A comparison of the isoenzymes of Trypanosoma (Duttonella) vivax isolates from East and West Africa

The genetic diversity in 13 stocks and clones of Trypanosoma vivax from East and West Africa was compared by isoenzyme analysis. The Ugandan and West African stocks and clones showed a very high degree of genetic similarity to each other but they differed from the Kenyan stocks and clones. Two haemorrhagic stocks, IL 2337 (Galana, Kenya) and IL 3067 (Bamburi, Kenya), showed a high degree of similarity in enzyme banding patterns in electrophoresed preparations. One of the Kenyan stocks, M1D 627, differed in most of its enzyme banding patterns from all the other stocks and clones used.     

Changes in soluble amino-N,soluble proteins and free amino acids in leaves and roots of salt-stressed maize genotypes

Abstract

The effects of salt stress on the contents of organic solutes and on the pattern of free amino acids were studied in leaves and roots of two maize genotypes, BR5033 (salt-tolerant) and BR5011 (salt-sensitive). In leaves and roots of salt-stressed plants, soluble amino-N increased with time when compared to the controls. Salt stress increased the soluble protein content only in leaves of BR5011. Salinity increased the content of the majority of the free amino acids in leaves and roots of genotypes studied. Results suggest the hypothesis of disturbances in translocation of N-containing compounds from shoot to root in the salt-sensitive genotype. Results also suggest that the accumulation of organic solutes, mainly in roots of BR5033, may have an important role in the tolerance of this genotype to salt stress.     

Comparative virulence of three Trypanosoma evansi isolates from water buffaloes in the Philippines

The virulence of three Trypanosoma evansi isolates in Luzon, Visayas and Mindanao water buffaloes was compared determining the mortality rate, parasitemia level, clinical signs, and lesions on mice. A total of 51 inbred Balb/c mice (5-6 weeks old) were used and divided into two sets. Set A had three groups corresponding to three trypanosomes isolates (Luzon, Visayas, and Mindanao) with seven mice each whose parasitemia level, clinical signs, and lesions were noted at necropsy. Set B had three groups corresponding to the three isolates with ten mice each whose mortality was monitored. Each infected mouse was inoculated with 0.2 ml of T. evansi intraperitoneally and blood was examined under high power magnification. Their parasitemia level was determined using "Rapid Matching Method". Dead mice were subjected to necropsy and the lungs, liver, spleen, brain and heart were subjected to histopathological processing. Results showed that the mortality rate was highest at Day 3 for the Visayas isolates (70%), while at Day 5 for Luzon (90%) and Mindanao (70%) isolates. The parasitemia level of Visayas isolates (1×10(8.7)) reached the earliest peak at Day 4 while Luzon isolates (1×10(9)) at Day 6 and Mindanao isolates (1×10(8.7)) at Day 8. Statistical analysis using Least significant difference (LSD) revealed significant difference among treatment means at Days 2 and 4. All of the affected mice showed rough hair coat, decreased body weight, and decreased packed cell volume. The most obvious gross lesions observed were pale liver with petechiations and pale muscles. Histopathological examination revealed depletion of the red pulp and extramedullary hematopoiesis in the spleen. Congestion, intralesional trypanosomes in blood vessel and extramedullary hematopoiesis were observed in the liver. In the lungs non-specific lesions observed were pulmonary edema, congestion and hemosiderosis.     

Homogeneity of Trypanosoma evansi isolates from domestic and sylvatic mammals from the Pantanal of Mato Grosso

'Mal de Cadeiras' is a disease which causes great mortality in horses in the Pantanal Matogrossense region, Brazil. The agent of this disease is Trypanosoma evansi, a kinetoplastid flagellate which belongs to the Trypanosomatidae family, classified into the Salivarian section. Transmission occurs mechanically by haematophagous Diptera, mainly by Stomoxys sp. and Tabanus sp. and vampire bats. Outbreaks of Mal de Cadeiras in horses result in economic losses, thus limiting their use in cattle raising. Ten isolates of T. evansi recently derived from coati (Nasua nasua, Carnivora, Procyonidae), horses and dogs were compared, using schizodeme analyses from DNA digested by the restriction enzyme Hin fl. The results showed similar electrophoretic profiles for all isolates from wherever the host came. Homogeneity of isolates from domestic and sylvatic animals suggested two hypotheses: (1) the parasites circulated in only one transmission cycle;, and (2) independent cycles were not established in sufficient time to modify the molecular profiles of the isolates.     

The electrophoretic movement of soluble proteins and the production of unusual amino acids in Rhizobium isolates as taxonomic criteria

Summary This paper shows that a characteristic and distinctive set of proteins can be recognised in the Rhizobium cultures isolated from the various host species examined, and allow the isolates to be placed into groups. The acid-producing isolates of Lotus form a homogeneous group, but the non-acid producers examined form a separate but rather heterogeneous group. The clover acid producers form another homogeneous group. There is a good correlation between groupings based on amino acid patterns and those based on protein patterns. These approaches, however, give little information on the relatedness of one group to another, a conclusion also valid for other bacteria, algae and fungi. The technique therefore cannot be used as major criteria in classification, but can be a useful aid in rhizobial taxonomy.     

