Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus)

Gerbils (Meriones unguiculatus) were intragastrically inoculated with axenic Giardia lamblia cultures from symptomatic and asymptomatic children. All isolates were able to colonize the duodenum. However, the colonization capacity of the symptomatic isolates was significantly higher compared to that of the asymptomatic ones. Despite the different colonization capacity of the isolates, the growth curves of infected animals were significantly lower than those of controls. The study demonstrates that acute giardia infections are capable of altering the corporal development of the host. These results may suggest that not only symptomatic, but also asymptomatic giardiasis in children, often unnoticed by parents and clinicians, could be causing a silent detriment in their nutritional status.     

Fast energy transfer between BChl d and BChl c in chlorosomes of the green sulfur bacterium Chlorobium limicola

We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of approximately 4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.     

Immunohistochemical expression of p53 in breast carcinoma is associated with the intron 1 BglII polymorphism of the p53 gene

Breast carcinoma is a public health problem worldwide. It is known that both genetic and environmental factors are important for breast carcinogenesis and that structural and/or functional alterations at p53 gene are commonly observed in breast tumors. In addition, polymorphisms of several genes in either their coding or non-coding sequences have been found related to cancer risk and/or clinicopathological characteristics of tumors. In this study we have evaluated the intron 1 BglII polymorphism of the p53 gene with a PCR-based approach in 117 cases of breast cancer and 102 healthy women and its association with the immunohistochemical expression of p53 in the tumors. The results showed that the presence of the polymorphism (allele 2) is highly associated with the tumor expression of p53 (p<0.0001) and that there is a trend for increased frequency of allele 2 in cases than in controls (p=0.2376). These data suggest that the germ-line variation in the intron 1 of the p53 gene could produce functional or structural changes of the protein that is reflected by its abnormal expression.     

Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene

The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.     

Effect of hydroquinone on meiotic segregation in Drosophila melanogaster females

The induction of sex chromosomes meiotic nondisjunction (ND) by hydroquinone (HQ) given orally was investigated in Drosophila melanogaster 2-7, 8-22, 24, 48, 72 and 96 h-old females. ND was assessed by a system where exceptional females (XXY) and only 1/4 of the expected regular progeny are viable. Oocytes were treated at different stages of development. 4% HQ tested only in 72 h-old females induced ND in oocytes sampled in brood I (mostly mature oocytes at metaphase I). 6% HQ increased ND in brood I of 8-22 h-old females, while other broods, (including cells treated at early prophase) were also affected in older flies, the highest significance being attained in the 48 h-old series. Newly hatched females (2-7 h-old) were refractory to the treatment, though oocytes sampled in the first three subcultures are comparable to cells showing enhancement of ND in series run with older females. Toxicity of 2, 4 and 6% HQ increased with concentration and females' age: (a) 2% was not toxic; (b) 4% was toxic only to 72 h-old females; (c) 6% was increasingly toxic to females 24, 48 and 72 h-old. The results indicate that age plays a significant role on both chromosomal segregation and toxicity and suggest that in Drosophila HQ is metabolized to its reactive species. The lack of toxic and aneugenic effect in very young females could reflect a more efficient detoxification due to the known high specific activity of glutathione-S-transferase (GST) after eclosion. The decline in GST activity around day 2 of adult life coincides with the high effect of HQ in 48 h-old females.     

Flavonoids from the stem bark of Lonchocarpus xuul

The stem bark of Lonchocarpus xuul (Leguminosae) has yielded four flavonoids which have been identified by spectroscopic methods as the novel 4beta,5-dimethoxy-6",6"-dimethyl-2H-pyrano-(2",3":7,6)-fl avan (xuulanin), 3beta,4beta,5-trimethoxy-6",6"-dimethyl-2H-pyrano-(2",3":7,6 )-flavan (3beta-methoxyxuulanin). 4beta-ethoxy-5-methoxy-6",6"-dimethyl-2H-pyrano-(2",3":7,6)- flavan (4beta-demethylxuulanin-4beta-ethyl ether), and the known 5,7-dihydroxy-6,8-di(3-methylbut-2-enyl)flavanone (spiniflavanone-B). The ethyl derivative is considered likely to be an artefact.     

Extensor digitorum brevis free flap: anatomic study and further clinical applications

The extensor digitorum brevis muscle flap is reliable, safe, and can be used either as a pedicle or as a free flap with minimal donor site morbidity. To increase the existing knowledge of this flap and to establish further anatomic basis for the design and elevation of the extensor digitorum brevis flap, 26 specimens from 13 fresh cadavers were dissected under 3.5x loupes. The lateral tarsal artery was found to be the main blood supply to the muscle. It has an average diameter of 1.83+/-0.35 mm and a length of 1.89+/-0.69 cm. The dorsalis pedis artery has, at the level of the lateral tarsal artery takeoff, a diameter of 3.25+/-0.62 mm. From this point to the origin of the deep plantar branch, the dorsalis pedis artery has minimal branching, and the surgeon has available an artery homogeneous in diameter that is 6.77+/-0.99 cm in length. Related neurovascular structures (anterior tibial artery and the venae comitantes, dorsalis pedis and first dorsal metatarsal artery, and deep peroneal nerve) were also studied. A safe and reliable harvesting technique and the "T interposed extensor digitorum brevis" technique for sparing the anterior tibial artery are presented, as are clinical case examples on the use of this flap as a flow-through, extensor digitorum brevis-vascularized nerve graft, a combined extensor digitorum brevis-deep peroneal nerve graft, and a bilobed extensor digitorum brevis-dorsalis pedis fasciosubcutaneous free flap.     

Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.