Detection of human TNF-alpha mRNA by NASBA.

A method to amplify and detect TNF-alpha mRNA from primed Mono Mac 6 cells is described. A silica-based extraction system was utilised for preparation of cell extracts and specific oligonucleotide primers were designed for amplification of TNF-alpha mRNA by the NASBA process. Amplification products were detected using either a liquid hybridisation assay, with analysis by polyacrylamide gel electrophoresis, or a plate hybridisation system. The method has many potential applications for the study of inflammatory cytokines and cellular mRNAs in cell culture and clinical samples.

Analysis of latent forms of renin using antibodies raised against the propart segment of human prorenin: validation with representative samples of ovarian cyst and follicular fluids.

Antisera were raised against synthetic peptides from the prosegment of human prorenin. The use of each of these for detection of the appropriate prosegment region of prorenin was validated by development of an ELISA protocol standardised with recombinant prorenin present in culture medium conditioned by myeloma cells transfected with a prorenin expression plasmid. Detection of the respective epitopes in the prosegment required prior exposure of the prorenin in the medium to acid pH in order to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. By these ELISA protocols, the form of latent renin present in representative samples from ovarian cyst and follicular fluids was analysed; one follicular cyst fluid was found to contain full-length prorenin whereas the fluid from a benign cyst and ovarian follicular fluid samples contained the precursor in truncated form.

Presence and possible significance of immunocytochemically demonstrable metallothionein over-expression in primary invasive ductal carcinoma of the breast.

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

Immunological distinction between piscine and mammalian metallothioneins.

1. Separate antisera to metallothioneins (MT) from rainbow trout and horse were produced in mice and their reactivity with the respective immunogen was confirmed using an ELISA. 2. The ELISA, used in a competitive mode, revealed that the anti-horse MT serum did not cross-react with trout MT. Reciprocally, the anti-trout MT serum did not show any reactivity with horse MT. 3. The anti-rainbow trout MT serum was shown to cross-react totally with MTs from plaice, flounder, turbot, perch, salmon and pike, but exhibited no reactivity towards MTs from human, mouse, rat, worm or crab. Partial reactivity with the proteins isolated from oyster and mussel was demonstrated. 4. The anti-horse MT serum cross-reacted totally with MTs from human, rat and rabbit but no reactivity was demonstrable when MT from either plaice or worm was tested. 5. The behaviour of apo-, holo- and recombinant rainbow trout MTs, in which the metal content was different, indicated that reactivity with anti-MT antibodies was not dependent on the presence or nature of the metals bound in the protein. 6. The patterns of reactivities were analysed in relation to the known amino acid sequences of MT.