Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entmoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoeLtric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP+-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADHi clone had an open reading frame that was 360 amino acids long and encoded a protein of -39 kDa (calculated size). EhADHi showed a 62% amino acid identity with the tetrameric NADP+-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADHi showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADHi contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADHi lacked a 23-amino acid stretch present in other alcohol dehydrogenases that indudes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-Stransferase fusion protein showed the expected NADP+-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADHi fusion protein were inhibited by pyrazole and 4-methylpyrazole.Entamoeba histolytica is a protozoan parasite that infects -500 million individuals in the developing world, resulting in -40 million cases of dysentery with a consequent annual mortality rate of ==40,000 (1, 2). Entamoebae reproduce under anaerobic conditions in the colonic lumen and cause dysentery when they invade the bowel wall. Because poor countries cannot afford to make the changes in sanitary conditions that might eliminate fecal-oral spread of amoebic infection, amebiasis is primarily controlled by drug treatment of persons sick with the parasite (1-3).Entamoebae are obligate fermenters because they lack mitochondria and the enzymes involved in respiration (4, 5). Amoebae use facilitated diffusion for the uptake of glucose, which is then metabolized to pyruvate via the Embden-

The mechanism by which trophozoites of the protozoan parasite, Entamoeba histolytica destroy mammalian cells is not clear. A variety of amoeba-associated properties have been correlated with virulence (reviewed in 1 and 2). In vitro studies (3-6) have shown that contact of the trophozoite with a target cell is required. Contact killing has been described (6) as occurring in the sequence: adherence, cytolysis, then phagocytosis (possibly) of the target or its remnants. Dependence of in vivo virulence upon the phagocytic competence of the amoeba has also been reported (7). All steps of the sequence are inhibited by cytochalasins (6,8,9), indicating that dynamic participation of the amoeba actin cytoskeleton is required. However, the role of actin in the attack process has not been defined.By fluorescence microscopy of glutaraldehyde-fixed and rhodamine-phalloidin-stained (10) trophozoites, we have revealed the organization and distribution of polymerized actin in Entamoeba involved in a variety of motility-related activities (11, 12), including target cell interactions. Furthermore, by methanol extraction and spectrofluorometric measurement of the bound fluorescence, we have been able to quantitate polymerized actin in trophozoites. In this report, we describe this latter procedure and studies of the organization and quantity of polymerized actin in E. histolytica trophozoites before and immediately after challenge with human red blood cells (RBC) 1. Within 5 s after challenge, polymerized actin appeared at the contact interface with many adherent RBC. A net increase in amoeba polymerized actin content was detectable 1 rnin after the challenge, and reached a maximum of approximately twice the value in unchallenged cells within 4 min. Latex beads, which were phagocytized by E. histolytica trophozoites, neither stimulated an actin response nor were able to diminish the interaction with RBC. RBC, on the other hand, inhibited uptake of latex beads.These results indicate that the initial interaction of E. histolytica trophozoites with target cells is a recognition-specific process that triggers rapid polymerization of amoeba actin at the site of target contact. This actin appears to be involved in phagocytosis of the target cells. The procedure developed to quanThis work was supported in part by grants AI 19023 and RR 08006 from the National Institutes of Health, Bethesda, MD.i Abbreviations used in this paper: BSA, bovine serum albumin; PBS, phosphate-buffered saline; RBC, red blood cell. 546J. ExP. M~D.

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IPurified a and 63 globin complementary DNAs (cDNAs) have been separated from total radioactively labeled human globin cDNA using mRNA purified' from liver of a hydrops fetalis (a thalassemia). The P cDNA hybridizes to the hydrops fetalis mRNA while the a eDNA remains single-stranded. The purified a and iB cDNAs were assayed for their purity by their hybridization to mRNA prepared from reticulocytes of nonthalas.semia, a thalassemia, and I thalassemia subjects. The results indicate that the separated cDNAs are selective in hybridization to a or j3 globin mRNAs, respectively. The previously reported deficiency of globin mRNA in thalassemia cells has been confirmed with these purified cDNAs.The purified a and # cDNAs were' hybridized to cellular DNA to determine the relative number of a-and i3-like genes in non-thalassemia, B+ thalassemia, and hydrops fetalis (a thalassemia) DNA. The a cDNA hybridized to hydrops fetalis liver DNA to a much lower extent than B cDNA, confirming the previously reported deletion of a globin genes in hydrops fetalis. By contrast, both the a and j5 eDNA probes hybridized to the same extent to spleen DNA from non-thalassemia and from ,B + thalassemia patients. Between two and five globin genes in non-thalassemia and .B + thalassemia DNA hybridize to is cDNA and one to five to a cDNA. These studies indicate that in s+ thalassemia, there is no detectable deletion in g3 globin genes. The genetic defect in , + thalassemia appears to be due to either repression of transcription of j3 globin genes or abnormal processing of B globin mRNA.Using radioactively labeled complementary DNA (cDNA) as a probe, we have recently reported that there are less than 10 copies of globin genes present per haploid genome in human DNA (1). In these studies, #+ thalassemia spleen DNA was found to have a complement of globin genes similar to that of non-thalassemia DNA when cDNA containing both a and , sequences were used. We report here the measurement of the relative numbers of a-and ,8-like globin genes in human DNA using purified human a and gB cDNA. Hydrops fetalis (a thalassemia) mRNA which contains no a mRNA has been used to separate a and j3 cDNA from total normal human cDNA (2). Hybridization of total globin cDNA (a plus P cDNA) with hydrops fetalis mRNA leads to # cDNA ,3 mRNA hybrids, whereas a cDNA remains single stranded. The single stranded and double stranded species were separated using hydroxylapatite chromatography (1), and g3 mRNA removed from j3 mRNA -, cDNA hybrids by alkaline hydrolysis. The specificity of each of the purified a and 8 cDNAs has been determined by hybridization to globin mRNA from normal, #+ thalassemia, and a thalassemia cells.Previous studies using cell-free systems (3-7) and hybridization to cDNA (8, 9) have shown that mRNA from a thalassemia cells contains decreased a mRNA, and mRNA from 16 thalassemia cells decreased amounts of # mRNA. In the present experiments, hybridization of the purified a and P cDNA probes to 5 thalassemia mRNA shows a 10-fold excess of a co...