Entamoeba dispar: infrastructure, Surface Properties and Cytopathic Effect

The cytological features of Entamoeba dispar , recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica. When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 760RR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition, E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect, E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica , both at the light microscopical and the ultrastructural levels. In addition, they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelial cell monolayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.     

Recombinant Klebsiella oxytoca Strains with Improved Efficiency in Removal of High Nitrate Loads

Klebsiella oxytoca CECT 4460 removes high nitrate loads from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose in K. oxytoca CECT 4460 was considered. To achieve this, we cloned and transferred into this strain the Klebsiella pneumoniae nasB gene, which encodes assimilatory nitrite reductase (Lin et al., J. Bacteriol. 176:2551–2559, 1994). The delivery vector was either the wide-host-range plasmid pUPE2, in which the nasB gene is expressed from the Escherichia coli P_lac promoter, or a mini-Tn5-Km vector, which upon random insertion in the host chromosome allowed expression of the nasB gene from an unidentified chromosomal host promoter. The effect of the increase in the dose of the nasB gene in K. oxytoca CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed that K. oxytoca CECT 4460 bearing pUPE2 accumulated 88% less nitrite than the wild-type strain, while the recombinant strain bearing the K. pneumoniae nasB gene in the host chromosome showed a 25% lower level of nitrite accumulation in the culture medium than that of the wild type.     

Fixed Versus Removable Microdialysis Probes for In Vivo Neurochemical Analysis: Implications for Behavioral Studies

Abstract: The levels of several neurochemicals, i.e., uric acid (UA), dopamine (DA), dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid, collected daily from the rat striatum with either fixed or removable microdialysis probes for 7 days after surgery were compared. The implantation of the fixed cannula was followed by a 10-fold increase in the UA content in the dialysates collected from the first day after surgery onward and by a steady decrease in dihydroxyphenylacetic acid levels, whereas those of DA remained fairly stable. With the removable cannula system, only a smaller, transient increase in UA during the first 3 days after surgery was observed, with no change in DA or monoamine metabolites. The glial reaction around the cannula tracks was assessed by both quantitative histological techniques and measuring the glutamine levels in the dialysates collected at the time of surgery and 7 days later. Both the glial cell number and nuclear size, as well as the glutamine outflow, were considerably larger in the animals implanted with the fixed probes. It is, therefore, likely that the UA levels in the dialysate reflect the glial reaction to the probe. The suitability of the removable probe system for behavioral experiments involving repeated microdialysis sampling was illustrated in an experiment showing that the DA release in the nucleus accumbens of male rats assessed daily at postsurgery days 5–10 was virtually identical in three alternating sessions of sexual behavior as was the smaller release of this neurotransmitter detected during intervening nonsexual social interactions.     

Adenovirus-Mediated Gene Expression In Vivo Is Enhanced by the Antiapoptotic Bcl-2 Gene

An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.     

Dew and its effect on two heliophile understorey species of a tropical dry deciduous forest in Mexico

 A series of horizontal and vertical measurements of dew deposition and dew duration were carried out in a tropical dry deciduous forest in western Mexico (19° 30′ N, 105° 03′ W). The effect of dew on transpiration in heliophile species was also investigated. The amount of dew was very variable with no temporal or spatial pattern. The amount of dew measured at two horizontal transects (maximum and minimum) (at heights of 0.20 m and 1.30 m above ground level) was from 0.014 to 0.203 mm and from 0.013 to 0.061 mm in the middle and at the end of the dry season, respectively. Dew deposition at different vertical levels (0.50–12.5 m height) ranged from 0.04 to 0.36 mm. The duration of dew formation ranged between 60 and 129 min after sunrise above the canopy (a height of 11 m), and between 259 and 290 min after sunrise at a height of 2 m. Daily transpiration rates were 883 and 632 g m^–2 d^–1 in Coccoloba liebmannii and 538 and 864 g m^–2 d^–1 in Jacquinia pungens in January and April, respectively. Transpiration was restricted from sunrise to early afternoon in April, as a result of the pronounced midday closure of stomata in both species. The reduction of transpiration by dew ranged from 13.2 to 50.1 g m^–2 d^–1 and from 4.5 to 77.7 g m^–2 d^–1 for C. liebmannii and J. pungens, respectively. Dew can play an important role in enhancing the survival of heliophile species in the dry season by reducing transpiration rates during the morning. Received: 30 April 1998 / Accepted: 14 December 1998     

Biodeterioration and restoration of a 16th-century book using a combination of conventional and molecular techniques: A case study

In this paper we deliver a report on the study of microbiological damage found on the pages of a 16th-century book. Our aim is to describe the procedures needed to ensure a conservative approach to the restoration of valuable books and objects of art made from, or supported on, paper. The techniques employed to evaluate and describe the damage observed, as well as the organisms responsible for biodeterioration, are discussed. A range of sampling techniques and instruments were utilised, including swabs and adhesive tape. Conventional methods, such as classic culturing and the direct microscopic observation of sampled material, were coupled with DNA-fingerprinting and phylogenetic analysis. We postulated that the purple stains which migrate through the pages with a felted consistency (Fig. 2), based on all the information obtained using traditional and molecular means, were caused by a cellulolytic fungus producing purple essudates, characterised by echinated conidia and Hülle cells. These elements were consistent with the discovery of both A. versicolor and A. nidulans using molecular techniques.     

Phenology of temperate trees in tropical climates

Several North American broad-leaved tree species range from the northern United States at 47°N to moist tropical montane forests in Mexico and Central America at 15–20°N. Along this gradient the average minimum temperatures of the coldest month (T _Jan), which characterize annual variation in temperature, increase from –10 to 12°C and tree phenology changes from deciduous to leaf-exchanging or evergreen in the southern range with a year-long growing season. Between 30 and 45°N, the time of bud break is highly correlated with T _Jan and bud break can be reliably predicted for the week in which mean minimum temperature rises to 7°C. Temperature-dependent deciduous phenology—and hence the validity of temperature-driven phenology models—terminates in southern North America near 30°N, where T _Jan>7°C enables growth of tropical trees and cultivation of frost-sensitive citrus fruits. In tropical climates most temperate broad-leaved species exchange old for new leaves within a few weeks in January-February, i.e., their phenology becomes similar to that of tropical leaf-exchanging species. Leaf buds of the southern ecotypes of these temperate species are therefore not winter-dormant and have no chilling requirement. As in many tropical trees, bud break of Celtis, Quercus and Fagus growing in warm climates is induced in early spring by increasing daylength. In tropical climates vegetative phenology is determined mainly by leaf longevity, seasonal variation in water stress and day length. As water stress during the dry season varies widely with soil water storage, climate-driven models cannot predict tree phenology in the tropics and tropical tree phenology does not constitute a useful indicator of global warming.