Biomedical subjects

Norton Heise

Publications and source records attributed to Norton Heise.

9 recordsLinked to original sources

PubMed2005

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant.

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.

Alternative Splicing↗

PubMed2005

Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi.

IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.

Animals↗

PubMed2005

Protozoan parasite-specific carbohydrate structures.

The carbohydrate moieties displayed by pathogenic protozoan parasites exhibit many unusual structural features and their expression is often developmentally regulated. These unique structures suggest a specific relationship between such carbohydrates and parasite pathogenicity. Studies of infected humans indicate that immune responses to protozoan parasites are elicited by glycan determinants on cell-surface or secreted molecules. Infections by protozoa are a major worldwide health problem, and no vaccines or efficacious treatments exist to date. Recent progress has been made in elucidating the structure and function of carbohydrates displayed by major protozoan parasites that infect man. These structures can be used as prototypes for the chemical or combined chemo-enzymatic synthesis of new compounds for diagnosis and vaccine development, or as inhibitors specifically designed to target parasite glycan biosynthesis.

Animals↗

PubMed2004

B cell response during infection with the MAT a and MAT alpha mating types of Cryptococcus neoformans.

In the present study, we compared the B cell response of BALB/c and C57Bl/6 mice during Cryptococcus neoformans infection. This response was investigated using virulent serotype D forms of mating types alpha and a (MAT alpha and MAT a). C57Bl/6 mice showed massive (mainly cerebral) infection by both types, while BALB/c were resistant to infection. Some resistance of C57Bl/6 mice was induced by previous immunization with the capsular polysaccharide from MAT alpha. Passive immunization of C57Bl/6 mice with purified antibody (Ab) obtained from capsular polysaccharide-immunized mice also increased resistance to infection. Both mouse strains showed comparable low IgM response to the capsular polysaccharide from MAT alpha, and only C57Bl/6 mice produced IgM to the polysaccharide of MAT a. Comparable levels of different immunoglobulin (Ig) isotypes against capsular components of MAT alpha and MAT a were detected, and the response of C57Bl/6 mice was higher when compared to that of BALB/c mice. FACS analysis indicated an increase in the percentage of a high-granulosity (side-scatter) splenic subpopulation and in the percentage of splenic Gr-1+ cells in infected C57Bl/6 mice. In addition, the percentage of follicular splenic B cells was decreased after C. neoformans infection of C57Bl/6 mice. This response was more pronounced when we investigated infection induced by the MAT a mating type. Taken together, our results indicate that capsular polysaccharide derived from MAT alpha and MAT a types of C. neoformans have a stimulatory effect upon B cells but that there is no correlation between resistance of BALB/c mice and Ab production. However, the increase in resistance of C57Bl/6 mice parallels the production of Abs and a major change in splenic cell populations.

Animals↗

PubMed2004

Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel yersiniose A-containing O-polysaccharide.

Burkholderia brasiliensis, a Gram-negative diazotrophic endophytic bacterium, was first isolated from roots, stems, and leaves of rice plant in Brazil. The polysaccharide moiety was released by ammonolysis from the B. brasiliensis lipopolysaccharide (LPS), allowing the unambiguous characterization of a 3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose (yersiniose A), an uncommon feature for Burkholderia LPS. The complete structure of the yersiniose A-containing O-antigen was identified by sugar and methylation analyses and NMR spectroscopy. Our results show that the repeating oligosaccharide motif of LPS O-chain consists of a branched tetrasaccharide with the following structure:-->2-alpha-d-Rhap-(1-->3)-[alpha-YerAp-(1-->2)]-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->.

Burkholderia↗

PubMed2004

Toxic effects of natural piperine and its derivatives on epimastigotes and amastigotes of Trypanosoma cruzi.

We describe herein an evaluation of trypanocidal effects of the natural alkaloid piperine and twelve synthetic derivatives against epimastigote and amastigote forms of the protozoan parasite Trypanosoma cruzi, the causative agent of the incurable human disease, Chagas' disease. The results obtained point to piperine as a suitable template for the development of new drugs with trypanocidal activity.

Alkaloids↗

PubMed2004

Glycoinositolphospholipid from Trypanosoma cruzi: structure, biosynthesis and immunobiology.

The pathogenic protozoan parasite Trypanosoma cruzi expresses on its surface an unusual family of glycoinositolphospholipids (GIPLs) closely related to glycosylphosphatidylinositol (GPI) anchors. Different parasite isolates express distinct GIPLs which fall into two series, depending on the substitution of the third mannosyl residue in the conserved glycan sequence Man4-(AEP)-GlcN-InsPO4 by ethanolamine phosphate or beta-galactofuranose. Although the exact role of these molecules in the cell biology and pathogenicity of T. cruzi remains unknown, the lipid and glycan moieties impart distinct responses to host T and B lymphocytes and phagocytes, overall favouring an immune response permissive to the parasite. The biosynsthesis of GIPLs follows a pathway similar to that observed for GPI anchors. However, a more detailed understanding might enable the development of specific inhibitors of parasite-specific enzymes and lead to novel drugs to ameliorate Chagas disease.

Animals↗

PubMed2002

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms.

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.

Carbohydrate Conformation↗

PubMed2002

Proinflammatory and cytotoxic effects of hexadecylphosphocholine (miltefosine) against drug-resistant strains of Trypanosoma cruzi.

The increased resistance of the protozoan parasite Trypanosoma cruzi to nitro derivatives is one of the major problems for the successful treatment of Chagas' disease. In the present study, we have tested the effects of 1-O-hexadecylphosphocholine (miltefosine) against strains of T. cruzi that are partially resistant (strain Y) and highly resistant (strain Colombiana) to the drugs in clinical use. As expected, epimastigotes of strain Colombiana showed higher levels of resistance to benznidazole than those of strain Y. However, the level of resistance to miltefosine was the same for both strains. This alkylphospholipid was also extremely toxic against intracellular amastigotes of both strains. This ether-lipid analogue induced in a dose-dependent manner the production of tumor necrosis factor alpha and nitric oxide (NO) radicals by infected and noninfected macrophages, suggesting that miltefosine may activate macrophages in vitro. Nevertheless, the cytotoxic effect of miltefosine against intracellular amastigotes was independent of the amount of NO produced by the infected macrophages since the same dose-response curves for miltefosine were observed when the NO production was blocked by the NO synthase inhibitor N(G)-monomethyl-L-arginine monoacetate. Preliminary in vivo studies with BALB/c mice infected with strain Y indicated that oral miltefosine promoted survival and reduced the parasitemia to levels comparable to those observed when benznidazole was used. Four months after treatment, no parasites were detected in the blood or spleen tissue sections maintained in culture. Together, these results support the hypothesis that miltefosine may be used for the treatment of Chagas' disease, including cases caused by resistant strains of T. cruzi.

Animals↗