The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis

Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from [1-14C]propionic acid and 35SO4(-2). With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.     

Characterization of the Oregon isolate of Neospora hughesi from a horse

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.     

Prevalence of Toxoplasma gondii antibodies in barren-ground caribou (Rangifer tarandus groenlandicus) from the Canadian Arctic

Prevalence of antibodies to Toxoplasma gondii was determined in 147 barren-ground caribou (Rangifer tarandus groenlandicus) from 5 herds in the Northwest Territories and Nunavut, northern Canada, by the modified agglutination test (MAT). In the mainland herds (Bluenose, Bathurst, and Beverly), antibodies were found in 43 (37%) of 117 caribou, and MAT titers were 1:25 in 10, 1:50 in 24, and 1:500 in 9. In the island herds, only 1 (4.3%) of 23 animals sampled from the North Baffin Island herd was positive (titer = 1:25) and no antibodies were detected in 7 caribou from the Dolphin and Union herd. The high prevalence of antibodies to T. gondii in the mainland caribou herds indicates that caribou meat may contain viable T. gondii.     

Reverse micelles: a novel tool for H2 production

Reverse micelles serve as a novel tool to entrap enzymes and microbial whole cells within aqueous pockets and can be of great use in enhancing the efficiency and sustainability of the biological system. Photosynthetic bacterium Rhodopseudomonas sphaeroides entrapped inside the aqueous pool of reverse micelles prepared from benzene-sodium lauryl sulphate exhibited 25-fold enhancement of H₂ photoproduction rate (1.67 ml H₂ [mg protein]^–1 h^–1) compared to cells suspended in normal aqueous medium. Hydrogen photoproduction by the bacterium was catalysed by the nitrogenase enzyme system which was supported at a low light intensity of 12 Em^–2 sec^–1 photon flux energy at a wavelength of 520 nm. The optimum temperature for the process was 40 °C.     

Prevalence of Toxoplasma gondii antibodies in sera of domestic pigs and some wild game species from Zimbabwe

Serum samples of domestic pigs (Sus scrofa), elands (Taurotragus oryx), sable antelopes (Hippotragus niger), warthogs (Phacochoerus aethiopicus), bushpigs (Koiropotamus [Potamochoerus] koiropotamus), white rhinos (Ceratotherium simus), African buffalos (Syncerus caffer), wildebeest (Connochaetas taurinus), and African elephants (Loxodonta africana) from Zimbabwe were tested for Toxoplasma gondii IgG antibodies by the modified agglutination test (MAT) with whole formalized tachyzoites and mercaptoethanol. Sera were diluted at 1:25, 1:50, and 1:500 for MAT testing. Sera with antibodies in a 1:25 dilution were considered to have T. gondii infection. Toxoplasma gondii antibodies were found in 9.3% of 97 domestic pigs, 36.8% of 19 elands, 11.9% of 67 sables, 0 of 3 warthogs, 0 of 3 bushpigs, 50% of 2 white rhinos, 5.6% of 18 buffalos, 14.5% of 69 wildebeest, and 10.5% of 19 elephants examined.     

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.     

Toxoplasma gondii antibodies in naturally exposed wild coyotes, red foxes, and gray foxes and serologic diagnosis of Toxoplasmosis in red foxes fed T. gondii oocysts and tissue cysts

Antibodies to Toxoplasma gondii were determined in sera from 222 coyotes (Canis latrans), 283 red foxes (Vulpes vulpes), and 97 gray foxes (Urocyon cinereoargenteus) from Indiana, Kentucky, Michigan, and Ohio during 1990-1993. Sera were examined in 1:25, 1:100, and 1:500 dilutions by the modified direct agglutination test (MAT) with formalinized whole tachyzoites plus mercaptoethanol. Antibodies were found in 131 (59.0%) of 222 coyotes, 243 (85.9%) of 283 red foxes, and 73 (75.3%) of 97 gray foxes. Antibodies were also measured by different serologic tests in 4 littermate T. gondii-free red foxes fed T. gondii tissue cysts or oocysts; the fifth littermate fox was not fed T. gondii. Antibodies were measured in fox sera obtained 0, 14, and 36-55 days after infection with T. gondii. All 4 foxes fed T. gondii developed MAT and dye test antibody titers of 1:200 or more 14 days later. The latex agglutination test (LAT) and indirect hemagglutination test (IHAT) were less sensitive than MAT for the diagnosis of T. gondii infection in foxes. Antibodies were not detected by LAT (titer 1:64) in the 2 foxes fed tissue cysts nor by IHAT in 1 of the foxes fed tissue cysts. Toxoplasma gondii was isolated by bioassay in mice from tissues of all 4 foxes fed T. gondii. The control fox had no T. gondii antibodies detectable by any of the serologic tests.     

Hepatic sarcocystosis in a horse

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.     

Synergistic effects in enzymic reactions

It has been shown that when two enzymes showing similar actions act in close proximity of each other they influence each other synergistically. The phenomenon of synergism is, however, not observed if the two enzymes are of dissimilar action type. The condition of closest proximity has been simulated by conducting the enzymic reactions inside the reversed micelles. In the present study we have experimented with alpha-amylase and invertase both hydrolysing enzymes and also with peroxidase and invertase which do not show similar actions.     

A gas chromatography/mass spectrometry assay to measure estradiol, catecholestradiols, and methoxyestradiols in plasma

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.