Morphological and molecular characterization of six Indian Tetracotyle type metacercariae (Digenea: Strigeidae Railliet, 1919), using ribosomal and mitochondrial DNA.

Strigeids have a cup-shaped fore body, containing a holdfast organ with two lobes and hind body. The species diversity of strigeids remains incomplete, especially in the Indian sub-continent. Here, we described six Tetracotyle type metacercariae (T. muscularis, T. fausti, T. lucknowensis, T. xenentodoni, T. mathuraensis and T. glossogobii) from five fresh water fish, collected at Lucknow (India). Next, we characterized these metacercariae using ribosomal (18S, 28S, ITS2) and mitochondrial DNA (COI) to determine their systematic and phylogenetic position. Molecular identification using inter-specific variation range for all four molecular markers revealed 1.9-4.9% (18S); 3.3-8.8% (28S); 6.8-12.9% (ITS2), and 3.5-9.4% (COI) among six Tetracotyle type metacercariae. In phylogenetic analysis, constructed by neighbour-joining (NJ) and maximum likelihood (ML) methods, T. fausti, T. glossogobii, T. xenentodoni, T. lucknowensis and T. mathuraenis nested as sister groups with the member of strigeids for all four markers used; T. muscularis, however, formed a basal clade. Furthermore, phylogenetic placement states the monophyly of the Tetracotyle type of metacercariae in all the markers (18S, 28S, COI), except ITS2.

Morphological and molecular characterisation of three Indian Neascus-type metacercariae (Digenea: Diplostomidae).

Diplostomid digeneans are important parasites whose larval stages infect freshwater snails and fish as first and second intermediate hosts respectively. Diplostomid digeneans as adults are parasitic in many fish-eating birds and mammals. Our understanding of the species diversity of diplostomid digeneans remains incomplete, especially in the Indian sub-continent. Here, we describe three Neascus-type metacercariae (N. hanumanthai, N. gussevi, and N. xenentodoni) from freshwater fish specimens of Channa punctata (Bloch 1793), Trichogaster fasciata (Bloch and Schneider 1801) and Xenentodon cancila (Hamilton, 1822) respectively, collected in India. Next, we characterised these metacecariae using nuclear (28S and ITS1) and mitochondrial DNA (cox1) to determine their systematic and phylogenetic position. Molecular identification using interspecific variation for all three molecular markers revealed a closer relationship between N. hanumanthai and N. gussevi (1.9%-2.4%) than either of N. hanumanthai and N. gussevi to N. xenentodoni (3.1%-3.7% and 4.4%-4.0% respectively). In phylogenetic analyses, estimated by neighbour-joining (NJ) and maximum likelihood (ML) methods, N. gussevi and N. hanumanthai nested as sister groups of Posthodiplostomum Dubois, 1936 for all three markers used; N. xenentodoni, however, nested as a sister taxa of all other diplostomes when using 28S and ITS1 and nested as a sister taxa of Bolbophorus, Alaria and Neodiplostomum using cox1. These findings suggest that N. hanumanthai and N. gussevi are members of Posthodiplostomum, but that N. xenentodoni belongs to a separate and unknown genus. Similarly, by proteomics, we found that the cox1 protein sequences and structures were similar between N. hanumanthai and N. gussevi but distinct for N. xenentodoni.

A redescription of Sundanonchus behuri (Agrawal & Singh, 1982) n. comb. (Monogenoidea: Dactylogyridea: Sundanonchidae) from the gills of the freshwater Gangetic leaffish, Nandus nandus (Perciformes: Nandidae) in India.

