Heterologous expression in the Archaea: transcription from Pyrococcus furiosus gdh and mlrA promoters in Haloferax volcanii.

Multicopy plasmids containing the promoter regions for gdh and mlrA genes from Pyrococcus furiosus were propagated in Haloferax volcanii. High-level expression was detected from gdh promoter sequences, with transcription initiating at the same start-site as that found in P. furiosus. For mlrA, several transcripts were detected, with one initiating at the P. furiosus start-site; removal or disruption of the likely P. furiosus boxA element resulted in the disappearance of this transcript, indicating that these sequences were utilized by the H. volcanii RNA polymerase for initiation. This is the first demonstration of the utilization of promoters from a hyperthermophilic archaeon in a mesophilic haloarchaeon and provides further evidence for the unity of transcription processes in the domain Archaea.

Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

Expression of the Epstein-Barr virus encoded EBNA-1 gene in stably transfected human and murine cell lines.

Five murine and 3 human tumor cell lines were transfected with a retroviral vector that carries the EBV encoded EBNA-1 gene. All cell lines expressed intranuclear EBNA-1 as detected by anticomplement immunofluorescence and Western blot assays. The cell lines differed in the level of EBNA-1 expression and the size of the protein. The internal major late promoter of adenovirus was efficient in directing the transcription of EBNA-1 in the human lymphoma line BJAB, the murine T-cell lymphoma Tikaut, RBL-5, EL-4 and in the mouse sarcoma line MSWBS but was less efficient in Ramos, an EBV negative Burkitt lymphoma line, the human T-cell leukemia line 1301TK and the P815-X2 mouse mastocytoma line. All transfected lines except MSWBS contained EBNA-1 in a truncated form. The truncated EBNA-1 polypeptide reacted with the conventional human antibody reagents in an EBNA specific fashion but failed to bind rabbit or human antibody directed against the glycine-alanine repeat sequence. MSWBS contained a truncated as well as a full size EBNA-1 polypeptide. It also reacted with antibody directed against the glycine-alanine repeat. This indicates that the repeat sequence is regularly affected by the truncation.

Isolation and dissociation of immune complexes from pleural effusions of lymphoma patients.

Immune complexes (IC) isolated from pleural effusions of lymphomas with favorable and unfavorable prognoses were of IgG type. These IC were further dissociated by ion exchange chromatography using 8 M urea. The antibody was found to be a high molecular weight protein (1.5 X 10(5) daltons) and reacted with antihuman IgG immunologically while a second peak obtained on ion exchange chromatography may be an antigen moiety with a molecular weight of 3.2 X 10(4) daltons as it reacted immunologically with the antibody. Strong cytoplasmic fluorescence was observed with various cell suspensions of lymphomas when reacted with the antibody preparations. The antisera raised against two different antigen fractions prepared from two lymphomas--nHL and LL showed positive fluorescence with both nHL and LL suspensions. The absorption of these rabbit antibodies with individual cell extracts or with antigen preparations also entirely blocked the cytoplasmic staining. The antigen moiety (PK-II) may have a common origin in the disease process. Pleural effusions from patients with unfavorable and favorable prognoses showed identical patterns of separation of IC components.

Evaluation of circulating immune complexes in lymphomas and leukemias using two different assays.

Circulating immune complexes (CICs) have been detected in the sera of patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease, chronic myeloid leukemia, and acute lymphoblastic leukemia by using C1q-binding and L1210-binding assays. Both assays gave broadly similar patterns of reactivity in terms of frequency and magnitude, though there are some differences. Significantly elevated CIC levels were observed in all pathologic groups. However, sera from NHL patients with an unfavorable prognosis consistently exhibited the highest frequency of positive values and mean CIC levels in both these assays. The two tests showed concordance in 66.6% of the NHL patients' sera and were significantly correlated. Of the sera from NHL patients 12.7% were positive in the C1q-binding assay only and 15.9% in the L1210-binding assay only. Both the assays gave positive results in some patients, and a degree of overlap indicates the presence of different types of CIC in cancer patients' sera. The combined use of two methods for detecting CICs may be useful for evaluation of the activity, the extent, and the prognosis of the malignant disease.

Circulating immune complexes in patients with lymphomas and leukemias.

Immune complexes (IC) were examined in the sera of 100 patients with histologically confirmed non-Hodgkin's lymphoma (NHL) and 80 leukemic patients by the EA-rosette forming cell inhibition assay. Sera from 55 healthy controls were also tested for the presence of IC. Using 9% rosette inhibition as a base-line, IC were observed to be present in 66 out of 100 sera from patients with NHL (66%), 35 out of 80 sera from patients with leukemia (43.7%) and 10 out of 55 control subjects (18%, p less than 0.001). The percentage of positive results was significantly lower in NHL patients with favorable prognosis (45%) than in patients with unfavorable prognosis (80%). IC from the sera of 7 ALL and 6 CML patients were investigated before chemotherapy, in remission and at relapse. The mean inhibitory rate of rosette inhibition was significantly higher in patients during the blastic stage of leukemia than during the complete remission (12.5%), and later it became higher again at the time of relapse. In CML patients, the previously normal serum rosette inhibition activity increased during the blastic crisis. These observations indicate that the follow-up studies of such patients may determine their prognosis accurately.