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Biomedical subjects

D Savva

Publications and source records attributed to D Savva.

At least 55 records · Page 3Linked to original sources
PubMed1991

Expression and partial purification of human prolactin in Escherichia coli.

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.

Blotting, Western↗
PubMed1991

Ovine trophoblast protein-one: evidence for possible glycosylation.

1. The polymerase chain reaction has been used to amplify specifically the cDNA coding for the secreted form of ovine trophoblast protein-one from a preparation of total cellular RNA extracted from sheep embryos removed from ewes 16 days after mating. 2. Cloning and sequencing of the amplified cDNA revealed two new sequence variants: SPW49 having 93% similarity with deduced amino acid sequences from published cDNA data, and SPW27 a variant coding for a deleted form of ovine trophoblast protein-one. 3. The gene for ovine trophoblast protein-one is intronless. 4. This study provides further evidence for the existence of an ovine trophoblast protein-one gene family. 5. Both variants contain a potential N-glycosylation site not apparent in published sequences for ovine trophoblast protein-one.

Amino Acid Sequence↗
PubMed1991

Expression of bovine inhibin beta subunit in Escherichia coli.

1. A DNA fragment encoding the beta subunit of bovine inhibin was amplified using the polymerase chain reaction and was cloned in plasmids pUC8 and pUR291. 2. Cultures of Escherichia coli TG2 harbouring pKDK37, a pUR291-derived recombinant plasmid, produced a novel protein with a molecular weight of 130,000 corresponding to a beta-galactosidase-inhibin beta fusion protein. 3. The fusion protein was purified from inclusion bodies by solubilization in 8 M urea followed by an ion-exchange and gel permeation chromatography. 4. Analysis by immunoblotting and competitive radioimmuno assay revealed that the fusion protein was recognized by a monoclonal antibody raised against a chemically synthesized peptide for amino acid residues from +82 to +114 of the beta subunit of the bovine inhibin thereby confirming its identity.

Activins↗
PubMed1991

Detection of foreign DNA in transgenic chicken embryos using the polymerase chain reaction.

Chicken primordial germ cells were infected with a defective retrovirus containing the Escherichia coli lacZ gene and injected into the heart of stage 15 embryos. DNA samples were isolated from various tissues of the injected embryos at different stages of development and were examined for the presence of the lacZ gene using the polymerase chain reaction. Integration of the retrovirus DNA was demonstrated with a 32P-labelled oligonucleotide in five-, 10- and 18-day embryos. This quick procedure provides an opportunity for the early detection of foreign DNA in small numbers of transfected cells and is a valuable tool in the detection of transgenic animals.

Animals↗
PubMed1991

New methods in the diagnosis and management of cerebral toxoplasmosis associated with the acquired immune deficiency syndrome.

Cerebral toxoplasmosis is a life-threatening condition associated with the acquired immune deficiency syndrome (AIDS). Current diagnostic and therapeutic methods have serious limitations. The diagnosis of cerebral toxoplasma infection in a patient with AIDS was assisted by the detection of specific IgM in a highly sensitive immunosorbent agglutination assay and by the demonstration of Toxoplasma gondii nucleic acid in a brain biopsy specimen by means of the polymerase chain reaction. Following initial failure of the patient to respond to treatment with sulphadiazine and pyrimethamine, clinical improvement was observed during treatment with clindamycin followed by dapsone. Further assessment of novel methods in the management of cerebral toxoplasmosis is required.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction.

Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

Alleles↗
PubMed1990

Genetic characterization of an alloalbumin, albumin Kashmir, using gene amplification and allele-specific oligonucleotides.

The molecular basis for albumin Kashmir was studied using the polymerase chain reaction to amplify a DNA fragment containing codon 501 in exon 12 of the human albumin gene. Southern blots of the amplified DNA were hybridized to oligonucleotide probes specific either for the normal allele of albumin or for albumin Kashmir. The results provide strong evidence that codon 501 in albumin Kashmir is AAG (lysine) instead of GAG (glutamic acid), thus confirming the protein sequences reported. This approach was used to characterize a bisalbuminaemic individual as a carrier for albumin Kashmir. Similar strategies may be devised to study the molecular basis and to identify carriers of other alloalbumins.

Albumins↗
PubMed1990

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification.

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Cloning, Molecular↗