Shape-based features for cat ganglion retinal cells classification

This article presents a quantitative and objective approach to cat ganglion cell characterization and classification. The combination of several biologically relevant features such as diameter, eccentricity, fractal dimension, influence histogram, influence area, convex hull area, and convex hull diameter are derived from geometrical transforms and then processed by three different clustering methods (Ward's hierarchical scheme, K-means and genetic algorithm), whose results are then combined by a voting strategy. These experiments indicate the superiority of some features and also suggest some possible biological implications.

Avaliação de diferentes sistemas de cultivo in vitro para a micropropagação de Psychotria ipecacuanha (Brot.) Stokes

This work was carried out with Psychotria ipecacuanha, a Brazilian medicinal plant the roots of which contain emetine. The main objective was to develop a protocol for the micro-propagation of these species, by testing different culture techniques, the temporary immersion system, and the semi-solid and liquid media systems. In the semi-solid system, experiments were developed in flasks of two different sizes containing MS, B5, and WP media to which were added different growth regulators. Innoculum density was also evaluated. The liquid medium system consisted of MS medium supplemented with different growth regulators. For the temporary immersion system, the MS medium received an addition of 1.5mg/L BAP and 0.5mg/L GA3, and a reverse digital apparatus and vacuum pump were used. The liquid medium system with MS medium supplemented with 1.5mg/L BAP and 0.5mg/L GA3 presented the best results for shoot proliferation in a period of 30 days in culture (2.37 ± 0.32 shoots/explant). Cultures carried out for 90 days in the semi-solid system, using 8.5 × 5.5cm flasks and 3 explants per flask, developed 1.80 ± 0.20 shoots/explant, achieving 3.06 ± 0.51 cm of height adn presented superior survival ratio (96%). Explants cultured in temporary immersion system for 90 days showed 2.30 ± 1.10 shoots/explant achieving a growth of 2.08 ± 0.12 cm and 52% survival.

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.

Analise citotoxica de esparfloxacino e seus produtos de degradaçao em células mononucleares humanas

Sparfloxacin, a difluorquinolone derivative, is a potent antibacterial agent active against a wide range of gram-positive and gram-negative organisms including Streptococcus pneumoniae, Staphylococcus aureus, methicillin resistant S. aureus, Legionella spp, Mycoplasma spp; Chlamydia spp. and Mycobacteria. A drawback of fluorquinolones is their photoreactivity. Sparfloxacin has been studied in terms of therapeutic activities. However, few reports about analytical methods of sparfloxacin are available in the literature. The aim of this study was to determine cytotoxic effects, using sparfloxacin reference substance (SPAX-SR), sparfloxacin tablets (SPAX-COMP) and sparfloxacin tablets submitted UV light during 36 hours (SPAX-COMP.36) solution, and two isolated products (7 and 9) of SPAX-SR submitted UV-C light, in concentrations of 31.25, 62.5, 125 and 250 μg/mL by in vitro mononuclear humane culture cells. The results, statistically analyzed by Teste de Tukey, showed SPAX, SPAX-COMP and SPAX-COMP.36 solutions could reduce the cells number in these conditions. These results could not be observed for products 7 or 9. These results can suggest that isolated product can be less cytotoxic than SPAX-SR, is method can also be used to identified products degradation of sparfloxacin in stability study. However, the low activity achieved with sparfloxacin submitted to UV-light is a source of concern and requires further investigation about its photodegradation mechanism.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aro A-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg-1. The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory. © 2002 Elsevier Science (USA). All rights reserved.

Efeito do alumínio no crescimento de brotações de Eucalyptus grandis x E. urophylla cultivadas in vitro

The aim of this work was to evaluate the effects of aluminum on the growth of Eucalyptus shoots cultivated in vitro through nutrient and total soluble protein content. The trial had a totally randomized design with four treatments and four replicates. The treatments were: 0.0; 0.25; 0.5 and 1.0 mM of AlCl 3.6H 2O. Shoots without roots of a Eucalyptus grandis x E urophylla clone were used for the in vitro culture. Evaluations were made on the 4th, 8th, 12th, 16th, 20th, 24th and 28th day of culture. The Al addition to the culture media reduced mainly Ca, P and K availability and absorptions by the shoots. The cellular metabolism was affected, conducted to morphological alterations in shoots (browning, mass calluses formation and shoots not friable), dry matter increased and a decreased in total protein soluble.

