Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 mu M and 10 mu M calcium ionophore A23187, 1 mM GABA, and 5 mu M progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm. (C) 1997 Elsevier Science B.V.

In vitro activities of small peptides from snake venom against clinical isolates of drug-resistant Mycobacterium tuberculosis

The recent re-emergence of tuberculosis, especially the multidrug-resistant cases, has highlighted the importance of screening effective novel drugs against Mycobacterium tuberculosis. In this study, the in vitro activities of small peptides isolated from snake venom were investigated against multidrug-resistant M. tuberculosis. Minimum inhibitory concentrations (MICs) were determined by the Bactec TB-460 radiometric method. A small peptide with the amino acid sequence ECYRKSDIVTCEPWQKFCYREVTFFPNHPVYLSGCASECTETNSKWCCTTDKCNRARGG (designated as vgf-1) from Naja atra (isolated from Yunnan province of China) venom had in vitro activity against clinically isolated multidrug-resistant strains of M. tuberculosis. The MIC was 8.5 mg/l. The antimycobacterial domain of this 60aa peptide is under investigation. (C) 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate

Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC50) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3 +/- 0.2, 4.5 +/- 2.0 and 8.5 +/- 3.8 mu M with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC50 against primary R5 HIV-1 isolate from Yunnan province in China was 13.1 +/- 5.5 mu M, with a Sl of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus-cell fusion likely through interacting with the HIV- I envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC50 of 88.3 +/- 0.1 mu M and a Sl of 30. The 50% cytotoxicity concentration (CC50) of SRS was >3.0 mM, as determined in human genital ME 180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0 mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development. (C) 2007 Elsevier B.V. All rights reserved.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129 bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments. (c) 2007 Elsevier B.V. All rights reserved.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3 h, and was maximal after 6 h of exposure to TCDD, B [alp and Aroclor 1254. The estimated 6 h EC50 for TCDD, B [a]P, and Aroclor 1254 was 1.2x10(-9), 5.7x10(-8) and 6.6x10(-6) M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24 h after exposure. The estimated 24 It EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4x10(-9), 8.1x10(-8) and 7.3x10(-6) M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. (c) 2006 Elsevier B.V. All rights reserved.

Assessment of estrogenic activity of leachate from automobile tires with two in vitro bioassays

In this study, a combination of enzyme-linked receptor assay (ELRA) and yeast estrogen screen (YES) assay was firstly applied to determine whether automobile tires immersed in fresh water can leach chemicals, which display estrogenic activity. We optimized ELRA substituting the chromogene substrate by a luminescent one, and found that luminescent ELBA was more sensitive to 17 beta-estradiol (17 beta-E2) with a detection limit of 0.016 mu g/l, compared to 0.088 mu g/l in the chromogene version. In ELRA, all tire leachates obviously showed estrogenic activity, which was increased with duration of immersion. Moreover, the leachate from hackled tires showed more potent estrogenicity than that from the whole ones. In comparison to ELRA, no detectable estrogenic activity was found in all tire leachates with YES assay. The results from YES assay further evidenced that antiestrogenic compounds can be leached from tires. As tire leachates contain estrogenic compounds, they could be important pollution sources, potentially harmful to wildlife and human health. Thus, use of shredded tires as road fill or in landfill sites should arouse our attention.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro

Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis in vitro and to develop approaches for germ-line transmission.

Comparative study of estrogenic potencies of estradiol, tamoxifen, bisphenol-A and resveratrol with two in vitro bioassays

This study was undertaken to compare the sensitivity of two in vitro screening test methods and to determine the accuracy of predicted response to spiked laboratory water samples. A newly developed enzyme-linked receptor assay (ELRA) and a widely used yeast estrogen screen (YES) assay were selected to evaluate the estrogenic responses. Four natural, pharmaceutical, xenobiotic or phytobiotic chemicals: 17beta-estradiol (E2), tamoxifen, bisphenol-A and resveratrol were examined, and 17beta-E2 was used as a positive control. 17beta-E2 can strongly induce estrogenic response in both test systems, however, ELRA was found to be more sensitive to 17beta-E2 with a detection limit of 0.07 mug/l compared to 0.88 mug/l in YES assay. Similar results were obtained for bisphenol-A and resveratrol, and their estrogen potencies relative to E2 (100%) determined by ELRA were at least 5.6 times greater than produced by YES assay. ELRA was unable to distinguish the anti-estrogen tamoxifen and YES assay is also poor at distinguishing. Comparison of response to spiked laboratory water samples show that ELRA can give accurate determination to all four chemicals with recoveries among 70-120%, while YES can only give accurate determination to 17beta-E2 and bisphenol-A with recoveries among 69-112%. The comparative results provide evidence that ELRA is more suitable for rapid screening estrogenic potency of the environmental samples. Combination of ELRA and mammalian cellular assay will constitute an advantageous test to specify agonistic or antagonistic effects. (C) 2003 Elsevier Ltd. All rights reserved.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural. observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

In vitro culture of embryonic hearts from guppy fish (Poecilia reticulata)

Forty embryonic hearts were taken out by anatomical needle from denuded embryos of the ovoviviparity guppy fish that were dechorioned by mechanic method or by trypsin digestion, and were in vitro cultured. In the cultured hearts, 80% have maintained beating in vitro for 4 weeks, and the longest record for beating was 142 d. Owing to fish embryo transparency, beating frequency and blood color changes are easily viewed from the embryonic hearts under a dissecting microscope. The current study established the in vitro culture method of embryonic hearts in guppy fish, which can be used as a model for the study of heart and cardiovascular system in vertebrates.