Uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida shows cold adapted features: A comparative analysis to Vibrio cholerae uracil-DNA N-glycosylase

The genes encoding uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida and the mesophilic counterpart Vibrio cholerae have been cloned and expressed in Escherichia coli. The purified proteins have been characterized in order to reveal possible cold adapted features of the V. salmonicida UNG (vsUNG) compared to the V. cholerae UNG (vcUNG). Characterization experiments demonstrated that both enzymes possessed the highest activities at pH 7.0–7.5 and at salt concentrations in the range of 25–50 mM NaCl. Temperature optima for activity were determined to approximately 30 °C for vsUNG and 50 °C for vcUNG. Temperature stability of the enzymes was compared at 4 °C and 37 °C, and vsUNG was found to be more temperature labile than vcUNG. Kinetic studies performed at three different temperatures, 15 °C, 22 °C and 37 °C, demonstrated higher catalytic efficiency for vsUNG compared to vcUNG due to lower K_M-values. The increased substrate affinity of vsUNG is probably caused by an increased number of positively charged residues in the DNA-binding site of the enzyme compared to vcUNG. Thus, activity and stability measurements reveal typical cold adapted features of vsUNG.     

Primary structure and properties of the formyltransferase from the mesophilic Methanosarcina barkeri: comparison with the enzymes from thermophilic and hyperthermophilic methanogens

The ftr gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from Methanosarcina barkeri was cloned, sequenced, and functionally expressed in Escherichia coli. The overproduced enzyme was purified eightfold to apparent homogeneity, and its catalytic properties were determined. The primary structure and the hydropathic character of the formyltransferase from Methanosarcina barkeri were compared with those of the enzymes from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri. The amino acid sequence of the enzyme from Methanosarcina barkeri was 64%, 61%, and 59% identical to that of the enzyme from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri, respectively. A negative correlation between the hydrophobicity of the enzymes and both the growth temperature optimum and the intracellular salt concentration of the four organisms was observed. The hydrophobicity of amino acid composition was +21.6 for the enzyme from Methanosarcina barkeri (growth temperature optimum 37° C, intracellular salt concentration ≈ 0.3 M), +9.9 for the enzyme from Methanobacterium thermoautotrophicum (65°C, ≈ 0.7 M), –20.8 for the enzyme from Methanothermus fervidus (83° C, ≈ 1.0 M) and –31.4 for the enzyme from Methanopyrus kandleri (98° C, > 1.1 M). Generally, a positive correlation between hydrophobicity and thermophilicity of enzymes and a negative correlation between hydrophobicity and halophilicity of enzymes are observed. The findings therefore indicate that the hydropathic character of the formyltransferases compared is mainly determined by the intracellular salt concentration rather than by temperature. Sequence similarities between the formyltransferases from methanogens and an open reading frame from Methylobacterium extorquens AM1 are discussed. Received: 7 September 1995 / Accepted: 7 November 1995     

Plasmodium fragile: Inhibition of cultures by serum from Rhesus monkeys immunized with homologous parasites

Rhesus monkeys, Macaco mulatto, that had previously been immunized with the Nilgiri strain of Plasmodium fragile grown in culture, together with control monkeys with and without inoculation of Freund's adjuvant, were challenged with cultured parasites. After treatment with chloroquine, the monkeys were rechallenged. Serum specimens from three immunized monkeys caused a specific, dose-dependent inhibition of parasite growth in culture. Fifty percent inhibition of in vitro growth was obtained using 5% immune serum combined with 10% normal rhesus serum. The specific inhibitory component of immune serum was shown to be IgG antibody. Results of the study demonstrated that there is good correlation between the inhibitory activity of immune serum, parasite growth in vitro, the in vivo response to challenge, and the indirect fluorescent antibody titer.     

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M_r and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.     

Schistosoma mansoni: A comparison of mouse and rat worms with respect to host antigens detected by the technique of transfer into hamsters

A significant proportion of schistosomes transferred from the mouse into the mesenteric veins of hamsters pre-immunized with mouse erythrocytes were rejected. The rejection was specific, could be passively transferred with immune serum and was demonstrable with mouse worms which had been ‘washed’ in normal hamsters for up to 24 h. On the contrary, schistosomes transferred from the rat into the mesenteric veins of hamsters pre-immunized with rat erythrocytes were generally not rejected to any significant extent. Rejection was not brought about by variations in the immunization procedure, or by passive transfer of immunity, or by the use of ‘older’ rat worms. Rat antigens could be detected on rat schistosomes with the use of mixed agglutination techniques, but they appeared to be present in relatively low amounts. The significance of these findings is discussed in relation to a possible protective function of host antigens.     

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: Plausible implications in the toxic activity of the protein

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89^th position was replaced by aspartic acid and in the other both glycine and valine at the 88^th and 89^th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.     

The blood respiratory, haematological, acid-base and ionic status of the Port Jackson shark, Heterodontusportusjacksoni, during recovery from anaesthesia and surgery: a comparison with sampling by direct caudal puncture

The effects of caudal cannulation on the blood physiology of the Port Jackson shark, Heterodontus portusjacksoni, were investigated in sharks given between 4 and 72 h to recover from surgery. Neither the Pa_O2–Pv_O2 difference nor the Ca_O2–Cv_O2 difference of cannulated sharks fluctuated throughout the sampling period. The plasma acidosis exhibited 4 h after surgery was partially compensated after 24 h by a respiratory (hyperventilatory) alkalosis and after 72 h by a marked metabolic alkalosis. Whilst H. portusjacksoni exhibited some cell swelling after surgery the haematological status of cannulated sharks generally varied little throughout the recovery period. In contrast, marked changes in plasma and erythrocyte ion concentrations were indicative of increased branchial and erythrocyte ion permeability. The blood status of H. portusjacksoni given 72 h to recover from surgery was also compared with sharks sampled by caudal puncture. The respiratory and acid-base status of sharks sampled by caudal puncture was comparable to that of cannulated sharks. In contrast, the plasma ion concentrations of the cannulated sharks were markedly elevated and the erythrocyte ion concentrations concomitantly reduced when compared with punctured sharks. The apparent increase in the water and ion permeability of cannulated sharks was reflected by the reduced [Hb] and mean cell haemoglobin concentrations (MCHC). Blood sampling by caudal puncture appeared to reduce the haematological and ionic perturbations that resulted from surgery and thus provided a less invasive and reliable method for obtaining samples from ‘non-disturbed’ elasmobranchs.     

A three- and two-dimensional documentation of structural elements in schwagerinids (superfamily Fusulinoidea) exemplified by silicified material from the Upper Carboniferous of the Carnic Alps (Austria/Italy): a comparison with verbeekinoideans and alveolinids

Silicified schwagerinids (superfamily Fusulinoidea v. Moeller 1878) from the Upper Carboniferous (Carnic Alps, Austria and Italy) were isolated from cemented carbonate rocks using hydrochloric acid. The shells show details of the wall texture and of internal structures in three dimensions which are illustrated with SEM pictures. Thin sections from hand specimens provided two-dimensional sections of the shell for comparison. The functional significance of fusulinoidean internal structures is discussed and compared with verbeekinoideans and alveolinids. Particular attention is paid on the disposition of the different openings within the shell and from the chamber lumen to the outside which reflects the direction of protoplasmic flow. Based on the knowledge of the nature of protoplasm ultrastructure in Recent foraminifera and its biological significance we draw some conclusions about the nature of protoplasm in fusulinoideans and its change within the Permian verbeekinoideans.