A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

A severe autosomal recessive acromesomelic dysplasia,the Hunter-Thompson type,and comparison with the Grebe type

Summary Two siblings with a short-limb dwarfing condition which we call acromesomelic dysplasia, Hunter-Thompson type are reported. Abnormalities are limited to the limbs and limb joints in this severe form of dwarfism. The middle and distal segments of the limbs are most affected. The lower limbs are more affected than the upper. We are aware of one previously published case of this entity reported by A. G. W. Hunter and M. W. Thompson in 1976. Dislocations of the elbows and ankles were present in all three patients and dislocations of the hips and knees in two. One of the siblings who did not have hip and knee dislocations clinically resembled Grebe chondrodysplasia, another severe acromesomelic dwarfing condition. However, radiological analysis suggests that while acromesomelic dysplasia, Hunter-Thompson type and Grebe chondrodysplasia are related, they are not identical. Grebe chondrodysplasia has been established as an autosomal recessive trait. It appears probable that the entity we describe has the same mode of genetic transmission.     

Red blood cells as an antigen-delivery system.

The use of adjuvants is usually required to induce strong immunological responses to protein antigens. However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation. We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant. Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses. Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies. Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.     

Fine root distribution of trees and understory in mature stands of maritime pine (Pinus pinaster) on dry and humid sites

Maritime pine (Pinus pinaster) is the main tree cropping species in the Landes of Gascogne forest range in south western France. Soils are nutrient poor, sandy podzosols and site fertility is determined essentially by organic matter content and depth of water table, which is known to limit root growth. We hypothesised, with an ultimate goal of constructing a nutrient uptake model applicable to this region, that the organic top horizons together with the depth of the water table should be the most important parameters related to fine root distribution and presence of associated mycorrhiza. To test this hypothesis, we compared two adult Pinus pinaster stands, contrasting in depth of water table and soil fertility and evaluated fine roots (diameter ≤2 mm) of understory species and fine roots and ectomycorrhizal morphotypes of Pinus pinaster down to 1.2 m, using a soil corer approach. Total fine root biomass of Pinus pinaster was not significantly different between both sites (3.6 and 4.5 t ha⁻¹ for the humid, respectively, dry site), but root distribution was significantly shallower and root diameter increased more with depth at the humid site, presumably due to more adverse soil conditions as related to the presence of a hardpan, higher amount of aluminium oxides and / or anoxia. Fine roots of Pinus pinaster represented only about 30% of total fine root biomass and 15% of total fine root length, suggesting that the understory species cannot be ignored with regards to competition for mineral nutrients and water. A comparison of the ectomycorrhizal morphotypes showed that the humid site could be characterised by a very large proportion of contact exploration types, thought to be more relevant in accessing organic nutrient sources, whereas the dry site had a significantly higher proportion of both long-distance and short-distance exploration types, the latter of which was thought to be more resistant to short-term drought periods. These results partly confirm our hypothesis on root distribution as related to the presence of soil mineral nutrients (i.e. in organic matter), point out the potential role of understory plant species and ectomycorrhizal symbiosis and are a valuable step in building a site-specific nutrient uptake model.     

Errors Related to Different Techniques of Intraperitoneal Injection in Mice

We found a 12% error in the placement of intraperitoneal injections of mice with the one-man procedure of injection. With a two-man procedure, the incidence of error was consistently reduced to 1.2%.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.