Primary structure of elongation factor 2 around the site of ADP-ribosylation is highly conserved from archaebacteria to eukaryotes.

Elongation factor 2 (EF-2) from eukaryotes and archaebacteria can be ADP-ribosylated by diphtheria toxin (DT) [(1977) Annu. Rev. Biochem. 46, 69-94; (1980) Nature 287, 250-251]. The primary structure of the ADP-ribose accepting region in EFs from the archaebacteria Thermoplasma acidophilum Halobacterium cutirubrum and Methanococcus vannielli was determined in order to elucidate the degree of conservation compared with 4 previously established eukaryotic sequences [(1971) FEBS Lett. 103, 253-255]. Within a 9-residue sequence including the site of ADP-ribosylation 5 positions were found to be occupied by the same amino acid in all the archaebacterial and eukaryotic factors studied. There were more differences among the 3 archaebacterial sequences than among the 4 eukaryotic ones.

Production of an antiserum specific to the ADP-ribosylated form of elongation factor 2 from archaebacteria and eukaryotes.

An antiserum to ADP-ribosylated elongation factor 2 (ADPR-EF-2) from S. acidocaldarius was raised in rabbits using stained, homogenized, ADPR-EF-2-containing slices from SDS-gels as a source of antigen. Elongation factor 2 (EF-2) from S. acidocaldarius was cloned in E. coli and the expressed gene product was used in order to adsorb all anti-EF-2 antibodies which do not contain the ADP-ribosyl group within their epitopes, as E. coli is unable to synthesize the ADP-ribosyl acceptor diphthamide. The remaining antibodies were specific to ADP-ribosylated EF-2 from Thermoplasma acidophilum, S. acidocaldarius and Desulfurococcus mucosus. ADP-ribosylated EF-2 from eukaryotic sources also reacted with the adsorbed antiserum as shown for EF-2 isolated from the killi-fish Cynolebias whitei, the mouse species BALB/c and Han/Wistar rats. The adsorbed antiserum did not cross-react with ADP-ribosylated actin or rho protein or with FAD-containing D-amino acid oxidase.

Ribosome specificity of archaebacterial elongation factor 2. Studies with hybrid polyphenylalanine synthesis systems.

Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus. By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom. In contrast, EF-1 from T. thermophilus significantly cooperated with ribosomes from T. acidophilum.

The role of macrophages in Acanthamoeba keratitis.

PURPOSE: Macrophages are thought to be the first line of defense in many infectious diseases and are present in high numbers in corneas with Acanthamoeba keratitis. Conjunctival macrophage depletion was performed in an animal model of Acanthamoeba infection to determine the importance of macrophages in this disease. METHODS: Selective elimination of macrophages was achieved by repeated subconjunctival injection of liposomes containing dichloromethylene diphosphonate in a Chinese hamster model of Acanthamoeba keratitis. RESULTS: Macrophage depletion affected the incidence, severity, and chronicity of keratitis. The incidence of infection in normal animals was approximately 60% but rose to 100% on day 4 in animals treated with liposomes containing dichloromethylene diphosphonate (C12MDP-LIP). Moreover, the clinical appearance of the keratitis in the C12MDP-LIP group was much more severe than in animals treated with liposomes containing phosphate-buffered saline at all time points. There was also a major change in the chronicity of keratitis, with an earlier onset and a prolonged and chronic course in the C12MDP-LIP treated hamsters. CONCLUSIONS: The profound exacerbation of Acanthamoeba keratitis in hamsters treated with C12MDP-LIP strongly suggests that macrophages play an important role in corneal infection with Acanthamoeba trophozoites, probably by acting as a first line of defense and eliminating significant numbers of Acanthamoeba trophozoites.

The role of contact lenses, trauma, and Langerhans cells in a Chinese hamster model of Acanthamoeba keratitis.

PURPOSE: To determine the role of contact lenses, corneal trauma, and Langerhans cells in the development of keratitis caused by Acanthamoeba organisms in Chinese hamsters. METHODS: Various methods were used to induce corneal infections in Chinese hamsters, including application of parasite-laden contact lenses. The role of corneal epithelial defects in promoting parasite binding was examined in vitro in a microscopic binding assay. The role of corneal abrasion in the development of Acanthamoeba keratitis was also examined in Chinese hamsters exposed to parasite-laden contact lenses. Other experiments evaluated the effect of infiltrating Langerhans cells on the incidence and severity of Acanthamoeba keratitis. RESULTS: Corneal epithelial defects promoted extensive parasite binding to abraded corneas compared to intact, nonabraded counterparts. Corneal abrasion was absolutely necessary for the induction of Acanthamoeba keratitis in hamsters infected with contaminated contact lenses. Infection was never detected unless the corneas were abraded before exposure to parasite-laden contact lenses. The presence of Langerhans cells in corneas prevented the development of Acanthamoeba keratitis. CONCLUSIONS: The highest incidence of Acanthamoeba keratitis occurs in corneas expressing epithelial defects and exposed to parasite-laden contact lenses. The presence of Langerhans cells in corneas exposed to parasite-laden contact lenses prevents the development of Acanthamoeba keratitis.

