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.

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.

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.

Powerful probes for glycosidases: novel, fluorescently tagged glycosidase inhibitors.

Amino-1,2,5-trideoxy-2,5-imino-D-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by attachment of a dansyl amide bearing spacer to this centre. Compounds obtained are highly potent inhibitors of beta-glucosidase exhibiting Ki values in the single figure nanomolar range. The 1-N-dansyl substituted inhibitor was successfully exploited for binding studies with beta-glucosidase from Agrobacterium sp. employing fluorescence spectrometric methods.

Characterization of a proteasome alpha-chain from Giardia lamblia.

To begin to characterize the components of the 20S proteasome of Giardia lamblia, we have cloned a genomic sequence encoding an alpha-chain (type alpha3/C9, predicted size 244 amino acid residues). Southern analysis indicated that a single gene codes for this protein, and a Northern blot exhibited a single signal at 850 nt. An antiserum against a C-terminal fragment of the alpha-chain expressed in Escherichia coli reacted with a single protein band of Mr 27,000 that was present at constant levels in trophozoites and encysting cells. On a 2D blot of the purified 20S proteasome, we identified the cross-reacting component as a single protein of IEP 6.0, in agreement with the IEP predicted by the coding sequence. Our data confirm that the G. lamblia 20S proteasome is typically eukaryotic in containing a set of diverged alpha-subunits.

Energy metabolism of cocks and broiler chickens fed on diets with different carbohydrate sources.

Energy balances of cocks and chickens were measured using the nitrogen-carbon-balance method. In Experiment 1 twelve adult White Leghorn cocks were fed alternately on a basal ration or on a supplemental ration composed of 75% basal diet and 25% carbohydrate source as a supplement. In Experiment 2 six groups of 12 male broiler chickens were fed successively on two diets each with different carbohydrate sources (40% of DM) and on two energy levels. The investigated carbohydrate sources were glucose, fructose, sucrose, maize starch, raw and steamed potato starch, dried sugar beet pulp, tapioca, wheat, maize, rye and barley. In both experiments the energy digestibility of the diets with raw potato starch, beet pulp and barley was significantly lower compared to the other diets. Digestibility of those ranged from 88 to 81%. By simple linear regression no significant differences in efficiency of utilisation of ME of the diets between the carbohydrate sources sugars, starches and cereal grains could be proved. The corresponding MEm values agreed very close among the diets (411 to 429 kJ.kg BW-0.75.d-1).

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.

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes.

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.

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.

Involvement of alpha 2-adrenergic activities in thermogenic responses to feeding, feeding level, and ambient temperature.

To investigate the alpha 2-adrenergic effect on the metabolic rate, young bulls were exposed to environmental variants (feeding levels of 1.0 and 1.6 times the MEm and ambient temperatures of 18 degrees C and 4 degrees C) and treated preprandially with a alpha 2-adrenergic agonist (clonidine) in each case. The heat production (HP) was continuously measured by indirect calorimetry using climatized respiratory chambers. Post-clonidine, the preprandial HP fell in all variants but the strongest decrease occurred at 4 degrees C, 1.6 times the MEm. The postprandial HP rose 1.3-fold the HP of animals received the carrier (saline) at 4 degrees C, 1.6 times the MEm. Animals exposed to 18 degrees C, 1.6-fold the MEm did not significantly increase the postprandial HP after clonidine administration, suggesting different sympathetic outflow corresponded to differing resting metabolic rate, occurring in the environmental variants. Circulating fuels (glucose, non-esterified fatty acids) responded to alpha 2-adrenergic reduction of the sympathetic outflow but did not parallel the HP changes. Studies on monocytes revealed a linear correlation (r2 > 0.9) between resting metabolic rate and expression of sulfonylurea receptors, the constitutive component of ATP-sensitive K+ channels (KATP) suggesting a function of KATP in coupling the systemic HP with cellular metabolism.

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.

Evidence for the existence of both proteasomes and a novel high molecular weight peptidase in Entamoeba histolytica.

To screen for high molecular weight proteases in Entamoeba histolytica, we subjected a soluble amebal extract to density gradient centrifugation and tested the fractions for activity against the chymotryptic peptide substrate, Suc-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide. Two peaks of activity, of approximately 11 and 20 S, were clearly separated. Based on SDS-electrophoretic pattern and immunoblot analysis, we ascribe the 20 S activity to proteasomes. The 11 S protein was purified from amebal homogenates by a series of chromatographic steps. As determined by molecular sieve chromatography and nondenaturing gel electrophoresis, the native complex had an apparent Mr of 385,000 +/- 10%. On SDS gels, the purified enzyme exhibited a single band of Mr 62,000 that yielded a single N-terminal sequence, indicating that the preparation was homogeneous and that the native complex consisted of six very similar or identical subunits. The enzyme preferred peptides with aromatic residues at the P1 position and had low but distinct activity toward azocasein. We conclude that the 11 S enzyme is a novel high molecular weight protease that is distinct from proteasomes.

[Results from measurements of nitrogen and energy metabolism in adaptive different cattle]. / Ergebnisse aus Stickstoff- und Energieumsatzmessungen an adaptiv differenten Rindern.

In experiments with Galloway (G), Highland (H) and Black-White Dairy (B) cattle no significant differences between the breeds were measured in the energy and nutrient digestibility and energy metabolizability of rations with high variation in the nutrient composition. In H and B cattle no differences existed in digestibility in relation to the environmental temperature (30, 18 and 3 degrees C). Lowering the environmental temperature from 18 to 12 and 4-6 degrees C resulted in no changes of heat production in G and H but in B heat production increased about 6% and 20% respectively.

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.

Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.

[Continuous measuring of heat production and activity during the course of the day in hungry growing rats]. / Kontinuierliche Messung von Wärmeproduktion und Aktivität im Tagesverlauf an wachsenden Ratten bei Hunger.

Heat production (indirect calorimetry) and motor activity were measured continuously with 4 repetitions at 8 male albino rats (24 h fasting) over 21 h (4 minute cycle of measured value registration). The average difference between maximum and minimum heat production in a 4-minute measuring period amounted to 82 +/- 14% (68 to 139%). There was a significant connection between heat production and activity. 7.9 +/- 1.2% of the heat production of the hungry rat could be ascribed to activity. In the course of the day of fasting (24 h) an average metabolism reduction of 15 kJ/kg LW0.75 could be observed.