Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal ptant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 celts, and the 50% inhibitory concentration (IC50) were 22.01 mu g/ml (selectivity index (SI) = 2.83) and 21.10 mu g/mi (SI = 2.95), respectivety. At the highest concentration of the drug (40 mu g/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains. (C) 2006 Elsevier GmbH. All rights reserved.

In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20°C and 40°C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A Tnpho A Insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.

Evaluation of the in vitro susceptibility and emergence of mutants resistant to ciprofloxacin among multidrug resistant clinical isolates.

Two hundred and seventy-seven multidrug resistant clinical isolates [K. pneumoniae, (N = 87); E coli, (N = 30); Salmonella typhimurium (N = 100); P. aeruginosa, (N = 30); S. aureus, (N = 30)] from hospitalized patients specimens, were tested in vitro for sensitivity to Ciprofloxacin. Application of the disk diffusion test and determination of the minimal inhibitory concentration by the microdilution method indicated that, almost all isolates were sensitive to the drug. Overall, S. aureus and P. aeruginosa were the less sensitive organisms. Ciprofloxacin-resistant mutants occurred at frequencies of > or = 10(-5)/CFU.

In vitro evaluation of the antimicrobial activity of a castor oil-based irrigant

The antimicrobial activity of irrigating solutions - Endoquil (castor oil detergent), 2% chlorhexidine gluconate solution, and 0.5% NaOCI solution - was evaluated against Gram-positive cocci (Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus sobrinus), Gramnegative rods (Escherichia coli and Pseudomonas aeruginosa), and the yeast Candida albicans. Activity was evaluated using the two-layer agar diffusion technique. The base layer was obtained by pouring 10.0 ml of Muller Hinton Medium or 10.0 ml of Brain Heart Infusion agar in a Petri dish. After solidification a 5.0 ml seed layer of Muller Hinton Medium or Brain Heart Infusion agar with inoculum (106/ml) was added. Absorbent paper disks (6.0 mm in diameter) immersed in the solutions were placed at equidistant points. Plates were maintained at room temperature for 2 h for prediffusion of the solutions and incubated at 37°C for 24 h. The candle jar system was used for the Brain Heart Infusion agar plates. All tests were performed in duplicate. After incubation the medium was optimized with 0.05 g% triphenyltetrazolium chlorate gel and inhibition halos were measured. All bacterial strains were inhibited by 2.0% chlorhexidine gluconate. Endoquil was effective against Grampositive microorganisms, and 0.5% NaOCI was effective only against S. aureus. Copyright © 2001 by The American Association of Endodontists.

Efeito do extrato aquoso de sete-sangrias [Cuphea mesostemon (Koehne) Lourt] sobre o fluxo de água, medido em bexiga urinária de sapo in vitro

The Cuphea mesostemon specie, known as sete-sangrias, is widely used as a diuretic substance in popular medicine. As the toad urinary bladder is an epithelium analogous to the distal nephron of mammals, it is used in order to study the transport water and electrolytes in many laboratories. This preparation permits excellent observation in water flow, from the urinary bladder lumen to the external side or the serosal one (water re-absorption), by means of gravimetrical measures. In the present work the hydrosmotic effect of aqueous extract (AE) of sete-sangrias leaf was studied. A 20% solution was added to the serosal side (S) of the bladder preparation, and the water flow was measured every 15 minutes after that. The results showed that 4mL of AE in the S side, increased the JH20 in a significant manner (p<0,05). This effect had a dose - response shape, with the volumes of 0,2mL, 0,4mL and 0,8mL of AE in the S bath. The hydro-osmotic effect of the anti-diuretic hormone (ADH) was studied as well and a significant stimulation (p<0,05) in the JH2O was observed with the magnitude of 150%. The AE effect was similar to the ADH one, and was not antagonized by this hormone. We concluded that Cuphea possesses an anti-diuretic activity similar to that presented by ADH, in toad urinary bladder, in vitro.

In vitro cytotoxicity of some natural and semi-synthetic isocoumarins from Paepalanthus bromelioides

Numerous natural compounds have a potential for therapeutic applications, but may have to be chemically modified to alter toxic side effects. We investigated structural parameters that could affect the cytotoxicity of isocoumarins similar to 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c)pyran-1-one (paepalantine 1). Paepalantine 1 has antimicrobial activity, as well as significant in vitro cytotoxic effects in the McCoy cell line. Two other natural and two semi-synthetic isocoumarins with similar structures obtained from the capitula of Paepalanthus bromelioides were tested on the same cell line by the neutral red assay. Substitution of the 9 and/or 10-OH group made these compounds less cytotoxic.

Efeito dose-resposta de uma formulação de dentifrício com concentração reduzida de fluoreto - estudo in vitro.

