Isolation and identification of strains of Bacteroides fragilis group from the digestive tract of Callithrix penicillata marmosets

Thirty-five strains of the Bacteroides fragilis group were isolated from oral and intestinal samples from 5 wild caught, captive Callithrix penicillata. Nine oral strains of Bacteroides fragilis (7) and Bacteroides distasonis (2), and 26 intestinal strains of Bacteroides fragilis (14) and Bacteroides distasonis (12) were identified.     

Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants.

In this paper we report the nucleotide sequence and predicted amino acid sequence of glycoprotein B of herpes simplex virus 1 strain F and the amino acid substitutions in the domains of the glycoprotein B gene of three mutants selected for resistance to monoclonal antibody H126-5 or H233 but not to both. Analyses of the amino acid sequence with respect to hydropathicity and secondary structure yielded a two-dimensional model of the protein. The model predicts an N-terminal, 29-amino-acid cleavable signal sequence, a 696-amino-acid hydrophilic surface domain containing six potential sites for N-linked glycosylation, a 69-amino-acid hydrophobic domain containing three segments traversing the membrane, and a charged 109-amino-acid domain projecting into the cytoplasm and previously shown to marker rescue glycoprotein B syn mutations. The nucleotide sequence of the mutant glycoprotein B DNA fragments previously shown to marker transfer or rescue the mutations revealed that the amino acid substitutions cluster in the hydrophilic surface domain between amino acids 273 and 305. Analyses of the secondary structure of these regions, coupled with the experimentally derived observation that the H126-5- and H233-antibody cognitive sites do not overlap, indicate the approximate locations of the epitopes of these neutralizing, surface-reacting, and immune-precipitating monoclonal antibodies. The predicted perturbations in the secondary structure introduced by the amino acid substitutions correlate with the extent of loss of reactivity with monoclonal antibodies in various immunoassays.     

Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes

A rabbit antibody to the neuraminidase of the infective form of Trypanosoma cruzi identifies a subpopulation of trypomastigotes that expresses neuraminidase. Complement-mediated lysis by the antibody selectively destroys 30 to 40% of the trypomastigotes, supporting the conclusion that the immune antibody binds to a subset of parasites. The trypomastigotes that react with the immune antibody are the only ones expressing neuraminidase because the trypomastigotes that survive complement-mediated lysis are depleted of neuraminidase activity. The enzyme seems to negatively modulate infection in vitro, since infection of host cells by trypomastigotes is enhanced when neuraminidase activity is blocked by antineuraminidase antibody; infection is also enhanced when the infecting trypomastigotes have been depleted of parasites that express neuraminidase. Addition of exogenous neuraminidase (from Vibrio cholerae) to trypomastigotes treated with immune antibody, reverts the enhancement observed when infection takes place in the presence of antibody to T. cruzi neuraminidase only. Addition of V. cholerae neuraminidase in the absence of immune antibodies has no effect on infection. These results show that T. cruzi neuraminidase depresses infection and also suggest that sialic acid is involved in the parasite-host cell interaction. The antibody to T. cruzi neuraminidase recognizes on the surface of live trypomastigotes a set of proteins with high m.w. (165,000 to 200,000) and also two antigens of 79,000 to 82,000. The high m.w. proteins appear to be associated with neuraminidase activity as shown by renaturation experiments of released enzyme fractionated on a sodium dodecyl sulfate-polyacrylamide gel.     

Morphological effects of mercury exposure on windowpane flounder gills as observed by scanning electron microscopy

Windowpane flounder, Scophthalmus aquosus Mitchill, were exposed for 60 days to 5 or 10 μg 1⁻¹ mercury and gill samples were examined by scanning electron microscopy. The response of the gill epithelium was different at the two levels of mercury exposure. The number of chloride cell apical pits and gill filaments bearing 'cratered' epithelial cells increased at the 5-μg 1⁻¹ level and decreased at the higher exposure level.
Focal swellings demonstrated a dose-dependent relationship, their numbers being greatest at the higher exposure level. Marked fragmentation of pavement cell microridge patterns and swelling of the respiratory epithelial cells was evident at the 10-μg 1⁻¹ exposure level.     

Quantitative and qualitative immunohistochemical detection of myc and src oncogene proteins in normal, nodule, and neoplastic rat liver

This study examined the possibility of using an immunohistochemical technique to detect the expression of myc and src oncogene proteins (ops) in livers of male Sprague-Dawley rats after treatment with the carcinogen diethylnitrosamine (with or without phenobarbital promotion) or untreated. We found that the majority of nodules and tumors from these livers stained for myc and src ops, indicating that myc and src expression did occur in these structures. These results were expected, since myc and src expression has been previously observed by others using different techniques. However, in our study, myc and src op staining was also noted in normal liver areas from rats in any of the four treatment groups (DENA, DENA + PB, PB alone, or untreated). The staining pattern of normal liver was different for each oncogene probe but was consistent within the four groups. In most cases, oncogene expression of normal liver occurred at sites of abnormal (but non-neoplastic) hepatocytes. The method reported here used both a qualitative technique of op expression analysis and a quantitative method using a Zeiss computer-driven image analysis system.     

Morphological and Neurochemical Effects of Diazepam and Phenobarbital on Selective Culture of Neurons from Fetal Rat Brain

The responses to diazepam (DZ) and phenobarbital (PhB) were studied in enriched neuronal primary cultures from rat embryo hemispheres. Cells were grown in chemically defined medium and the drugs were added for 3 days to cultures, at pharmacologically active concentrations. Following exposure to DZ or to PhB, morphological changes, such as less prominent neuronal processes, were observed in neurons. It was also shown that each drug reduced the specific uptake of 2-deoxy-D-glucose by the cells and interfered with protein and RNA metabolism. It was concluded that both DZ and PhB might affect, at least transiently, the normal growth of neurons in culture.     

3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.     

Blockade of transgenic gamma delta T cell development in beta 2-microglobulin deficient mice.

The gamma delta T cell receptor (TCR) of the hybridoma KN6 recognizes the self molecule encoded by a class I gene which maps within the TL region of the major histocompatibility complex (MHC) of H-2b mice. Mice transgenic (Tg) for this TCR were crossed with mice genetically deficient in beta 2-microglobulin (beta 2m). No mature Tg gamma delta T cells were detected in the thymus or the spleen of the beta 2m- gamma delta Tg mice. We conclude that interaction between the Tg gamma delta TCR and a beta 2m-associated molecule (probably an MHC class I molecule) is required for the generation of mature Tg gamma delta T cells.     

Antiproliferative Activity and VEGF Expression Reduction in MCF7 and PC-3 Cancer Cells by Paclitaxel and Imatinib Co-encapsulation in Folate-Targeted Liposomes

Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.