Technical note: a new facility for continuous respiration measurements in lactating cows.

An open-circuit indirect calorimetry system consisting of 4 climate-controlled respiration chambers for cattle has been constructed and validated. The system allows for the continuous monitoring of O(2), CO(2), and CH(4) concentrations in chamber air, and the simultaneous determination of feed and water intake, overall physical activity, position changes, standing and lying times, and animal behavior. For complete balance trials, feces, urine, and milk can be collected quantitatively. Most importantly, lactating cows can be milked in the chamber, and blood samples can be drawn from permanent catheters without disruption of the measurements. The investigator, on entering the chamber, wears a facemask connected to the ambient air during the whole milking process. Data are routed to a data acquisition system with appropriate data evaluation software developed in our research unit. Thus, dynamic changes of the above-named parameters during the course of the day or of longer time periods can be monitored. Such data are critical for understanding the complex regulation and interplay of feed intake, energy metabolism, climatic conditions, and milk production.

Studies on aconitase species from Saccharomyces cerevisiae, porcine and bovine heart, obtained by a modified isolation method.

Aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) was isolated from Saccharomyces cerevisiae, porcine and bovine heart by a simplified method including affinity chromatography on Blue Dextran-Sepharose. Partial characterisation reveals that the aconitase species are all similar due to molecule size, amino acid composition, isoelectric point and enzymatic activity. Aconitase appears as a single polypeptide chain with a small carbohydrate content. A molecular weight of 79000 +/- 2000 and a Svedberg constant of s20,w = 4.75 +/- 0.2 S indicate a compact structure of aconitase. Due to different properties among the yeast aconitase species concerning isoelectric point and enzymatic activity a coherence between net charge of the protein and redox state of the Fe-S cluster can be expected.

Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica.

Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.

Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica.

We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.

Cysteine proteinase of Entamoeba histolytica. I. Partial purification and action on different enzymes.

A method for a 50-60-fold purification of a cysteine proteinase from trophozoites of Entamoeba histolytica using 35-80% ammonium sulphate fractionation, gel chromatography on Sephadex G-75, and preparative isoelectric focusing is described. The enzyme was examined for its proteolytic potencies towards native enzyme substrates. The amebic proteinase directly inactivates aldolase and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle as well as glucose-6-phosphate dehydrogenase from yeast. The inactivation of citrate synthase from porcine heart proceeds rather slowly, whereas malate dehydrogenase from porcine heart is not affected by the amebic proteinase under the condition used. With the exception of aldolase all inactivated enzyme substrates have been cleaved by limited proteolyses yielding major cleavage products. The inactivation of aldolase probably functions by the release of a small segment from a terminus being essential for aldolase activity.

A weakly acidic protease has a powerful proteolytic activity in Entamoeba histolytica.

A thiol dependent protease from homogenates of the parasite Entamoeba histolytica has been identified and partially purified by means of ammonium sulphate fractionation, gel filtration and isoelectric focusing. The protease, having a molecular mass of 21 +/- 2 kDa as judged by gel chromatography, represents a highly potent proteolytic capacity. The protease shows maximal activity against azocasein around the slightly acidic pH of 4.4 at 37 degrees C, but is also active at pH 3.4 and 8.5. At optimal pH, the turnover increases with increasing temperature up to 85 degrees C. The enzyme possesses a thiol group essential for activity, which is inhibited by the thiol blocking reagent p-chloromercuribenzoate. Solutions containing low concentrations of free Hg2+ cause conservation of protease activity. The native protein exhibits an isoelectric point of 4.9. This protease resembles the thiol endopeptidases of mammalian lysosomes, and appears to be a major proteolytic enzyme in Entamoeba histolytica.

Cysteine proteinase of Entamoeba histolytica. II. Identification of the major split position in bovine insulin B-chain.

The substrate specificity of a cysteine proteinase of Entamoeba histolytica was tested using the oxidized B-chain of bovine insulin. 28 proteolytic peptides were formed after different intervals of incubation and could be separated by high-performance liquid chromatography. Two dominant peptides appearing after 10 min of incubation were isolated and analyzed for amino acid composition. The results obtained indicate that these two peptides were formed by cleavage of the peptide bond between the amino acids 23 and 24. Thus as major split position the Gly-Phe bond in the insulin B-chain could be identified.

Specificity of a cysteine proteinase of Entamoeba histolytica towards the alpha 1-CB2 peptide of bovine collagen type I.

The cysteine proteinase of Entamoeba histolytica was shown to hydrolyze the cyanogen bromide peptide alpha 1-CB2 of calf skin collagen type I yielding two split products. Amino acid analyses of the formed peptides and estimation of the amino-terminal residues revealed that the alpha 1-CB2 peptide was exclusively cleaved between Gly10 and Leu11 within the sequence -Arg9-Gly10-Leu11-yielding two peptides with 7 and 29 amino acids, respectively. Under identical conditions the ratio of hydrolysis of the Gly10-Leu11 bond in the alpha 1-CB2 peptide to the Gly23-Phe24 bond within the internal sequence -Arg22-Gly23-Phe24- of bovine insulin B-chain was 100:65. The enzyme was found to split both benzyloxycarbonyl-Arg-Arg-methoxy-2-naphthylamide and benzyloxycarbonyl-Arg-Gly-2-naphthylamide. The ratio of hydrolysis of these substances was 100:11.6. Benzyloxycarbonyl-Gly-Arg-2-naphthylamide was a very poor substrate for the enzyme.

