The polymerase chain reaction applied to identification of specific alleles of the bovine milk protein genes.
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Alleles↗
Biomedical subjects
D Savva
Publications and source records attributed to D Savva.
Polymerase chain reaction for detection of Toxoplasma gondii.
A DNA-based assay has been developed for the detection of Toxoplasma gondii. The assay makes use of the polymerase chain reaction (PCR) to amplify part of the P30 gene on the parasite's DNA. Following gel electrophoresis, the amplified DNA can be detected either directly on the gel or by Southern hybridisation with radioactive or non-radioactive DNA probes. The assay has been used to detect the DNA from different isolates of T. gondii in a background of human or mouse DNA. Together with other information such as clinical data, CT scans and serology, the PCR assay should improve the diagnosis of toxoplasmosis in immunosuppressed and immunocompromised patients as well as in fetal tissues.
Animals↗
Diagnosis of toxoplasmosis using DNA probes.
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DNA↗
Diagnosis of cerebral toxoplasmosis in association with AIDS using the polymerase chain reaction.
Two AIDS patients were provisionally diagnosed as having cerebral toxoplasma infection on the basis of compatible clinical presentation, serological evidence of exposure to toxoplasma and the detection of multiple space occupying lesions on CT scan. Initial response to conventional antitoxoplasma therapy was poor. Brain biopsy was performed and toxoplasma nucleic acid detected in the cerebral tissues utilising the polymerase chain reaction (PCR). Specific therapy was continued and a satisfactory clinical and radiological response was achieved in each case. The PCR represents a method of potential value for the diagnosis of cerebral toxoplasmosis associated with AIDS. Further studies are required to assess the sensitivity, specificity and prognostic value of this technique in comparison with established diagnostic methods.
Acquired Immunodeficiency Syndrome↗
Diagnosis of ovine toxoplasmosis: an alternative to serology?
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Animals↗
Treatment of ovariectomized heifers with bovine follicular fluid specifically suppresses pituitary levels of FSH-beta mRNA.
To investigate the inhibin-induced suppression of FSH secretion by the anterior pituitary, chronically ovariectomized heifers (three per group) were treated for 56-58 h with either steroid-free bovine follicular fluid (bFF; 8 ml i.v. every 8 h) or 0.9% (w/v) NaCl (8 ml i.v. every 8 h). Blood was withdrawn at 8-h intervals for analysis of plasma concentrations of FSH and LH by radioimmunoassay. At the end of the treatment period, heifers were slaughtered and pituitary glands recovered for determination of gonadotrophin contents and levels of mRNA encoding FSH-beta, LH-beta, TSH-beta and common alpha glycoprotein hormone subunits using [32P]cDNA probes in total RNA dot and Northern blot assays. Treatment with bFF markedly suppressed plasma FSH by 85% (P less than 0.001 compared with pretreatment period), but did not affect plasma LH concentrations. Plasma FSH and LH concentrations did not vary significantly in the saline-injected control heifers. The level of FSH-beta subunit mRNA was reduced by 60% (P less than 0.001) in heifers treated with bFF, whereas no significant differences between control and bFF-treated heifers were observed in the levels of mRNA encoding LH-beta, TSH-beta or common alpha subunits. Treatments with bFF, however, did not affect pituitary content of either FSH or LH. These results support the conclusion that inhibin exerts its selective suppressive effect on the secretion of FSH by the bovine pituitary, at least in part, by directly inhibiting expression of the gene encoding the FSH-beta subunit.
Animals↗
Expression and partial purification of human pro-opiomelanocortin in Escherichia coli.
A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59-241 has been cloned and expressed in Escherichia coli. A 1.0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a beta-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The beta-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-amino-benzyl-1-thio-beta-D-galactopyranoside agarose.
Blotting, Western↗
Isolation of a potential DNA probe for Toxoplasma gondii.
A genomic DNA library for Toxoplasma gondii has been constructed in plasmid pUC8. Some recombinant plasmids (pJMBG1-pJMBG55) were analysed by restriction endonuclease mapping and by hybridization to DNA isolated from T. gondii and from human tissue culture cells. A number of recombinant plasmids, or DNA fragments derived from these, hybridizing only to Toxoplasma DNA have been identified. These are currently being characterized for the development of diagnostic DNA probes for Toxoplasma.
Animals↗
Identification of the promoters of the Bacillus subtilis spoIIA and spoVA operons.
Nuclease S1 mapping was used to identify the transcription start points of the spoIIA and spoVA operons of Bacillus subtilis. The spoIIA operon has been shown to be transcribed from two promoters separated by about 850 bp of DNA. The downstream of the two promoters has -10 and -35 regions characteristic of promoters recognised by the sigma-B form of RNA polymerase and it gives rise to a transcript with a 45-base 5' untranslated leader sequence. Studies on the upstream spoIIA promoter are still in progress and preliminary results indicate the presence of an open reading frame between the two promoters. In the case of the spoVA operon the promoter shows homology to promoters recognised by the sigma-E form of RNA polymerase; the 5' untranslated sequence on the spoVA mRNA is 14 bases.
Bacillus subtilis↗
Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis.
The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.
Bacillus subtilis↗
Cloning of the Bacillus subtilis spoIIA and spoV A loci in phage phi 105DI:1t.
