Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.

Mechanism study on the frequent variations of cell-bound microcystins in cyanobacterial blooms in Lake Taihu: Implications for water quality monitoring and assessments

Although Microcystis-based toxins have been intensively studied, previous studies using laboratory cultures of Microcystis strains are difficult to explain the phenomenon that microcystin concentrations and toxin variants in natural blooms differ widely and frequently within a short-term period. The present study was designed to unravel the mechanisms for the frequent variations of intracellular toxins related to the differences in cyanobacterial colonies during bloom seasons in Lake Taihu, China. Monitoring of Microcystis colonies during warm seasons indicated that the variations in microcystins in both concentrations and toxin species were associated with the frequent alteration of Microcystis colonies in Lake Taihu. High concentration of microcystins in the blooms was always associated with two Microcystis colonies, Microcystis flos-aquae and Microcystis aeruginosa, whereas when Microcystis wesenbergii was the dominant colonial type, the toxin production of the blooms was low. Additionally, environmental factors such as temperature and nutrition were also shown to have an effect on the toxin production of the blooms, and may also potentially influence the Microcystis species present. The results of the present study provides insight into a new consideration for quick water quality monitoring, assessment and risk alert in cyanobacterium- and toxin-contaminated freshwaters, which will be beneficial not only for water agencies but also for public health. (C) 2009 Elsevier Ltd. All rights reserved.

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.

Studies on the toxic effects of La3+ to Tetrahymena thermophila by microcalorimetry

The toxic effects of La3+ on Tetrahymena thermophila have been studied by microcalorimetry at 28 degrees C. The metabolic rate constant (r) and peak time were linked to the concentration of La3+. The changes of metabolic rate constant indicated that low-concentration La3+ (0-75 mg/L) had no significant effects on the metabolism of Tetrahymena cells but high-concentration La3+ (100-175 mg/L) could inhibit their metabolism. From the results obtained by cell counting and fluorescence depolarization measurements, the inhibition of metabolism resulted from the decrease in cell number and the reduction in cell membrane fluidity. According to the results, it is clear that the metabolic mechanism of Tetrahymena cells has been changed with the addition of high-concentration La3+. In addition, microcalorimetry of Tetrahymena could be a sensible, easy-to-use, and convenient method for monitoring the potential effects of rare earth elements on cells and the freshwater ecosystem.

Field and laboratory studies on pathological and biochemical characterization of microcystin-induced liver and kidney damage in the phytoplanktivorous bighead carp

Field and experimental studies were conducted to investigate pathological characterizations and biochemical responses in the liver and kidney of the phytoplanktivorous bighead carp after intraperitoneal (i.p.) administration of microcystins (MCs) and exposure to natural cyanobacterial blooms in Meiliang Bay, Lake Taihu. Bighead carp in field and laboratory studies showed a progressive recovery of structure and function in terms of histological, cellular, and biochemical features. In laboratory study, when fish were i.p. injected with extracted MCs at the doses of 200 and 500 mu g MC- LReq/kg body weight, respectively, liver pathology in bighead carp was observed in a time dose-dependent manner within 24 h postinjection and characterized by disruption of liver structure, condensed cytoplasm, and the appearance of massive hepatocytes with karyopyknosis, karyorrhexis, and karyolysis. In comparison with previous studies on other fish, bighead carp in field study endured higher MC doses and longer-term exposure, but displayed less damage in the liver and kidney. Ultrastructural examination in the liver revealed the presence of lysosome proliferation, suggesting that bighead carp might eliminate or lessen cell damage caused by MCs through lysosome activation. Biochemically, sensitive responses in the antioxidant enzymes and higher basal glutathione concentrations might be responsible for their powerful resistance to MCs, suggesting that bighead carp can be used as biomanipulation fish to counteract cyanotoxin contamination.

