 87 ± 13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.     

Arvoredol—An unusual chlorinated and biofilm inhibiting polyketide from a marine Penicillium sp. of the Brazilian coast

Penicillium sp. F37 has been isolated from the marine sponge Axinella corrugata and shown to be closely related to Penicillium maximae. From the culture of Penicillium sp. F37 arvoredol, a novel chlorinated polyketide with 6,7-dihydro-4(5H)-benzofuranone moiety has been isolated and characterized by spectroscopic methods Arvoredol prevented biofilm formation of the human pathogen Staphylococcus epidermidis at a concentration of 125 μg mL⁻¹ by 40%. It was also active against colorectal carcinoma HCT116 cells with a MIC of 7.9 μg mL⁻¹.     

Phorbaketals L–N,cytotoxic sesterterpenoids isolated from the marine sponge of the genus Phorbas

Three new sesterterpenoids, phorbaketals L–N (1–3), were isolated from a marine sponge of the genus Phorbas and their complete structures were elucidated via analysis of HRFABMS and NMR spectroscopic data. Phorbaketal N (3) showed potent cytotoxicity against human pancreas cancer cells (IC₅₀ = 11.4 μM).     

Molecular cloning,structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k_cat = 153 s⁻¹, 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.     

Shear effects on suspended marine sponge cells

Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Müller protocol [W.E.G. Müller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205–219]. Supplementation of the standard Ca²⁺- and Mg²⁺-free artificial seawater of the Müller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75 rpm essentially killed all the viable cells within 2.5 h, but no loss of viability occurred at a higher agitation speed of 100 rpm for up to 6 h when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3 °C instead of the normal 17 °C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within ±10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell–cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact).     

Assessment of the reproductive parameters,laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Canindé goats (Capra hircus)

The Canindé breed of goats (Capra hircus) is currently endangered. The aims of this study were to characterize the estrus behavior, ovulatory responses and progesterone profiles, and to evaluate the in vitro embryo production (IVP) in this breed. In Experiment 1, ten nulliparous and seven pluriparous females received medroxyprogesterone acetate (MAP)-containing sponges (60 mg) plus 75 μg d-cloprostenol for estrus synchronization and their reproductive parameters were evaluated. In Experiment 2, oocytes obtained by laparascopy from hormonally stimulated females (n = 15) were used for IVP. There was no difference (p > 0.05) between nulliparous and pluriparous goats in terms of estrus response (40.0% vs. 85.7%), time from progestagen sponge removal to the onset of estrus (62.0 ± 15.5 vs. 50.7 ± 19.2 h; mean ± SEM), duration of estrus (25.0 ± 16.1 vs. 30.0 ± 15.1 h), percentage of ovulating animals (60.0% vs. 85.7%), number of ovulations (1.2 ± 0.4 vs. 1.3 ± 0.8), and diameter of the preovulatory follicle (5.8 ± 0.5 vs. 6.1 ± 0.3 mm). Progesterone concentrations were also similar (p > 0.05) in both groups. During laparoscopic recovery, there were average 12.2 aspirated follicles and 9.1 oocytes per goat, resulting in a high recovery rate (74.3%, 182/245). A total of 78 embryos were produced (51.0%). The mean number of cells in the blastocysts at day 7 of in vitro culture was 170.3 ± 12.5. In conclusion, nulliparous and pluriparous Canindé goats exhibited similar reproductive profiles. It was possible to produce embryos in vitro, allowing the instigation of an embryo bank for preservation of this breed.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.     

Antifouling activity exhibited by secondary metabolites of the marine sponge,Haliclona exigua (Kirkpatrick)

Bioassay guided purification of the acetone extract of the marine sponge, Haliclona exigua, (Gulf of Mannar, India) yielded a fraction rich in bis-1-oxaquinolizidine alkaloids, active against seven strains of fouling bacteria as well as cyprids of the cosmopolitan barnacle, Balanus amphitrite. The major alkaloids in the mixture have been tentatively identified as nor-araguspongine C (33.76%), araguspongine C (6.49%), dihydroxy araguspongine (36.36%), methyl and dimethyl derivatives of the latter (12.98 and 10.38%, respectively) from HRMS studies. The lower EC₅₀ (6.6 μg/ml as against the US Navy standard of 25 μg/ml for NPAs) and low toxicity (LC₅₀ 18 μg/ml as compared to 0.00001 μg/ml for TBT) values, coupled with its favourable therapeutic ratio (2.7 as against the requirement of >1) makes these compounds ideal NPAs in environmentally compatible antifouling coatings.     