Functional identification of alpha 1-giardin as an annexin of Giardia lamblia.

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III.

Subcellular distribution of amebapain, the major cysteine proteinase of Entamoeba histolytica.

Amebapain, the major cysteine proteinase of E. histolytica, appears to be important both for digestion and as a cytotoxic factor. By immunocytology we established that amebapain was bound to matrix-like structures both on the cell surface and within subcellular vesicles; the amebapain co-localized with pinocytized iron oxide, suggesting that the enzyme recycles between plasma membrane and pinocytic vesicles. We conclude from these data that (i) the pinocytic vesicles in E. histolytica perform functions that in higher eukaryotes are taken over by specialized organelles such as lysosomes and cytotoxic granules, and (ii) the vesicles contain a matrix that helps retain the protease upon exocytosis of vesicle contents.

Analysis of leaves by using the laser ICP-MS with isotope dissolution method.

The use of laser ablation inductively coupled mass spectrometry (laser ICP-MS), in combination with a calibration procedure involving the addition of enriched isotopes, for the determination of trace elements in birch leaves and their ashes is described. Samples are pressed into pellets without binder materials and analyzed using the ICP-MS spectrometer ELAN 5000 with the laser sampler type 320 (Perkin Elmer). The analytical results obtained by two methods are compared with the values obtained after digestion of the same samples and analysis of the resulting solutions by ICP-MS. The results are discussed in terms of precision, accuracy and limits of detections.

alpha14-Giardin (annexin E1) is associated with tubulin in trophozoites of Giardia lamblia and forms local slubs in the flagella.

In a previous study, we reported that the novel annexin XX1 (annexin E1), identical to alpha14-giardin, is specifically localized to the flagella and to the median body of the trophozoites. However, the mode of interaction and the direct partners involved remained unclear. In the present study, we show that alpha4-giardin obviously does not evenly distribute over the full length of the axonemes, but rather, resides at local slubs near the proximal part and the ends of the flagella. In immunocytochemical co-localization studies, the anti-giardin primary antibody exclusively reacted with distinct regions of the flagella in permeabilized cells, whereas the anti-tubulin antibody bound to all areas of the axonemes in the cells and to isolated cytoskeletons. Isolated cytoskeletons did not react with anti-giardin antibodies. alpha14-Giardin itself is able to assemble to multimeric structures. Taken together, our findings suggest that alpha14-giardin adheres to microtubules of the flagella via self-assembly that may regulated by Ser/Thr-phosphorylation.

The second cysteine protease inhibitor, EhICP2, has a different localization in trophozoites of Entamoeba histolytica than EhICP1.

The genome of Entamoeba histolytica contains two genes encoding inhibitors of cysteine proteases of the chagasin family. In contrast to that of EhICP1, the derived primary structure of the second inhibitor, EhICP2, possesses a typical N-terminal signal sequence. Processed EhICP2 is as weakly related to amoebiasin-1 (27% identity) as to chagasin (identity 30%), indicating a different evolutionary origin of both amebic genes. By Northern blots, we confirmed the expression of the ehicp2 gene, and in Western blots, the presence of the 11.5-kDa protein in trophozoite extracts was demonstrated. The inhibitor localized to large intracellular structures clearly differs from those containing EhICP1 as shown by indirect immunofluorescence. Recombinant EhICP2 significantly inhibited the cysteine protease activity of the amebic cell extract but with a lower extent than EhICP1. An overlay assay using a crude trophozoite extract demonstrated binding affinity of the amebic cysteine protease EhCP1 to EhICP2.

On the specificity of a cysteine proteinase from Entamoeba histolytica.

A cysteine proteinase has been isolated from the virulent strain HM1:IMSS of Entamoeba histolytica by two gel chromatography steps, ion exchange chromatography on DEAE-cellulose and affinity chromatography on organomercurial-Sepharose. The purified proteinase was homogeneous, as judged by polyacrylamide gel electrophoresis in the presence and in the absence of sodium dodecyl sulphate, and was a monomeric protein with a relative molecular mass of Mr 27000 +/- 2000 possessing an N-terminal alanine. The enzyme was inhibited by natural and synthetic inhibitors against cysteine proteinases. Split experiments employing blocked and unblocked peptide analogues with -2-naphthylamide moieties indicate that the cleavability was enhanced by the presence of basic residues, such as arginine or lysine, near the acyl end of the substrate. With unblocked tetrapeptides as substrates, exopeptidase activity of the amebic enzyme required an arginine at the P2-position, lysine could not substitute for arginine. The protease was able to digest native collagen type I, with an initial attack at the alpha 2-chain of the molecule.

Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix.

The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.