Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis

The gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA-related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 +/- 2.5 ng per mg of larval body weight.     

Six year follow up of infants with bacteriuria on screening.

OBJECTIVE--To determine the value of screening for bacteriuria in infants with special emphasis on the natural course of untreated asymptomatic bacteriuria, renal growth, and renal damage. DESIGN--Prospective six year follow up of infants with bacteriuria on screening in an unselected infant population. SETTING--Paediatric outpatient clinic. PATIENTS--50 Infants (14 girls, 36 boys) with bacteriuria on screening verified by suprapubic aspiration from an unselected population of 3581 infants in a defined area of Gothenburg. INTERVENTIONS--Children with asymptomatic bacteriuria and normal findings on initial urography were untreated, although other infections were treated. MAIN OUTCOME MEASURES--Culture of urine and determination of C reactive protein concentration every six weeks for the first six months after diagnosis, every three months from six months to two years, and every six months between two and three years; thereafter yearly urine culture. Evaluation of renal concentrating capacity with a desmopressin test; radiological examination, including first and follow up urography and micturition cystourethrography without antibiotic cover; and measurement of renal parenchymal thickness and renal surface area. RESULTS--Of the original 50 infants, 37 (12 girls, 25 boys) were followed up for at least six years. Two infants developed pyelonephritis within two weeks after bacteriuria was diagnosed; the others remained free of symptoms. 45 Infants were untreated; the bacteriuria cleared spontaneously in 36 and in response to antibiotics given for infections in the respiratory tract in eight. Recurrences of bacteriuria were observed in 10 of the 50 children, of whom one had pyelonephritis. No child had more than one recurrence. At follow up urography in 36 of the 50 children (9 girls, 27 boys) after a median of 32 months no child had developed renal damage. First samples tested for renal concentrating capacity showed significantly higher values than those from a reference population (mean SD score 0.50, 95% confidence interval 0.21 to 0.79; p less than 0.001), but the last samples showed no significant difference (mean SD score 0.08, -0.24 to 0.40; p greater than 0.05). CONCLUSIONS--Mass screening for bacteriuria in infancy results primarily in detection of innocent bacteriuric episodes and is not recommended.     

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.     

Age- and sex-related differences in lipid peroxidation of mouse cardiac and skeletal muscles

1. The purpose of the present study was to characterize age- and sex-related changes in lipid peroxidation capacities and enzymatic antioxidants of cardiac and skeletal muscles in NMRI-mice (Mus musculus). 2. Lipid peroxidation rates (unstimulated and enzymatic/iron-stimulated) strongly decreased in skeletal muscle during ageing. 3. Unstimulated lipid peroxidation rate but not that of stimulated, also decreased in cardiac muscle. 4. The total level of Fe2+/ascorbate-stimulated non-enzymatic lipid peroxidation was not, however, affected by ageing. 5. The activity of catalase slightly increased in cardiac muscle and that of glutathione peroxidase in skeletal muscle during ageing. 6. Unstimulated lipid peroxidation rate was significantly higher in the skeletal muscle of male than female mice. 7. Correspondingly, the Fe2+/ascorbate-stimulated lipid peroxidation capacities of microsomal and mitochondrial fractions of skeletal muscle were significantly higher in male mice. 8. The activity of glutathione peroxidase as well as the concentration of lipofuscin were higher in the cardiac muscles of female than male mice.     

Immunocytochemical investigations on the presence of neuron-specific antibodies in the CSF of Parkinson's disease cases

The CSF and sera of 7 patients with Parkinson's disease were investigated immunocytochemically, in order to see if antibodies were present which recognized DA-ergic cell bodies in glutaraldehyde fixed rat brain. In 2 patients a marked labeling of DA-ergic neurons in the substantia nigra was observed, identified by anti-DA antiserum and by 6-OHDA induced degeneration, but also other neurons in the ventral mesencephalon were recognized. The other patients were weakly positive or negative. Sera gave unspecific labelling of all neurons. In one patient the sub-classes of IgG were investigated and found to be of IgG₃ (labeling nerve terminals) and of IgG_{1–2}, low affinity type (recognizing perikarya). The epitopes recognized have not been identified, but are unlikely to be DA-like, since blocking experiments and ELISA-tests gave negative results. The possible clinical importance of the results are discussed.Special Issue dedicated to Prof. Holger Hydén.     

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.     

Infections with Francisella tularensis biovar palaearctica in hares (Lepus timidus, Lepus europaeus) from Sweden

The occurrence of tularemia was studied in 1,500 hares submitted to the National Veterinary Institute, Uppsala, Sweden for postmortem examination during 1973 through 1985. A total of 109 tularemia cases was recorded based on the fluorescent antibody (FA) test for Francisella tularensis and on the gross and microscopic pathology. Tularemia was diagnosed only in the varying hare (Lepus timidus) and not in the European brown hare (Lepus europaeus). The geographical distribution of the 109 cases indicates that tularemia has not spread in Sweden during the last 45 yr, with the exception of an endemic occurrence of the disease on the island of Stora Karls? in the Baltic sea. The disease was most frequent in the autumn and only a few cases were recorded during winter. Cases were not seen in the spring. The annual prevalence varied, with several cases in 1974 and 1981, but there were no cases in 1976 and 1980. The postmortem findings in hares dying of tularemia in the autumn were characterized by focal coagulative necrosis in liver, spleen and bone marrow, with high numbers of bacteria FA-positive for F. tularensis. In hares dying during winter months, the most characteristic findings were hemorrhagic enteritis and typhlitis, although necrotic lesions could occur in liver, spleen and bone marrow. Diseased hares on the island of Stora Karls? were demonstrated to be infected with ticks, while hares on the mainland of Sweden generally were fed upon by mosquitoes. Twenty-six of the 109 hares with tularemia were examined bacteriologically and F. tularensis biovar palaearctica was isolated from eight. The lung extract antibody test for F. tularensis was performed in 18 of the 109 hares. All were negative. In addition to the field study, an experimental study with F. tularensis biovar palaearctica was performed. Four varying hares and three European brown hares were inoculated. None of the hares died from tularemia, and generalized infection was not demonstrated.     

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

Summary A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3mg src="/content/g5n431272g760p52/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3mg src="/content/g5n431272g760p52/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.     

Simulated nitrogen dynamics and nitrate leaching in a perennial grass ley

A soil nitrogen model was used for a 4-year simulation of nitrogen dynamics and nitrate leaching, both during grass ley growth and after ploughing a grass ley. Model results were compared with field measurements of soil mineral-N status and leaching. A soil water and heat model provided daily values for abiotic conditions, which were used as driving variables in the nitrogen simulation. Simulated values for mineral-N levels in the soil agreed well with field data for the first 3 years of the simulation. During the final year the model predicted considerably higher levels of soil mineral-N content compared with measurements. To reach the mineral-N level measured at the time of ploughing the ley, the simulated N-uptake by plants had to be increased by 8 g N m^−2. Simulations of nitrate leaching suggested that estimates of leaching based on measurements in tile-drained plots can be considerably underestimated. Accurate quantification of leaching in tile-drained plots often requires additional information on water-flow paths. A substantial increase in simulated and measured values for the mineral-N content of the soil occurred after ploughing the ley. In the simulation, most of the increase was due to a high crop residue input and the absence of a growing crop after ploughing. Litter accumulations in the soil during the 4-year period contributed little to the increase in soil mineral-N.