Spongiacidin C,a pyrrole alkaloid from the marine sponge Stylissa massa,functions as a USP7 inhibitor

USP7, a deubiquitylating enzyme hydrolyzing the isopeptide bond at the C-terminus of ubiquitin, is an emerging cancer target. We isolated spongiacidin C from the marine sponge Stylissa massa as the first USP7 inhibitor from a natural source. This compound inhibited USP7 most strongly with an IC₅₀ of 3.8 μM among several USP family members tested.     

Marine spongian sesquiterpene phenols,dictyoceratin-C and smenospondiol,display hypoxia-selective growth inhibition against cancer cells

In the course of our search for hypoxia-selective growth inhibitors against cancer cells, a sesquiterpene phenol, dictyoceratin-C (1), was isolated from the Indonesian marine sponge of Dactylospongia elegans under the guidance of the constructed bioassay. Dictyoceratin-C (1) inhibited proliferation of human prostate cancer DU145 cells selectively under hypoxic condition in a dose-dependent manner at the concentrations ranging from 1.0 to 10 μM. The subsequent structure–activity relationship study using nine sesquiterpene phenol/quinones (2–10), which were isolated from marine sponge, was executed. We found that smenospondiol (2) also exhibited the similar hypoxia-selective growth inhibitory activity against DU145 cells, and the para-hydroxybenzoyl ester moiety would be important for hypoxia-selective growth inhibitory activity of 1. In addition, the mechanistic analysis of dictyoceratin-C (1) revealed that the 10 μM of 1 inhibited accumulation of Hypoxia-Inducible Factor-1α under hypoxic condition.     

Isolation of a sulphated polysaccharide from a recently discovered sponge species (Celtodoryx girardae) and determination of its anti-herpetic activity

Exopolysaccharides (EPS) were extracted from a sponge, Celtodoryx girardae recently discovered in the Golfe du Morbihan in 2000. Sponge samples were collected monthly from November 2007 to May 2008. SEC analysis of EPS samples showed that they exhibit a unique molecular weight of ≈800 kDa. However, infrared analysis revealed that structural seasonal variations occur. EPS fractions also exhibit significant sulphate contents and were screened in vitro for a potential antiviral activity against Herpes simplex virus type 1 (HSV-1). The best result was obtained with a sample collected in January which exhibits an EC₅₀ of 5.9 μg/mL without cytotoxicity on the Vero cell line. Experiments carried out to elucidate the mechanism of the EPS showed that the sulphated groups of EPS interact with the glycoproteins on the surface of the virus’ membrane.     

Ts14 from Tityus serrulatus boosts angiogenesis and attenuates inflammation and collagen deposition in sponge-induced granulation tissue in mice

We have previously described a 25 mer anti-hypertensive peptide, previously named TsHpt-I (Tityus serrulatus Hypotensin-I), now Ts14, as an agonist of B2 kinin receptor. Bradykinin is known to play physiological roles in angiogenic, inflammatory, and fibrogenic processes, mostly mediated by B2 receptor. Therefore, we investigated whether Ts14 could modulate key events (neovascularization, inflammatory cell recruitment, and extracellular matrix deposition) of the fibrovascular tissue, induced by polyether-polyurethane sponge implants in mice. Sponges were implanted in the dorsum of 7-week-old C57Bl/6 male mice that received daily intrasponge treatment with Ts14 (27.25 μg/sponge/day in 10 μL PBS) or vehicle (10 μL PBS/sponge/day) and were assessed on day 7 after surgery. Hemoglobin content, blood flow (laser Doppler perfusion imaging), and VEGF levels in the implants, used as indices of vascularization, indicated that Ts14 enhanced angiogenesis in implants relative to the PBS-treated group. Interestingly, Ts14 reduced TNF-α levels and neutrophil infiltration, although stimulated macrophage infiltration into implants, as determined by myeloperoxidase (MPO) and N-acetyl-β-d-glucosaminidase (NAG) enzyme activities, respectively. Regarding the fibrogenic component (soluble collagen content and Sirius-red histological staining), we observed that Ts14 inhibited collagen deposition in the implants. Overall, our results suggest that Ts14 exerts proangiogenic, anti-inflammatory, and anti-fibrogenic activities. These effects may indicate a therapeutical potential of this peptide in conditions where angiogenesis, inflammation, and fibrogenesis contribute to disease progression and chronicity.     