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.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Cytokines and dietary energy restriction in stable chronic obstructive pulmonary disease patients

Tumour necrosis factor (TNF)-α has been found to be increased in malnourished chronic obstructive pulmonary disease (COPD) patients; however, the main cause of this phenomenon remains undetermined. In normal subjects, TNF-α production may be induced by dietary energy deprivation. The aim of this study was to investigate if stable COPD patients present alterations of inflammatory mediators after 48 h of dietary energy restriction. Fourteen COPD patients were admitted to the hospital while receiving an experimental diet with an energy content of approximately one-third of their energy needs. Clinical evaluation, nutritional assessment and serum levels of interleukin (IL)-6, TNF-α and C-reactive protein, and secretion of TNF-α by peripheral blood monocytes were assessed on admission and after the experimental diet. For reference values of the laboratory parameters, blood was collected from 10 healthy, elderly subjects. COPD patients showed significantly higher serum concentrations of IL-6 than control subjects, however, the experimental diet was not associated with statistically significant changes in the inflammatory mediators. The findings of this study, although preliminary because of the limited degree and duration of the energy restriction, suggest that the elevated levels of tumour necrosis factor-α, previously described in undernourished or weight-losing chronic obstructive pulmonary disease patients, may not be linked to a decrease of dietary energy intake.

Secretion of tumor necrosis factor-alpha by mouse peritoneal macrophages in the presence of dental sealers, sealapex and endomethasone

After filling root canals, the healing process depends on the chemical composition or physical-chemical properties of the material used, among other factors. All root canal sealers, whether solid or plastic, are foreign matter for the body if they remain in permanent contact with apical and periapical tissues. As a result, the first organic reaction that occurs is an attempt to phagocytize the material. During phagocytosis, macrophages release a large number of cell mediators into the area, among which are cytokines that are essential in intercellular communication and in many physiological and pathophysiological processes. One of these cytokines is tumor necrosis factor-alfa (TNF-α), which acts through links to specific receptors on the cell membrane initiating a cascade of events leading to induction, activation, or inhibition of numerous cytokine-regulated genes in the cell nucleus. The release of TNF-α in a cell culture of mouse peritoneal macrophages incubated with three concentrations (25, 50, and 100 mg/ml) of two endodontic sealers was measured. The solutions containing the calcium hydroxide-based root canal sealer (Sealapex) released fewer units of TNF-α than solutions containing the zinc oxide and eugenol-based sealer (Endomethasone).

Hypochlorous acid inhibition by acetoacetate: Implications on neutrophil functions

Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl- system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate. © 2004 Pharmaceutical Society of Japan.

In vitro propagation of Maytenus ilicifolia (Celastraceae) as potential source for antitumoral and antioxidant quinomethide triterpenes production. A rapid quantitative method for their analysis by reverse-phase high-performance liquid chromatography

Cell culture of Maytenus ilicifolia were established in order to produce and to quantify the antitumoral and antioxidant quinonemethide triterpenes. In vitro calli were induced from leaf explants of native plants and cultured in semi-solid medium under controlled conditions of humidity, temperature and photoperiod. The quinonemethide triterpenes showed maximum accumulation in the logarithmic phase growth of the cell culture. A rapid, sensitive and reliable reverse-phase HPLC method was used for quantitative determination of the antitumoral and antioxidant quinonemethide triterpenes, 22β-hydroxymaytenin and maytenin in callus of Maytenus ilicifolia. Well resolved peaks with good detection response and linearity in the range 1.0 - 100 μg/mL were obtained. This quantitative work was performed by an external standard method.

Synthesis, characterization, and biological activity of a new palladium(II) complex with deoxyalliin

Synthesis, characterization, and biological activity of a new water-soluble Pd(II)-deoxyalliin (S-allyl-L-cysteine) complex are described in this article. Elemental and thermal analysis for the complex are consistent with the formula [Pd(C6H10NO2S)2]. 13C NMR, 1H NMR, and IR spectroscopy show coordination of the ligand to Pd(II) through S and N atoms in a square planar geometry. Final residue of the thermal treatment was identified as a mixture of PdO and metallic Pd. Antiproliferative assays using aqueous solutions of the complex against HeLa and TM5 tumor cells showed a pronounced activity of the complex even at low concentrations. After incubation for 24 h, the complex induced cytotoxic effect over HeLa cells when used at concentrations higher than 0.40 mmol/L. At lower concentrations, the complex was nontoxic, indicating its action is probably due to cell cycle arrest, rather than cell death. In agreement with these results, the flow cytometric analysis indicated that after incubation for 24 h at low concentrations of the complex cells are arrested in G0/G1. © 2005 NRC Canada.