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     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Selection of a Borrelia burgdorferi antigenic variant by cultivation in the presence of increasing amounts of homologous immune serum

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.     

Synthesis of thiol-functionalized N-acetylmuramyl peptide congeners suitable for their conjugation to target molecules

N-Acetylmuramyl-dipeptide and tripeptide derivatives containing at the C-terminus a masked thiol function, i.e. the S-tert-butylthiocysteamine residue, were synthesized via direct condensation of N-acetylmuramic acid with the peptide moiety using the dicyclohexylcarbodiimide/N-hydroxysuccinimide procedure. Reduction with tributylphosphine in aqueous organic media generates the free thiol function for a selective conjugation of these immunomodulants with target molecules via unsymmetrical disulfide bridging with a second thiol group by the sulfenohydrazide procedure or via thio-ether linkage by the addition to maleimido--or aziridine-derivatives.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria.

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.