Urocleidus behuri [Agrawal N., Singh H.S., On a known and three unknown monogenetic Urocleidus Mueller, 1934. Pranikee 1982; 3: 22-34], described from Nandus nandus in India, is reassigned to Sundanonchus Lim & Furtado [ Lim L.H., Furtado J.I., Sundanonchus g.n. (Monogenea, Tetraonchoididae) from two Malaysian freshwater fishes. Fol Parasitol 1985; 32: 11-19]. Sundanonchus triradicatus Lim & Furtado [Lim L.H., Furtado J.I., Sundanonchus g.n. (Monogenea, Tetraonchoididae) from two Malaysian freshwater fishes. Fol Parasitol 1985; 32: 11-19] is determined to be a junior subjective synonym of U. behuri and a new nomenclatural combination, Sundanonchus behuri is proposed. This article provides a redescription of this new combination, S. behuri [ Agrawal N., Singh H.S., On a known and three unknown monogenetic Urocleidus Mueller, 1934. Pranikee 1982; 3: 22-34], including the previously undescribed egg morphology, based on newly collected specimens from N. nandus in India-a new geographical record.

Distribution of HER2(V655) genotypes in breast cancer cases and controls in the United States.

The minor variant frequency of a HER2 polymorphism (HER2(V655)) has been determined for 471 United States women enrolled in a multiracial case-control study. Allelic frequencies varied significantly by race. Genotypic distributions showed no excess breast cancer risk associated with inheritance of HER2(V655) either as carriers (OR=1.2, 95% CI=0.8-1.9), heterozygotes (OR=1.2, 95% CI=0.8-1.9), or homozygotes (OR=1.4, 95% CI=0.4-4.2). Nor was there a significant association when each racial group was considered separately. The current study suggests the HER2(V655) allele is not a breast cancer risk factor for Caucasians, African-Americans, or Latinas.

Overexpression of human catalase gene decreases oxidized lipid-induced cytotoxicity in vascular smooth muscle cells.

Reactive oxygen metabolites such as hydrogen peroxide (H(2)O(2)) and oxidized fatty acids are proinflammatory and are involved in the pathophysiology of various diseases including atherosclerosis. The effects of these oxidants could be inhibited by the external addition of an antioxidant, suggesting the promotion or propagation of further oxidation. In this study, we describe the stable overexpression of human catalase in smooth muscle cells and the resistance of these cells to cytotoxicity induced not only by the addition of H(2)O(2) but also by the addition of 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results pose an intriguing possibility of the generation of H(2)O(2) from a peroxidized fatty acid. Accordingly, incubation of cells with both 13-HPODE and 13-hydroxyoctadecadienoic acid resulted in the generation of intracellular H(2)O(2). To explain the observed results by which catalase could overcome the effects of 13-HPODE, we propose that oxidized fatty acids are degraded in the cellular peroxisomes, resulting in the generation of H(2)O(2). In other words, the cellular effects of peroxidized fatty acids could be attributed to the generation of H(2)O(2).

Uptake of 13-hydroperoxylinoleic acid by cultured cells.

Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids.

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis.

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.

Protective effect of vanillin on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Vanillin (VA), an anticlastogen, has been demonstrated to inhibit gene mutations in both bacterial and mammalian cells. However, the data on its effect against radiation-induced cytogenetic damage are limited. The aim of this study was to investigate the protective effect of VA on radiation-induced chromosomal damage in V79 cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2) cm-2 and posttreated with 3 concentrations of VA (5, 50 or 100 micrograms ml-1 for 16 h for micronucleus (MN) and 18 h for structural chromosomal aberration (SCA) analyses. MN and SCA assays were performed concurrently according to standard procedures. Results indicate that there was a dose related increase in the percent of micronucleated binucleated cells (MNBN) (5.6 to 79.6) and percent of aberrant cells (Abs) (12 to 98) with X-ray treatment alone. Inhibition studies showed that the addition of VA at 100 micrograms ml-1 significantly reduced the percent of MNBN (21 to 48) induced by X-ray at 1, 2, and 4 Gy. There was a slight decrease in percent MNBN at 5 and 50 micrograms VA ml-1. All three concentrations of VA decreased percent Abs (15.7 to 57.1) induced by X-rays at all doses. UV radiation alone significantly increased percent MNBN (3.5 to 14.8) and percent Abs (17 to 29). Addition of 50 or 100 micrograms VA ml-1, significantly decreased percent MNBN (31.7 to 86.2) and percent Abs (54.5 to 90.9) at all doses of UV radiation. A decrease in percent MNBN (2.8 to 72.4) and percent Abs (34.8 to 66.7) was also noted at 5 micrograms VA ml-1. These data clearly indicate the protective effect of VA on radiation-induced chromosomal damage, suggesting that VA is an anticlastogenic agent.

Inhibition of methotrexate-induced chromosomal damage by folinic acid in V79 cells.

Methotrexate (MTX), an anticancer compound, is widely used in the treatment of leukemia. It induces cytogenetic damage as well as cytostatic effects on a variety of cell systems. Folinic acid (Leucovorin) is generally administered along with MTX as a rescue agent to decrease MTX-induced toxicity. However, information regarding the inhibitory effect of folinic acid against cytogenetic damage caused by MTX is limited. This study was conducted to assess the cytogenetic effect of MTX and its inhibition by folinic acid (FA) using the micronucleus and chromosomal aberration assays concurrently. Exponentially growing V79 cells were treated with MTX at five different concentrations (5-100 micrograms ml-1) with S9 microsomal fraction for 6 h and post-treated with two concentrations of FA (5 or 50 micrograms) for 40 h. Results indicate that MTX alone induced a concentration-related increase in % micronucleated binucleated cells (MNBN) and % aberrant cells (Abs). There was a decrease in nuclear division index (NDI) with increase in MTX concentration. Similarly, the mitotic index (MI) also decreased in all concentrations of MTX tested. The addition of FA at 50 micrograms ml-1 significantly reduced % MNBN (40-68%) and % Abs (36-77%). Inhibition was also seen at 5 micrograms FA (12 to 54% for MNBN and 20 to 61% for Abs). These results indicate that FA is capable of reducing the cytogenetic damage induced by MTX and appears to be an anticlastogenic agent.

Preneoplastic potential of morphologically distinct transformed foci induced by 3-methylcholanthrene.

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2-3, 3-4, and > or = 4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3-methylcholanthrene in Balb/c-3T3 cells. The transformed focal cells were used in in vitro studies including anchorage-independent analysis, focal reconstruction, gene transfection using NIH-3T3 host cells, and Southern blotting to assess amplification of five proto-oncogenes (K-ras, H-ras, c-fos, c-jun, c-myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage-independent growth of all five types of foci ranging from 7-12%; 2) all five morphological types of transformed foci showed 8-15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH-3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K-ras and H-ras proto-oncogenes; however, c-fos, c-jun, and c-myc did not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3-fold over the normal Balb/c-3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size > or = 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay.

Fresh mouse peritoneal macrophages have low scavenger receptor activity.

Peritoneal macrophages are easily isolated by lavage, suggesting that they are either nonadherent or weakly adherent in situ. Cultured macrophages express class A scavenger receptors (SCR), which mediate Ca2+-independent adhesion in vitro. We examined fresh peritoneal macrophages from mice and from women with endometriosis to determine whether the adherence of these cells was associated with increased expression of class A SCR. Fresh human macrophages were not immunoreactive to SCR antibodies; however, SCR immunoreactivity increased with time in culture. Fresh mouse and human macrophages took up minimal amounts of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-acetyl-low density lipoproteins (Ac-LDL), a class A SCR ligand. Murine macrophages in culture for 24-72 h internalized four times more Ac-LDL than fresh cells. Cells cultured for 2 days incorporated 3.2 times more [14C] oleate than freshly isolated cells (55.7 +/- 7.9 versus 17.6 +/- 3.0 nmol/mg cell protein). In contrast to SCR activity, mouse macrophage SCR mRNA expression was similar in freshly isolated macrophages and those cultured for 3 days. These results suggest that peritoneal macrophages express only low levels of SCR activity in situ and that posttranscriptional regulation after isolation leads to an increase in SCR activity that correlates with adherence of the macrophages in vitro.

