Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.     

Enzymatic activities of house dust extracts

Ten house dust extracts were examined for enzymatic activity using the Api-Zym System. Comparisons were also made with extracts of mites and molds. The results show a high degree of heterogeneity between dust extracts from different companies and also to a lesser extent between lots of dust extracts from the same company, indicating variability of source material for extraction. It seems possible to detect enzymatic activity in house dust extracts associated with molds but more difficult to do the same with mites. The correlation between enzymatic activity and allergenic activity of house dust cannot be made from these data.     

Immunoblot studies to analyze antibody to the Rickettsia typhi group antigen in sera from patients with acute febrile cerebrovasculitis.

In 1986, an unusual syndrome of acute febrile cerebrovasculitis in the Piedmont Region of Virginia was reported. All patients had encephalopathy and prior exposure to both a sylvan environment and flea-infested animals. The initial serological studies suggested a rickettsial origin, corroborating clinical, epidemiological, and histopathological findings. Sera from four of five patients were subsequently studied by immunoblotting. Unabsorbed and absorbed sera were tested with electrophoresed and electroblotted Rickettsia typhi, Legionella bozemanii, and Proteus vulgaris OX19 antigens. The unabsorbed sera reacted with all three antigens. The P. vulgaris- and L. bozemanii-absorbed sera reacted with R. typhi only and without significantly less intensity. In contrast, the reactivity of R. typhi-absorbed sera was significantly lower with all three antigens. These results indicate that these patients had specific antibodies to a typhus group antigen. Although our findings suggest that a rickettsia of the typhus group may have caused this syndrome, no definitive diagnosis could be achieved because a rickettsial organism was not isolated.     

Enterotoxic, cytotoxic, necrotic and lethal activities in cell-free extracts of Salmonella strains isolated from humans

Unconcentrated cell-free sonic extracts from thirty Salmonella strains isolated from the faeces and blood of humans were investigated for the production of enterotoxins in various tests (Vero cell, infant mouse, rabbit skin permeability and rabbit ileal loop), as well as for lethal activity in adult mice. Sonic extracts from 23 (76.7%) strains were lethal for mice, 21 (70%) increased skin permeability and 3 (10%) showed necrotizing activity for the rabbit skin. No Salmonella strain producing typical Escherichia coli toxins, such as thermolabile (LT) or thermostable (STa) enterotoxins, Verotoxin (VT) or cytotoxic necrotizing factor (CNF) cytotoxins, were detected. Non-repetitive fluid accumulation in rabbit loops was obtained when unconcentrated sonic extracts from 10 selected strains were assayed in seven rabbits. Growth of Salmonella in casamino acid yeast extract medium, followed by treatment of bacterial cells with polymyxin B, was demonstrated to be a rapid and sensitive method for releasing the delayed permeability factor.     

Growth of Rickettsia typhi in irradiated L cells enhanced by lysosomal stabilization.

The growth of some obligate intracellular parasites is contingent upon avoidance of lysosomal activation during growth in their host cells. This is accomplished by the various parasites by different mechanisms and with different degrees of efficiency. The possibility was tested that the lysosomal stabilizer cortisone acetate might protect and thus enhance the growth of Rickettsia typhi in mouse L cells irradiated 6 days earlier. Beginning 2 days before infection of the L cells with a multiplicity of 10 rickettsiae, 20 microgram of cortisone per ml was added in medium 199 containing 5% fetal calf serum. This concentration of cortisone was below the cytotoxic level, as determined by viability staining, but was sufficient to significantly alter the ratios of cellular and released acid phosphatase and beta-glucuronidase in uninfected and infected cells, as shown by spectrophotometric analysis. Rickettsial replication, measured by hemolytic activity at 96 h and confirmed by microscopic observations at earlier stages of infection, was increased by the cortisone. Cortisone concentrations of 10 or 40 microgram/ml were less effective, and cortisone was ineffective when the rickettsial multiplicity per L cell was 2 or lower. These results indicate that amounts of cortisone that increase lysosomal stabilization in L cells favor rickettsial multiplication when the multiplicity of infection is relatively high.     

Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi.