Synchronization of estrus with short- and long-term progestagen treatments and the use of GnRH prior to short-term progestagen treatment in ewes

The objective of the present study was to determine the efficacy of the synchronization of estrus using short- and long-term progestagen treatments in ewes at the onset of the breeding season, and to evaluate the effect of the exogenous GnRH administration immediately prior to short-term progestagen treatment on the reproductive performance. A total of 240 Tahirova cross-bred ewes, aged 18–24 months, and 40 rams, aged 2–4 years-old, were used in the trial. Ewes were divided equally into 3 groups (n = 80 per group). Intravaginal progestagen sponges containing FGA (30 mg) were inserted in the ewes for 7 d in the FGA1 (short-term) and GnRH treatment groups, and for 12 d in the FGA2 group (long-term). The ewes in the GnRH group received 10.5 μg busereline acetate i.m. at the time of sponge insertion. Tiaprost tromethamol (PGF_2α; 0.294 mg) and eCG (400 IU) were injected i.m. on the 6th day of progestagen treatment in the GnRH and FGA1 groups, and on the 11th day in the FGA2 group following sponge insertion. All ewes were hand-mated once at the detection of estrus. The estrous response, fertility rate, multiple birth rate and litter size recorded was 88.7, 87.3, 51.6% and 1.6 in the FGA1 group, 92.5, 71.6, 50.9% and 1.5 in the FGA2 group, and 96.2, 89.6, 71.0% and 1.8 in the GnRH group, respectively. No significant difference in estrous response between the groups was recorded, but the fertility rate in the FGA1 and GnRH groups was significantly (P < 0.05) higher than in the FGA2 group. The occurrence of multiple births and litter sizes were significantly (P < 0.05) higher in the GnRH group, compared to both the FGA1 and FGA2 groups, with the number of single lambs being significantly (P < 0.05) higher in the FGA1 (48.4%) and FGA2 (49.0%) groups than in the GnRH (29.0%) group. However, the differences recorded between any of the groups in terms of the number of twin and triplet lambs were insignificant. In conclusion, it can be said that estrous synchronization using the 12-d-FGA-eCG-PGF_2α regimen could be replaced with the 7-d-FGA-eCG-PGF_2α regime in sheep at the onset of the breeding season. However, the combination of GnRH with the latter regimen (7-d-GnRH-FGA-eCG-PGF_2α) increased the multiple birth rate and litter size in the ewes.     

Effect of time of artificial insemination on fertility of progestagen and PMSG treated indigenous Greek ewes,during non-breeding season

A total of 2567 indigenous Greek ewes (Chios, Vlachiki and Vlachiki×Chios breeds) were used to determine the optimum time for insemination, following synchronization of oestrus with MAP-impregnated intravaginal sponges and PMSG during non-breeding season. Within each breed group, the ewes were divided into three subgroups and submitted to a double blind cervical artificial insemination 48 and 60 h (subgroup I), 60 and 72 h (subgroup II) and 48 and 72 h (subgroup III) after sponge withdrawal. From the results of the present investigation it can be concluded that the conception rate in the Chios island breed is better than that in the Vlachiki×Chios breed, with the latter being better than that in the Vlachiki breed. Using fixed time for AI, a better conception rate is obtained when applied 48 and 72 h after sponge withdrawal for Chios and Chios×Vlachiki breeds, while for Vlachiki breed a better conception rate is obtained when fixed AI is applied 48 and 60 h after sponge withdrawal.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Decolorization of synthetic and real textile wastewater by the use of white-rot fungi

Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor.