The pyruvate:ferredoxin oxidoreductase enzyme is located in the plasma membrane and in a cytoplasmic structure inEntamoeba

This work investigated the cellular location of the pyruvate:ferredoxin oxidoreductase (PFO) enzyme inEntamoeba. A 1.9 kb fragment located at the 3 end of theEhpfogene was cloned in the pRSETB vector and expressed. The recombinant peptide was purified and inoculated in rabbits. By Western blot assays the antibodies detected a single 130 kDa band in allE. histolyticastrains tested and inE. moshkovskii. By immunofluorescence, the antibodies showed the presence of PFO in the plasma membrane and in a cytoplasmic structure that appeared as a ring or as a compact small body inE. histolyticastrains. InE. invadensandE. moshkovskii(strains FIC and Laredo) PFO was located in the plasma membrane showing different fluorescence patterns. Immunofluorescence onE. histolyticasynchronized cultures showed that the cytoplasmic structure appeared in 85, 60, 20 and 10% of the trophozoites in mitosis, G1, S and G2 phases, respectively. Byin situhibridization theEhpfogene was found in the nuclei and the trophozoites of the clone A, strain HM1:IMSS, differed in theEhpfogene content.     

Human and mouse cellularmyc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer 1

A molecular clone of viralmyc (v-myc, the oncogene of avian myelocytomatosis virus, MC29, detected homologous human, mouse, and Chinese hamster cellularmyc (c-myc sequences by Southern filter hybridization. A v-myc probe, containing sequences from the 3′ domain of the gene, hybridized to single human HindIII and mouse EcoRI genomic DNA fragments of the cellular myc genes whose segregation could be followed in interspecies somatic cell hybrids. Human c-myc segregated concordantly with the enzyme marker glutathione reductase and with a karyotypically normal chromosome 8. A rearrangement of human c-myc was observed in Burkitt's lymphoma cells possessing the t(8;14) translocation. These results suggest that human c-myc is located close to the breakpoint on chromosome 8 (q24) involved in the t(8;14) translocation. The mouse c-myc gene segregated concordantly with chromosome 15 in mouse-Chinese hamster cell hybrids. These gene assignments are noteworthy, as structural and numerical abnormalities of human chromosome 8 and mouse chromosome 15 are associated frequently with B-cell neoplasms.     

Trophozoite and nuclear size,DNA base composition,and nucleotide sequence homology of several Entamoeba strains in axenic culture

We carried out a comparative study of nuclear and trophozoite diameters and of DNA thermal denaturation in eight Entamoeba strains cultured axenically (four of them E. histolytica, two initially designated as E. invadens, one E. moshkovskii, and one E. histolytica-like), as well as an analysis of the overall DNA sequence homology of the non-E. histolytica strains. The average nuclear (N) and trophozoite (T) diameters (in m) were, respectively: global averages ±SD for E. histolytica strains, 6.5±2.5 and 28.8±3.7; E. invadens IP101, 5.8 and 27.5, and PZ, 7.8 and 33.6; E. moshkovskii FIC, 4.1 and 12.9; E. histolytica- like Laredo strain, 5.0 and 20.6. The GC content of DNA, estimated by thermal elution in hydroxyapatite, was around 23% in HK9 and its clone HK9-1 and around 27% in the HM2 and HM3 E. histolytica strains; it was 37% in the Laredo strain, 26% in IP101, 35% in PZ, and 33% in FIC. The reassociation kinetics of PZ strain DNA showed that it consists of 40% repeated sequences and 60% unique sequences. By means of DNA association experiments in which one of each pair of DNAs tested had been labeled in vitro with ¹²⁵I, we found the following overall sequence homology among the strains tested: PZ-FIC, 38%; IP101-Laredo, 38%; IP101-FIC, 47%; PZ-Laredo, 49%; Laredo-FIC, 69%; and IP101-PZ, 83%. We conclude that trophozoites of different E. histolytica strains have similar nuclear size and GC content, whereas these parameters and the nucleotide sequences are clearly different in every other Entamoeba species. Our data also suggest that PZ and IP101 strains do not belong to the same species.     

Characteristic protein electrophoretic patterns of fourEntamoeba strains

Summary Total proteins from trophozoites of four axenized strains of the histolytica group ofEntamoeba (twoE. histolytica, oneE. invadens, and oneE. moshkovskii) were analyzed by disc-electrophoresis in polyacrylamide sodium dodecyl sulphate slab gels and by radial immunodiffusion. Each strain had a characteristic and reproducible electrophoretic pattern with about 30 major protein bands distributed between molecular weights 12,000 and 110,000. A striking similarity was found between the protein patterns of bothE. histolytica strains. Immunodiffusion also resulted in characteristic patterns, in which the greatest resemblance occurred in the same strains. The sensitivity and high resolution of the electrophoretic method for protein analysis and its correlation with antigenic characterization suggests its use as a taxonomic criterion of the histolytica group of the genusEntamoeba.     

Lectin-induced agglutination of trophozoites of different species and strains ofEntamoeba

In vitro agglutinability of trophozoites of threeEntamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains ofE. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains ofE. histolytica-like Laredo-type amebae, a strain ofE. terrapinae, and a strain ofE. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.     