A commercial enzyme immunoassay kit called the Dip-S-Ticks (DS) for the detection of total immunoglobulin (Ig) G and IgM human antibodies to Rickettsia typhi was evaluated. In tests with 340 serum samples from patients with diagnosed cases of rickettsial diseases, patients suffering from other febrile illnesses, and normal subjects, the DS compared favorably with the standard indirect fluorescent-antibody (IFA) test. At IFA cutoff titers of > or = 1.64 and > or = 1:128, the DS showed sensitivities of 88.2 and 91.4% and specificities of 91.8 and 87.7%, respectively. The DS test correlated significantly with both the IFA IgG (r = 0.84, P < 0.0005) and IgM (r = 0.63, P < 0.0005) titers. Only 80% of IgG and 82% of IgM IFA readings determined by two technicians were within one dilution, while the DS was more reliable, with 100% within one dot. The rapidity, reliability, and simplicity of the DS suggest that it is a suitable test for use in clinical laboratories unable to perform the IFA test.     

Structural Properties of Lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and Their Chemical Similarity to the Lipopolysaccharide from Proteus vulgaris OX19 Used in the Weil-Felix Test

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-d-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (β-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.The typhus group (TG) rickettsiae possess at least two different types of antigens. One type of antigen is sensitive to sodium metaperiodate, resistant to trypsin, stable in 0.2 M NaOH, and thermostable and includes erythrocyte-sensitizing substance (24), lipopolysaccharide (LPS) (26, 27), and OX19-like antigens (20). These antigens appear to be the group-specific antigens of TG rickettsiae. On the other hand, the species-specific antigens of both Rickettsia typhi and Rickettsia prowazekii are destroyed by incubation at 56°C for 45 min (15), suggesting that they are proteins.LPS from Coxiella burnetii (3, 10, 14, 28, 29), a related species of rickettsia, has been identified and chemically described, but the exact structure of this LPS is unknown. Previously, Amano et al. also reported the chemical properties of the spotted fever group (SFG) rickettsial LPS, which contains 3-deoxy-d-manno-octulosonic acid (KDO), glucosamine, 6-deoxyglucosamine (quinovosamine), ribose, glucose, phosphate, and palmitic acid (2). On the other hand, endotoxic activity analogous to LPS endotoxin was found in R. prowazekii by Olitzki et al. (22, 23), and Schramek et al. (26, 27) extracted a hydrophobic LPS-like endotoxin from R. typhi and R. prowazekii. Smith and Winkler (30) have provided evidence that R. prowazekii contains KDO, a marker for LPS. Although these data suggest the presence of LPS in TG rickettsia, the structure of this LPS has remained obscure.In attempt to ascertain the structure of LPS from TG rickettsiae, we extracted LPSs from R. typhi and R. prowazekii and analyzed their chemical components in terms of the heterogeneity of their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, glucose, KDO, fatty acids, glucosamine, quinovosamine, and phosphate were all identified as components of the LPS from TG rickettsiae.     

Comparison of a latex agglutination procedure with the microimmunofluorescence test for Rickettsia typhi.

Sera submitted to the Texas Department of Health for the serodiagnosis of Rickettsia typhi were tested by the microimmunofluorescent antibody technique and a new latex agglutination procedure. Results indicated that the latex agglutination test was sensitive and specific and would serve well as a first-line screening test for murine typhus.     

The temporal dynamics of humoral immunity to Rickettsia typhi infection in murine typhus patients

ObjectivesThis study examined individuals with Rickettsia typhi infection in the Lao People's Democratic Republic (Lao PDR) to (a) investigate humoral immune dynamics; (b) determine the differences in reference diagnostic results and recommend appropriate cut-offs; (c) determine differences in immune response after different antibiotic treatments; and (d) determine appropriate diagnostic cut-off parameters for indirect immunofluorescence assay (IFA).MethodsSequential serum samples from 90 non-pregnant, adults were collected at seven time-points (days 0, 7, 14, 28, 90, 180 and 365) as part of a clinical antibiotic treatment trial. Samples were tested using IFA to determine IgM and IgG antibody reciprocal end-point titres against R. typhi and PCR.ResultsFor all 90 individuals, reciprocal R. typhi IgM and IgG antibody titres ranged from <400 to ≥3200. The median half-life of R. typhi IgM was 126 days (interquartile range 36–204 days) and IgG was 177 days (interquartile range 134–355 days). Overall median patient titres for R. typhi IgM and IgG were significantly different (p < 0.0001) and at each temporal sample collection point (range p < 0.0001 to p 0.0411). Using Bayesian latent class model analysis, the optimal diagnostic cut-off reciprocal IFA titer on patient admission for IgM was 800 (78.6%, 95% CI 71.6%–85.2% sensitivity; 89.9%, 95% CI 62.5%–100% specificity), and for IFA IgG 1600 (77.3%; 95% CI 68.2%–87.6% sensitivity; 99%, 95% CI 95%–100% specificity).ConclusionsThis study suggests suitable diagnostic cut-offs for local diagnostic laboratories and other endemic settings and highlights antibody persistence following acute infection. Further studies are required to validate and define cut-offs in other geographically diverse locations.     