Role of mucosal IgA in the resistance to Acanthamoeba keratitis.

PURPOSE: To determine whether oral immunization with Acanthamoeba castellanii antigens elicits mucosal antibodies of the IgA isotype and whether mucosal antibodies affect parasite adhesion to the corneal epithelium. METHODS: Chinese hamsters were immunized with 100 microg aqueous Acanthamoeba antigen mixed with cholera toxin (Ac-CT) and subsequently challenged with parasite-laden contact lenses that were applied to abraded corneal surfaces. Tears and stool samples were examined for the presence of Acanthamoeba-specific IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The effect of mucosal antibody on trophozoite binding to corneal epithelium and viability of trophozoites was examined in vitro. RESULTS: Hamsters immunized orally with Ac-CT showed significantly lower infection rates than did control groups (21.4% versus 72.6%). ELISA analysis of mucosal specimens showed the presence of parasite-specific IgA in stool samples and tears from hamsters orally immunized with Ac-CT, but not in control animals. In vitro assays showed that anti-Acanthamoeba IgA did not affect parasite viability. However, mucosal anti-Acanthamoeba IgA profoundly inhibited (>75%) the binding of parasites to corneal epithelial cells in vitro. CONCLUSIONS: Oral immunization with Ac-CT induces the production of parasite-specific IgA in mucosal secretions and prevents corneal infection. Mucosal antibody does not affect the viability of Acanthamoeba trophozoites but seems to prevent infection by inhibiting parasite binding to the corneal epithelium.

Uterine contraction intervals and transcutaneous levels of fetal oxygen pressure.

Continuous intrauterine transcutaneous PO2 (tcPO2) measurement was performed in 30 deliveries. The various characteristics of labor (peak of contractions, duration, and contraction interval) were correlated with the tcPO2 integral. The best correlation was seen between the tcPO2 integral and that of the contraction intervals. As the data show significant correlations with the pattern of labor, continuous tcPO2 measurement is a reliable and sensitive method for recognizing PO2 alterations early, especially in high-risk deliveries.

Cloning and sequencing of the gene coding for the elongation factor 1 alpha from the archaebacterium Thermoplasma acidophilum.

The gene which encodes the elongation factor 1 alpha (EF-1 alpha) of the archaebacterium Thermoplasma acidophilum (tuf-gene) has been cloned and sequenced. The gene coding for elongation factor EF-2 was found downstream from the 3' end of the tuf-gene. Comparison of the predicted amino acid sequence of Thermoplasma EF-1 alpha with EF-1 alpha sequences of other organisms showed that the highest similarity values were found between T. acidophilum and Methanococcus vannielii.

Cloning and sequencing of the fus-gene encoding elongation factor 2 in the archaebacterium Thermoplasma acidophilum.

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.

A novel eubacterial phylum: comparative nucleotide sequence analysis of a tuf-gene of Flexistipes sinusarabici.

A tuf-gene of Flexistipes sinusarabici has been cloned and sequenced. The primary structure of the predicted elongation factor protein was compared with available sequences of homologous genes and elongation factors Tu or 1 alpha of eubacteria, archaebacteria and eukaryotes. Based on elongation factor Tu data Flexistipes sinusarabici belongs to the eubacterial kingdom but no specific relationship to any phylum was detected. The amino acid of the elongation factor Tu of F. sinusarabici exhibits no striking pecularities. Among the highly conserved positions only two are different. Sites of known or postulated functions are conserved.

Effect of smoking on cadmium and lead concentrations in human amniotic fluid.

The amniotic fluids of 155 pregnant women, non-smokers (n = 128) and smokers (n = 27), were investigated on their cadmium (Cd) and lead (Pb) concentrations. The mean +/- s range of Cd in the amniotic fluid of non-smokers amounted to 2.58 +/- 1.36 ng/l, that of smokers to 7.29 +/- 2.39 micrograms/l. Moreover, there was a correlation between the extent of daily cigarette consumption and Cd levels. With Pb, higher concentrations were found ranging between 23.98 +/- 9.41 ng/l for non-smokers and 21.53 +/- 7.16 micrograms/l for smokers. No correlations were seen between age, week of pregnancy, blood pressure, disorders of pregnancy and the amniotic Cd or Pb concentrations. Thus, the maternal and fetal risks of the higher Cd levels in the amniotic fluid of smoking women remain unanswered.

Characterization and pathogenic potential of a soil isolate and an ocular isolate of Acanthamoeba castellanii in relation to Acanthamoeba keratitis.