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced byEntamoeba histolytica

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 ofEntamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (<1000 leukocytes/mm³), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains ofE. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulentE. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, andE. invadens) was generally proportional to their proteinase activity; however,E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential ofEntamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.     

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.     

Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E. coli sigma factor using phage display libraries

Background: During the epitope mapping of monoclonal antibodies specific for myc proteins, two E. coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified. One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass. The molecular masses of these cross-reactive proteins suggested that they may be subunits of E. coli RNA polymerase. Objectives: We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E. coli RNA polymerase. In addition, we have attempted to determine the epitope of MYC-X-5/1. Study design: The reactivity of MYC-X-5/1 antibody was tested against highly purified E. coli RNA polymerase halo-enzyme preparations and the cell lysate made from E. coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E. coli sigma subunit. The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries. Results: On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E. coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E. coli carrying the plasmid with the rpoD insert. In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235–245 of the human c-myc protein. A highly similar sequence to this was also identified in residues 62–72 of the sigma subunit of E. coli RNA polymerase. Conclusion: These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E. coli RNA polymerase. Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.     

DNA polymerase activity in encysting Entamoeba invadens

Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, we examined the level of DNA polymerase activity in E. invadens during encystation induced in vitro. We first characterized the DNA polymerase activity of trophozoites of E. invadens, comparing it with that of E. histolytica, and found that the activity of E. invadens was lower than that of E. histolytica at pH 2, 4, and 6 and was higher at pH 8 and 10. The activity of E. invadens was completely inhibited by high concentrations of K⁺. Among inhibitors of mammalian DNA polymerases, aphidicolin and N-ethylmaleimide inhibited the activity, but 2′,3′-dideoxythymidine-5′-triphosphate did not. Thus, the sensitivity of the E. invadens activity to salt and inhibitors of mammalian DNA polymerases was basically the same as that recorded for E. histolytica in our previous results. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs during cyst maturation in the absence of DNA synthesis. Received: 28 November 1998 / Accepted: 16 December 1998     

Increase of DNA content in tissue stages ofEntamoeba histolytica strain SFL 3

The DNA content of culture forms and tissue stages of pathogenicE. histolytica strain SFL 3 were measured photometrically after the nuclei had been stained with the fluorochrome BAO. As a control, the DNA guartity ofE. histolytica strain HK 9 andE. invadens were determined by the same method and compared with reference values. Tissue stages were obtained from hamsters experimentally infected by intrahepatic injection of SFL 3 amoebae. Further studies concerning possible changes in the DNA content of tissue stages involved the following methods: (a) isolation of tissue stages from the liver, followed by distinct suspension periods. (b) Infected liver pieces were directly transferred into culture medium; amoebae emigrating therefrom were cultivated. The study demonstrated that tissue stages contained up to 4 times more DNA than did culture forms. After 3 h cultivation, the DNA content of tissue stages decreased to the level of culture forms. Possible reasons for this change are discussed.Supported by Deutsche Forschungsgemeinschaft (DFG)     

Cloning and molecular analysis of genes encoding two immunodominant antigensofEhrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens ofE. risticiiin strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed inE. coli.Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a 10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues ofE. coliGroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains ofE. risticii.The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a 2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed inE. coliusing a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S.A.). The purified recombinant proteins cross-reacted with antisera toE. canisandE. sennetsu.     

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction

Previous studies of c-myc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and elecrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher that than of small cell carcinoma (Wilcoxon rank sum test, Z=2.06, P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in non-small cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma). Received: 17 October 2000 / Accepted: 7 May 2001     

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.     

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene''s coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.     

Expression of oncoproteins in primary human non-small cell lung cancer and incidence of metastases

In the current study the relationship between the incidence of metastatic spread and expression (at the protein level) of various proto-oncogenes was investigated in 217 human non-small cell lung carcinomas. Tumors with an overexpression of proteins encoded by the oncogenes c-jun and c-myc showed a significantly increased formation of metastases (c-jun: P = 0.008; c-myc: P = 0.018). No significant correlations were found between the expression of the c-fos, c-erbB1, c-neu and c-ras products and metastatic spread.     

Analysis of c-fos,c-jun,c-erbB1, c-erbB2 and c-myc in primary lung carcinomas and their lymph node metastases

The purpose of this study was to identify possible alterations in proto-oncogenes (c-fos, c-jun, c-erbB1, c-erbB2 and c-myc) at the protein level in primary lung carcinomas and simultaneous metastatic lymph nodes of 21 patients. The analysis showed that proteins of c-jun and c-myc were expressed in a significantly higher frequency in metastases than in primary lung tumors. Gross differences were not found between primary tumors and metastatic tumors with regard to the expression of c-erbB1, c-erbB2 and c-fos. The finding of cases with a higher expression of c-jun and c-myc in lymph nodes suggests that metastatic capability may be higher in certain cell populations.     

Comparison of age-associated changes of c-myc gene methylation in liver between man and mouse

Age-associated changes in DNA methylation of the c-myc gene in lever were compared between humans and mice which have large differences in aging rate. Although the overall methylation profiles of the gene and their age-related changes were found to be similar, the rate of change was much slower in humans than in mice. It suggests that the age-associated alteration of c-myc gene methylation is closely related to biological aging rather than to chronological aging.     

Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica

We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.