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.     

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.     

Enzymatic activities in mitochondria isolated from the rat brain during development.

In an attempt to discern whether mitochondrial changes during ontogeny are due to increases in number of mitochondria or to increases in number plus changes in their organization, we investigated the enzymatic markers of the inner and outer membranes of mitochondria from cerebral cortex and subcortical material in young and adult rats. We conclude from the data gathered that significant changes do occur in mitochondrial organization, that gradual maturation occurs in the inner membrane during development, especially in the cerebral cortex and considerably less so in the subcortical structures. We found no such changes in the sturucture of the outer membrane during the periods of development examined.     

Antigenotoxic activities of crude extracts from Acacia salicina leaves

For centuries, plants have been used in traditional medicines and there has been recent interest in the chemopreventive properties of compounds derived from plants. In the present study, we investigated the effects of extracts of Acacia salicina leaves on the genotoxicity of benzo[a]pyrene (B(a)P) and nifuroxazide in the SOS Chromotest. Aqueous, total oligomers flavonoids (TOF)-enriched, petroleum ether, chloroform, ethyl acetate, and methanol extracts were prepared from powdered Acacia leaves, and characterized qualitatively for the presence of tannins, flavonoids, and sterols. All the extracts significantly decreased the genotoxicity induced by 1 microg B(a)P (+S9) and 10 microg nifuroxazide (-S9). The TOF-enriched and methanol extracts decreased the SOS response induced by B(a)P to a greater extent, whereas the TOF-enriched and the ethyl acetate extracts exhibited increased activity against the SOS response produced by nifuroxazide. In addition, the aqueous, ethyl acetate, and methanol extracts showed increased activity in scavenging the 1,1-diphenyl- 2-picrylhydrazyl (DPPH) free radical, while 100-300 microg/ml of all the test extracts were active in inhibiting O2-production in a xanthine/xanthine oxidase system. In contrast, only the petroleum ether extract was effective at inhibiting nitroblue tetrazolium reduction by the superoxide radical in a nonenzymatic O2- -generating system. The present study indicates that extracts of A. salicina leaves are a significant source of compounds with antigenotoxic and antioxidant activity (most likely phenolic compounds and sterols), and thus may be useful for chemoprevention.     

Enzymatic activities of Legionella pneumophila and Legionella-like organisms.

The API ZYM system was used to investigate enzymatic activities of Legionella pneumophila and other Legionella-like organisms. Leucine aminopeptidase, alkaline and acid phosphatase, butyrate and caprylate esterase, and phosphoamidase activities were consistently detected in all strains tested. No evidence of myristate lipase, trypsin, chymotrypsin, or glycosidase activity was found.     

Failure of globin mRNA to stimulate globin synthesis in cell-free extracts of interferon-treated globin-synthesizing mouse erythroleukemic cells.

Exogenous globin mRNA or encephalomyocarditis virus mRNA can be efficiently translated by S30 lysates of mouse erythroleukemic cells and also by lysates of similar cells in which hemoglobin synthesis had been induced by dimethylsulfoxide. Cell-free extracts from interferon-treated cells had not only lost their translation capacity for viral but also for globin mRNA. The indiscriminate inhibition of the in vitro translation capacity of natural mRNA often seen in extracts from interferon-treated murine cells seems therefore to be caused by a defect of the cell-free systems. The defect in the S30 lysates of interferon-treated cells could be repaired by the addition of tRNA.     

Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants

Background  

Owing to their roles in tissue remodelling in health and disease, several studies have reported investigations on plant extracts as inhibitors of proteinases and as anti-oxidants.