Harvey Eisen

Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes.

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.

Sulfated lipids represent common antigens on the surface of Trypanosoma cruzi and mammalian tissues.

Cross-reacting lipid antigens were isolated from Trypanosoma cruzi and the mammalian brain with the monoclonal antibody VESP 6.2. Chemical reactions indicated that the sulfate group of the lipids is an important part of the epitope recognized by the monoclonal antibody. Lipid extracts of mouse brain contained all the antigenic species present in the parasite. One of the antigens was demonstrated by three different methods: (i) high performance thin layer chromatography immunostaining, (ii) solid phase radioimmunoassay, (iii) lysis of artificial liposomes. The T. cruzi sulfated lipid antigens were shown to be of parasite origin rather than scavenged from the culture medium. They could be radiolabelled with [35S]sulfate. Furthermore, lipid extracts from two T. cruzi strains grown in different media contained the same antigens while the media contained either no antigens or different species.

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins.

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Mimicry in Trypanosoma cruzi: fantasy and reality

Chronic infection of mammals by Trypanosoma cruzi often results in severe autoimmune and inflammatory pathology. Extensive antigen cross-reactivity between the parasite and its mammalian hosts has also been reported. These findings have stimulated speculation that Trypanosoma cruzi uses antigenic mimicry as a mechanism for escaping the host immune system. This may not be the case and the observed antigen cross-reactivity may be a result of perturbations of the immune system such that common, normally tolerated antigens are recognized in infected animals. The parasite, however, does appear to use functional mimicry to survive in the immune competent host.

SA85-1 proteins of Trypanosoma cruzi lack sialidase activity

Trypanosoma cruzi infects a wide range of mammalian species, and replicates within many different cell types [1]. Numerous reports indicate that trypomastigote surface proteins facilitate adhesion and invasion of mammalian cells [1]. Recently, several genes that are expressed specifically by mammalianstage parasites have been shown to encode surface proteins with homology to sialidases [2]. Two of these, genes have been shown to encode functional sialidases when expressed in Escherichia coli [3,4]. Other proteins, with homology to sialidase, apparently lack intrinsic sialidase activity, and their function remains unclear [4]. In addition, sialidase and trans-sialidase activities appear to be catalyzed by the same protein, and to represent coupled steps of the same reaction; the former representing sialic acid transfer to water, and the latter transfer to another carbohydrate [4]. We have described the SA85-1 family of surface proteins expressed by mammalianstage parasites that have homology to sialidases [5,6]. Hybridization to a cDNA named 1.1 (cl.1), or reactivity to antibodies purified with a recombinant protein generated by expression of cl. 1 (anti-cl. 1 antibodies) define

Evaluation of recombinant trypomastigote surface antigens of Trypanosoma cruzi in screening sera from a population in rural Northeastern Brazil endemic for Chagas' disease

A perfect serologic test for infection with Trypanosoma cruzi does not exist. This study uses recombinant T. cruzi surface proteins in the antibody capture enzyme linked immunoabsorption assay (ELISA); and compares this approach to the more standard immunofluorescence assay (IFA). Three recombinant antigens are studied: F1-160 corresponding to the 160 kDa flagellar associated surface protein of trypomastigotes (the motile form of T. cruzi in mammalian infections); and SA 85-1.1 and 1.2 corresponding to different members of the 85 kDa family of surface proteins expressed by trypomastigotes and amastigotes (the replicative, non-motile form of T. cruzi in mammalian infections). Each recombinant antigen is found to be highly specific (range 86-94%) but relatively insensitive (range 36-52%) when used to screen for antibodies to T. cruzi. Defining seropositivity as reactivity to any of the three recombinant antigens markedly increases the sensitivity (72%) with only a minor reduction in specificity (82%). Thus, employing recombinant T. cruzi antigens to screen for T. cruzi infection has promise, but improvements in sensitivity must be made before widespread utilization is recommended.