The utilization of dentifrices with low fluorine concentration, for children under 6 years of age, has been suggested to reduce the risks of dental fluorosis. However, in order to have anticariogenic potential, the dentifrice should form loosely-bound fluorine (CaF2) on dental enamel. Considering that the formation of CaF2 is a function inversely related to pH, dentifrices with pH 5.5, with 275, 550 and 1,100 ppm F (NaF/silica) were developed in order to assess dose-response effects. A comparison between those dentifrices, a placebo product and the Crest toothpaste (positive control - standard) was carried out. Furthermore, the bioavailability of dentifrices, in terms of formation of total fluorine (TF), CaF2, and fluorapatite (FA) on human dental enamel, was evaluated. An ion-specific electrode was utilized for the determination of the dosage of fluorine. The results revealed that the dentifrice with 550 ppm F was more effective than both the placebo and the dentifrice with 275 ppm, presenting no difference in relation to the positive control (p > 0.05). A dose-effect correlation was observed as to the CaF2 formed. In conclusion, the modified formulation with 550 ppm F can be considered as effective as the standard dentifrice with 1,100 ppm, and its utilization would be safer with regard to dental fluorosis.

Anti-genotoxic effect of aqueous extracts of sun mushroom (Agaricus blazei Murill lineage 99/26) in mammalian cells in vitro

The sun mushroom is the popular name for the Agaricus blazei Murill fungus, a mushroom native to south-eastern Brazil, which has been frequently used in popular medicine mainly in the form of tea to treat various ailments (stress, diabetes, etc.). In the present study, the genotoxic and/or anti-genotoxic effects ofA. blazei on mammalian cells in culture was assessed by checking the increase or reduction of micronucleus (MN) frequency and comets. The sun mushroom (lineage 99/26) was used as aqueous extracts prepared (2.5%) at three different temperatures (60, 25 and 4°C). The in vitro micronucleus (MN) test in binucleated cells and comet assay were used in V79 cells cultivated in HAM-F10+DMEM medium (1:1), supplemented with 10% of fetal bovine serum. The experiments were divided into four treatment types: 1. Negative control; 2. Positive control with MMS; 3. Treatments with the three forms of extracts (60, 25 and 4°C); and 4. Treatments with the extracts in different associations (simultaneous, pre-treatment, post-treatment and simultaneous after pre-incubation for 1 h) with MMS. None of the A. blazei extracts show genotoxic activity. In the comet assay no protecting effect was found. The results obtained in the MN test showed that the three forms of extracts used had protective activity, suggesting that the compound or active ingredients of A. blazei are always present in these extracts. The greater protective efficiency of the simultaneous treatment and simultaneous treatment with pre-incubation mixture with MMS suggests that the extracts have an antimutagenic action of the desmutagenic type. © 2002 Elsevier Science Ltd. All rights reserved.

LH Response (in vivo and in vitro) to an LHRH Agonist Administered to Domestic Male Cats

We investigated plasma luteinizing hormone (LH) concentration in domestic male cats challenged with Luteinizing Hormone Releasing Hormone Analog (LHRH-A) [des Gly10, (DTrp6)-LHRH ethylamide] that mediates the function of the hypothalamic-piruitary-gonadal axis (HPG). Plasma LH concentrations in cats treated daily with LHRH (10 μg/ 100 μl/kg/day, subcutaneously - sc) for 19 days (LHRH group) and in controls treated with saline (NaCl - 0.9%, same volume - SAL group) were chronically studied. LHRH administration (sc) for 15 days induced a significant fall (P < 0.05) in plasma LH concentrations during the chronic study. After the 15th day of treatment the groups were divided once more into animals treated with LHRH (10 μg/100 μl/kg) or saline (iv), and a time course study (300 min) was performed (acute study). Next, four groups of cats were compared in an acute study involving the sc/iv administration of SAL/SAL, SAL/LHRH, LHRH/SAL, and LHRH/LHRH. The responses of the SAL animals challenged by acute iv administration of LHRH (group SAL/LHRH) were significantly higher (P < 0.01) than those of animals treated with LHRH (sc) (group LHRH/LHRH). LH release was also significantly increased in the latter group (P < 0.05), although the effect was short lasting, being recorded only at the first observation (45 min). An in vitro study with the pituitaries was also performed on day 20. Mean (±SEM) LH concentrations in the culture medium containing pituitaries with LHRH (10-7 M) or saline were determined. In vitro analysis of these pituitaries demonstrated a significantly reduced response (P < 0.05) by animals treated sc with LHRH for 19 days. This study represents a source of data for the domestic cat going beyond its own physiology. Serving as a model, this animal provide important information for the study of reproductive physiology in other members of its family (Felidae), almost all of them threatened with extinction.