Alteration of free serum amino acids in voles infected with Trypanosoma brucei gambiense.

Free serum amino acid pools of field voles, Microtus montanus, were determined over a 24 hr period, and compared to values obtained from voles infected with Trypanosoma brucei gambiense. The majority of amino acids in the control animals demonstrated a diurnal variation, peaking predominantly during the dark portion of the photoperiod. This trend was not evident in the infected animals. In addition, infected voles possessed an apparent state of hypoaminoacidemia, with levels of threonine, serine, valine, isoleucine, leucine, tryosine, and tryptophan typically below uninfected values. Alanine and proline, in contrast, were markedly increased at certain time points. Tyrosine (reduced by approximately 50%) and tryptophan (reduced to levels below detection) underwent the most pronounced drop in trypanosome-infected animals, indicating the possibility of a related alteration in pools of derivative biogenic amines in other tissues. This suggests a role for the latter 2 amino acids in the neuropsychiatric syndromes of African trypanosomiasis.     

Studies of the proteins, peptides and free amino acids of mature bovine enamel

1. The organic matrix of enamel from erupted bovine teeth has been found to be composed mostly of small peptides containing principally aspartic acid, glycine, glutamic acid and serine. 2. A small amount of higher-molecular-weight components has been isolated by various procedures. One non-diffusible fraction was found to be heterogeneous in the ultracentrifuge, and composed principally of material that by gel filtration indicated a molecular weight greater than 30000. These components were largely carbohydrate in nature (glycoproteins and glycopeptides), containing only small amounts of amino acids.     

Effect of salinity on soluble protein,free amino acids and nicotine contents inNicotiana rustica L.

Nicotiana rustica plants were grown under two grades of salinity conditions, 50 mM and 100 mM NaCl. High levels of NaCl added to the nutritive solution produced a progressive absorption of Na in detriment of K, thereby causing an ionic disequilibrium, particularly in leaves. The weight and longitudinal growth of plants grown under salinity conditions were lower than control plants, while their protein content was higher. Deficit of K induced by salinity increased the levels of free amino acids, especially of aspartic acid, glutamic acid and proline. On the other hand, the levels of nicotine in leaves of treated plants were lower than controls. In contrast, treatment of the plants with 100 mM NaCl induced in general an increase of nicotine in the roots. These results indicate that there was an unclear effect of salinity either on synthesis or on translocation of nicotine from roots to leves.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

The phosphoglycerate kinases from Trypanosoma brucei. A comparison of the glycosomal and the cytosolic isoenzymes and their sensitivity towards suramin

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2',3'-ATP-Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga. Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308----His and Ala 306----Glu in the adenine-binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogues, but probably prevent phosphoryl-group transfer. Both isoenzymes displayed an activity optimum at pH 6.0-9.0 similar to that for the enzyme of yeast. Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Ki app. = 8.0 microM) to Suramin than either the cPGK (Ki app. = 20 microM) or the enzymes from rabbit muscle (Ki app. = 55 microM), yeast (Ki app. = 167 microM) or S. platensis (Ki app. = 250 microM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.     

Effect of neocarzinostatin on the nucleus, kinetoplast and microtubules of Trypanosoma gambiense and Trypanosoma evansi.

The antibiotic neocarzinostatin (NCS) induces the anucleate form, not the dyskinetoplastic form, of Trypanosoma gambiense and Trypanosoma evansi. Light and electron microscopic studies indicated that production of the anucleate form is due to delay or inhibition of nuclear division. Excess pellicular microtubules are formed after treatment of trypanosomes with NCS, suggesting that in trypanosomes the microtubules replicate by induction, not by division. NCS also causes deformation of the axonemal and spindle microtubules. The K clone of T. evansi is more sensitive than the AK clone to the effects of NCS in inhibiting nuclear division and inducing the anucleate form.     

Terahertz time-domain spectroscopy of amino acids and polypeptides

Frequency-dependent absorption coefficients and refractive indices of amino acids (glycine and l-alanine) and polypeptides (polyglycine and poly-l-alanine) in the wavenumber region from 7 to 55 cm⁻¹ were measured by terahertz time-domain spectroscopy. A vibrational band was observed at 45.5 cm⁻¹ for polyglycine, which was assigned as an interchain mode. The reduced absorption cross sections of the amino acids and polypeptides show power-law behavior. The exponents are different between the monomers and polymers, and those of the two polypeptides suggest that the time dependences of the total dipole moments are similar in the timescale of subpico- to picoseconds.     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi.

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.