A 6.95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage phi 105DI:1t. The recombinant phage (phi 105DS1) contained DNA of 33.8 kb as compared with 35.2 kb for phi 105DI:1t and 39.2 kb for the wild-type phage. In the presence of helper phage, phi 105DS1 complemented both spoIIA and spoV A mutations in B. subtilis.
Bacillus subtilis↗
Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA.
The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.
Bacillus subtilis↗
Cloning and restriction enzyme analysis of a Neisseria gonorrhoeae plasmid.
A 4.2-kb cryptic plasmid (pDS1) isolated from Neisseria gonorrhoeae FA101 has been cloned in Escherichia coli using the plasmid pUC9. Restriction enzyme maps of both native pDS1 and cloned pDS1 have been constructed; these indicate that the external C may be modified in some of the 5' CCGG 3' sites of the gonococcal plasmid.
Chromosome Mapping↗
Spontaneous mutation rates in continuous cultures: the effect of some environmental factors.
The rate of spontaneous mutation from tryptophan auxotrophy to prototrophy has been determined under different growth-limiting and environmental conditions in continuous cultures of Escherichia coli WP2. The growth rate, aeration rate, pH and temperature were found to affect the rate of mutation. In glucose-limited cultures the mutation rate was found to be directly proportional to both the growth rate and the aeration rate. A temperature coefficient of 2 was observed and there was an optimum temperature and pH for mutation.
Energy Metabolism↗
Mutation in Escherichia coli during substrate-accelerated death.
Substrate-accelerated death was studied in lactose-limited cultures of Escherichia coli WP2 trp- and E. coli WP2 trp+. During starvation of E. coli WP2 trp- the viable count decreased while the number of trp+ revertants increased. Addition of 7.5 mM-cAMP to the starvation medium prevented the death of the trp-cells but not the increase in the number of trp+ revertants. As starvation of pure cultures of trp+ revertants did not result in death it suggests that the level of cAMP in trp- cells was lower that in trp+ cells. Addition of benzyl penicillin, nalidixic acid, novobiocin or rifampicin did not affect the viable counts thus indicating that neither cryptic growth not DNA replication occurred; however, in the presence of novobiocin there was no increase in the number of trp+ revertants. The possibility of a constitutive error-prone DNA repair mechanism is considered.
Cyclic AMP↗
Toxoplasma serology in Zambian and Ugandan patients infected with the human immunodeficiency virus.
In the USA and Europe, toxoplasmosis is well recognized as an important cause of morbidity and mortality among immunocompromised individuals. Toxoplasma gondii has been shown to be a common opportunistic infection in patients infected with the human immunodeficiency virus (HIV) in the USA and Europe with published estimates ranging from 20% to 80%. The importance of Toxoplasma infection in East Africa has not yet been defined. The seroprevalence rates of toxoplasmosis in Zambian and Ugandan patients were determined using the dye test (DT) and the latex agglutination test (LAT). The geographical variation in seroprevalence rates noted in western countries was also found in these African countries, with Zambia showing significantly lower rates than Uganda. 34% of Ugandan (64/186) and 4% of Zambian (8/187) patients infected with HIV, compared with 27% of Ugandan (26/93) and 11% of Zambian (20/189) HIV-negative persons, had anti-Toxoplasma immunoglobulin G antibodies. With the LAT, 13% of the Ugandan and 7% of the Zambian sera gave a false positive result. The relevance of Toxoplasma serology in Africa is discussed.
Antibodies, Protozoan↗
Effect of administration of a novel recombinant bovine interferon on length of oestrous cycle in cattle.
In ruminants, extensive reproductive loss occurs during the process of maternal recognition of pregnancy and it has been suggested that trophoblast interferons may be potential therapeutic agents. This paper reports results from a trial using eight first lactation Holstein-Friesian heifers to test the efficacy of a novel recombinant bovine interferon produced in bacteria in extending the life of the corpus luteum. Oestrus was synchronised in these animals and 0.1 mg of this non-glycosylated interferon was infused into the uterus twice daily for 13 days starting approximately 12 days after oestrus. This treatment resulted in an extension of the lifespan of the corpus luteum by 5.5 days (P=0.028) compared with untreated controls. In these animals the interovulatory period was extended by 6.4 days (P=0.009). Administration of this protein did not have any adverse effects either on body temperature or on daily milk yields. The results indicate that this novel interferon may have potential therapeutic application for reducing embryo mortality.
Animals↗
Effect of a novel recombinant bovine interferon and trophoblast secretory products on protein metabolism by endometrial explants from cattle and sheep.
The effect of a novel recombinant bovine interferon on the rate of protein synthesis, as measured by the rate of leucine incorporation, in endometrial explants from cows and ewes was investigated and compared with the effect of trophoblast secretory proteins and of a recombinant bovine interferon-alpha1. Exposure of cultured bovine endometrial tissue to the novel recombinant bovine interferon mimicked the effect of pregnancy in reducing the rate of protein synthesis in both caruncular and intercaruncular endometrium. This effect was similar to that seen with naturally produced trophoblast secretory proteins and it was more pronounced than that seen when endometrial tissue was treated with a related recombinant bovine interferon-alpha1. Ovine endometrial cultures were unaffected by exposure to this novel recombinant bovine interferon. This novel recombinant interferon exhibited potent antiviral properties (an activity of 1.6 x 10(10) international reference units/mg protein) and may prove to be a valuable reagent in studying the maternal recognition of pregnancy in the cow and reducing embryo mortality.
Animals↗