Inhibition of methotrexate-induced chromosomal damage by vanillin and chlorophyllin in V79 cells.

Methotrexate (MTX), a chemotherapeutic agent used to treat cancer, produces cytogenetic damage and has a cytostatic effect in a variety of test systems. Several antigenotoxic agents have been studied in various in vitro and in vivo systems. However, data are limited regarding their ability to modulate MTX-induced genotoxicity. In the present study, vanillin (VA) and chlorophyllin (CHL) were used as antigenotoxic agents to study their ability to minimize the DNA damage caused by MTX. Exponentially growing V79 Chinese hamster lung cells were treated with MTX at five different concentrations (5-100 micrograms/ml) with S9 activation for 6 h and post-treated with two concentrations of either VA (50 or 100 micrograms/ml) or CHL (50 or 100 micrograms/ml) for 40 h. Cytochalasin B was added for the micronucleus (MN) assay along with antigenotoxic agents to evaluate MN in binucleated cells. Chromosomal aberrations were also evaluated in parallel cultures. Results indicate that MTX alone induced a dose-dependent decrease in the nuclear division index (NDI) and the mitotic index (MI). A significant increase in percent micronucleated binucleated cells (MNBN) and percent aberrant cells (Abs) was observed. Studies using VA as an antigenotoxic agent showed a decrease in the number of MNBN (26.3-83.1%) and Abs (16.0-87.5%) with the addition of either 50 or 100 micrograms VA/ml. The addition of CHL also significantly reduced the number of MNBN (53.0-91.5%) at both concentrations tested. Chromosomal aberrations were also significantly reduced (41.0-83.0). These studies indicate that both VA and CHL are capable of effectively minimizing MTX-induced chromosomal damage.

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2)/cm2) and post-treated with 5 or 50 micrograms FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. The results showed that X-ray treatment alone produced dose-related cytotoxicity as measured by nuclear division index (NDI) and mitotic index (MI). X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5-79%) and percent of aberrant cells (11-92%). FA at 5 micrograms/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12-46% of MNBN, 14-48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 micrograms FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5-10.3%) and percent aberrant cells (6-35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 micrograms/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 micrograms of FA. These data demonstrate the beneficial effect of FA in decreasing radiation-induced chromosomal damage.

Inhibition of tumor necrosis factor induced human aortic endothelial cell adhesion molecule gene expression by an alkoxybenzo[b]thiophene-2-carboxamide.

The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl-ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF-induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation.

Comparative studies on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Induction of micronuclei (MN) and structural chromosomal aberrations (SCA) by physical agents such as X-rays and UV radiation has been studied extensively in a variety of cell lines for genotoxicity assessment. However, comparative data on the relationship between these two cytogenetic endpoints are limited. This study compares SCA and MN formation in V79 Chinese hamster lung cells treated with X-rays and UV radiation. Four replicate cultures of exponentially growing cells were exposed to four doses of X-rays (100-800 rads). For two replicate cultures, cytochalasin B (3 micrograms/ml) was added and cells were harvested 16 h later for MN and nuclear division index (NDI) assessment. For the remaining two replicate cultures, colcemid (0.025 micrograms/ml) was added 16 h post-treatment and cells were harvested 2 h later for SCA and mitotic index (MI) analyses. This experiment was duplicated using four doses of UV radiation (100-800 microJ x 10(2)/cm2). In the X-ray experiment, generally, a decrease in the NDI and MI was noted with increasing dose. Also, there was a clear dose-related increase in percent micronucleated binucleated (MNBN) and aberrant cells. A similar dose response, but with lower frequencies, was observed in the UV radiation treatment. These data suggest a good correlation between chromosome damage as measured by percent MNBN and aberrant cells and cytotoxicity as measured by NDI and MI.