Hematological and plasma biochemical responses of crucian carp (Carassius auratus) to intraperitoneal injection of extracted microcystins with the possible mechanisms of anemia

Alterations in hematological indices such as decreases in blood cell counts (RBC), hematocrit (Ht) and hemoglobin (Hb) concentrations are key symptoms of anemia. However, few experiments were conducted to examine changes in hematological indices of fish exposed to microcystins that are believed to be fatal to circulatory systems of vertebrates. An acute toxicological experiment was designed to study hematological changes of crucian carp injected intraperitoneally (i.p.) with extracted microcystins at two doses, 50 and 200 mu g MC-LReqkg(-1) body weight. After being i.p. injected with microcystins, the fish exhibited behavioral abnormity. There were significant decreases in RBC in the high-dose group, and in Ht and Hb concentrations in both dose groups, while erythrocte sedimentation rate (ESR) significantly increased, indicating the appearance of normocytic anemia. There were no prominent changes in the three red cell indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH,), and mean corpuscular hemoglobin concentration (MCHC). Increases in blood urea nitrogen (BUN) and creatinine (CR) in both dose groups suggest the occurrence of kidney impairment. Alteration in blood indices was reversible at the low dose group. Conclusively, anemia induced by kidney impairment was a key factor to cause abnormity of swimming behaviors and high mortality of crucian carp. (c) 2007 Elsevier Ltd. All rights reserved.

Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID50 ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID50 ml-1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell line

Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genescytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3 beta-hydroxysteroid dehydrogenase (3PHSD2), 17 beta-hydroxysteroid dehydrogenase (17 beta HSD1 and 17 beta HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3PHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption.

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 mu m(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them. (c) 2005 Elsevier B.V. All rights reserved.

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes : Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post-hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood-thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.

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.

Electron diffraction and photoemission studies of clean and water-covered Si(5,5,12) and Si(112) surfaces

The structure of silicon surfaces in the orientation range (113)-(5,5,12)-(337)-(112) has been investigated using high resolution LEED and photoemission both on a spherical and on flat samples. We find that Si(5,5,12) [5.3 degrees from (113) and 0.7 degrees from (937)] is the only stable orientation between (113) and (111) and confirm the result of Baski et al. [Science 269, 1556 (1995)] that it has a 2 x 1 superstructure with a very large unit cell of 7.68 x 53.5 Angstrom(2). Adsorption measurements of water on Si(5,5,12) yield a mobile precursor kinetics with two kinds of regions saturating at 0.25 and 0.15 ML which are related to adsorption on different sites. Using these results, a modified structure model is proposed. Surfaces between (113) and (5,5,12) separate into facets of these two orientations; between (5,5,12) and (112), they separate into (5,5,12) and (111) facets. (337) facets in this range may be considered as defective (5,5,12) facets.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Studies on advantages of heavy ions in radiotherapy compared with gamma-rays

Hepatoma and melanoma cells were exposed to C-12(6+) beams generated by HIRFL facility and gamma-rays and the cell response was studied by colony assays as well as the analysis of RBE of carbon ions was evolved. The survival curves of cells irradiated by heavy ions were different from those of cells irradiated by gamma-rays. And two kinds of cell showed the obvious discrepancy in response to the photon and ion irradiation. The results showed that heavy ions have special physical properties and mighty potency to kill cell in both single and fractional irradiation meanwhile it can kill tumor cells with high radioresistance more efficiently. When involved in clinical therapy, heavy ions will enhance the therapy efficiency and decrease the suffering of patients because it can impair the repair for sublethal damage of cells which can lead to fewer irradiation fractions.

Correlation between initial chromatid damage and survival of various cell lines exposed to heavy charged particles

The biophysical characteristics of heavy ions make them a rational source of radiation for use in radiotherapy of malignant tumours. Prior to radiotherapy treatment, a therapeutic regimen must be precisely defined, and during this stage information on individual patient radiosensitivity would be of very great medical value. There are various methods to predict radiosensitivity, but some shortfalls are difficult to avoid. The present study investigated the induction of chromatid breaks in five different cell lines, including one normal liver cell line (L02), exposed to carbon ions accelerated by the heavy ion research facility in Lanzhou (HIRFL), using chemically induced premature chromosome condensation (PCC). Previous studies have reported the number of chromatid breaks to be linearly related to the radiation dose, but the relationship between cell survival and chromatid breaks is not clear. The major result of the present study is that cellular radiosensitivity, as measured by D-0, is linearly correlated with the frequency of chromatid breaks per Gy in these five cell lines. We propose that PCC may be applied to predict radiosensitivity of tumour cells exposed to heavy ions.