Acanthamoeba castellanii, one isolate from the eye and one from the soil, were compared on the basis of: (a) pathogenic potential; (b) plasminogen activator activity; (c) chemotactic activity; (d) cytopathic effects; (e) collagenolytic activity; (f) binding ability to contact lenses; and (g) and binding ability to corneal buttons. The ocular isolate of A. castellanii was found to be pathogenic based on its ability to produce corneal infections in Chinese hamsters. By contrast, the soil isolate produced only mild lesions in a single Chinese hamster. Amoebae from the ocular isolate bound to corneal epithelium in greater numbers than the soil isolate counterparts. Moreover, ocular isolate organisms displayed plasminogen activator activity that was not detected in cultures from soil isolates of A. castellanii. Although neither the soil isolate nor the ocular isolate amoebae responded chemotactically to epithelial or stromal components, the ocular isolate displayed a curious and reproducible positive chemotactic response to endothelial extracts. Both A. castellanii isolates produced cytopathic effects on pig corneal epithelium, however the cytotoxicity from the ocular isolate was significantly greater than that of the soil isolate. The results indicate that the pathogenic potential of A. castellanii is correlated with the parasite's capacity to bind to corneal epithelium, respond chemotactically to corneal endothelial extracts, elaborate plasminogen activators, and produce cytopathic effects on corneal epithelium.

Systemic immune response to Acanthamoeba keratitis in the Chinese hamster.

Recrudescence is a common and troubling feature of Acanthamoeba keratitis and suggests that corneal infection with this organism fails to stimulate the systemic immune apparatus. The present study examined the cell-mediated and humoral immune responses to Acanthamoeba keratitis in the Chinese hamster. Corneal infection with A. castellanii failed to induce either delayed-type hypersensitivity (DTH) or serum IgG antibody against parasite antigens. The failure to induce cell-mediated and humoral immunity did not result in anergy or tolerance since subsequent intramuscular (i.m.) immunization with parasite antigens elicited robust DTH and IgG antibody responses. The inability of corneal infections to induce primary cell-mediated immune responses was due to the absence of resident antigen-presenting cells in the central cornea because induction of Langerhans cell (LC) migration into the central cornea prior to infection with Acanthamoeba promoted the development of parasite-specific DTH. Although the presence of resident LC did not promote the development of a primary humoral immune response, subsequent i.m. immunization elicited heightened parasite-specific IgG antibody production which was indicative of an anamnestic response. Collectively, the results indicate that in the absence of resident antigen-presenting cells, corneal infection with Acanthamoeba fails to stimulate primary cell-mediated or humoral immunity. Induction of peripheral LC into the central corneal epithelium promotes the development of parasite-specific DTH, but does not exacerbate corneal disease.

The transcutaneous PO2 measurement as an additional evaluation index to the physiological and pathological conditions under delivery.

Transcutaneous PO2 was continuously recorded from 17 newborns sub partu by normal deliveries and four cases with umbilical cord complications. These data were correlated with the corresponding fetal heart rate alterations and the maximum of contraction directly and 30 s thereafter. During the course of delivery three stages are differentiated: first stage of labor, full cervix dilatation, second stage of labor. The statistical evaluation succeeded in the X2-test (P less than 0.01). Deriving from this result the tcPO2 measurement is an additional aiding procedure beside the cardiotocographic control for subpartal supervision.

An exploratory study on the characterization of A1 and A2 bloodstains using a fluorescence immunoassay.

A fluorescence immunoassay method is used in the differentiation of A1 and A2 bloodstains. Anti-A antiserum is first absorbed onto the stain. The IgM content in the eluate is then quantified by commerical Immuno-Fluor kits. The binding difference of A1 and A2 cells is retained in stains. This difference and the quantitative evaluation method provide a potential basis for blood typing or subtyping application.

Archaebacterial elongation factor is ADP-ribosylated by diphtheria toxin.

Archaebacteria have been defined as a 'third primary kingdom' of cells in addition to the urkaryotes and the eubacteria. While the latter two correspond approximately to the conventional categories eukaryotes and prokaryotes respectively, the Archaebacteria have up to now comprised four groups of microorganisms: the methanogenic bacteria, the extremely halophilic bacteria and the two thermoacidophilic genera Sulfolobus and Thermoplasma. Based on ribosomal RNA sequence homologies and lipid composition, they apparently form a distinct group. Furthermore they possess or lack typical biochemical markers of both the eukaryotes and the prokaryotes, as well as having unique properties not found elsewhere. Altogether, this indicates that they are not closer to either one of the classical categories. One clear-cut difference between prokaryotes and eukaryotes is the diphtheria toxin reaction, which catalyses the covalent binding of adenosine diphosphate-ribose (ADPR) to the eukaryotic peptide elongation factor EF2 in contrast to the homologous prokaryotic factor EF-G. We report here that diphtheria toxin also catalyses the ADP-riboslation of archaebacterial elongation factors. In this respect, these factors have to be assigned to the EF2 type; we suppose that the ADP-ribosylatable structure arising so early in evolution is of fundamental importance for the elongation process.