Effects of human presenilin 1 isoforms on proliferation and survival of rat pheochromocytoma cell line PC12.

Missense mutations in human presenilin 1 gene (hPS1) cause an autosomal dominant, early onset form of Alzheimer's disease (AD). To study effects of mutant presenilin on processes of cell growth, differentiation, and susceptibility to apoptotic signals, we produced a series of rat pheochromocytoma PC12 poly- and monoclonal cell lines stably expressing wild type hPS1 and hPS1 with mutations in amino (N-) and carboxyl (C-) terminal regions of the PS1 protein. Employing a heterologous rat PC12 cell system, we demonstrated that: 1) AD mutations inhibit, in part, processing of hPS1 holoprotein; 2) negative selection against highly expressed hPS1 may occur in polyclonal cell cultures; 3) expression of N-terminus mutant (M146V) hPS1 increases susceptibility to apoptosis in differentiated neuronal PC12 cells under deprivation conditions; 4) monoclones with hPS1 C-terminal AD mutation (C410Y) have lower proliferation rates than monoclones expressing wild type hPS1 under deprivation conditions and during NGF-induced neuronal differentiation. The data demonstrate deleterious effect of PS1 AD mutations. The effect depends on the level of expression of the hPS1 isoforms, the number of passages, and trophic and differentiation conditions used for growing PC12 cells.

A reporter system for prokaryotic and eukaryotic cells based on the thermostable lichenase from Clostridium thermocellum.

The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.

[Characteristics of arrhythmia and late ventricular potentials in patients with cardiological syndrome X]. / Kharakteristika narushenii serdechnogo ritma i pozdnie potentsialy zheludochkov u bol'nykh s kardiologicheskim sindromom X.

AIM: A comparative study of late ventricular potentials (LVP) in patients with cardiological syndrome X (SX) and stenotic atherosclerosis of coronary arteries (ACA) as well as their relations with arrhythmia, cardiac contractility, lipid metabolism and morphological characteristics of the myocardium. MATERIAL AND METHODS: The examination of 52 SX and 77 ACA patients as well as 17 healthy subjects included coronaroventriculography, bicycle exercise, 24-h ECG monitoring, echocardiography, signal-averaged high-resolution (SAHR) ECG, investigation of blood lipoproteins. Endomyocardial biopsy was made in 5 ACA and 5 SX patients. RESULTS: No differences were registered between SX and ACA patients by frequency and severity of arrhythmic episodes, percentage of patients with registered LVP, quantitative parameters of SAHR ECG. Frequency of high-gradation ventricular arrhythmia episodes was significantly higher in SX and ACA patients with LVP than such patients free of LVP. SX patients had correlation between parameters of SAHR ECG, myocardial contractility and lipid metabolism. Foci of diffuse cardiosclerosis are most probable anatomic substrate of LVP. CONCLUSION: The risk of prognostically unfavourable high-gradation ventricular arrhythmia episodes in SX and ACA patients is the same. LVP may predict severe ventricular arrhythmia episodes both in SX and ACA patients.

[Effects of human immunodeficiency virus type 1 nef and tat genes on rat PC12 pheochromocytoma cells]. / Effekty genov nef i tat virusa immunodefitsita cheloveka tipa 1 na kletki feokhromotsitomy krysy PC12.

The regulatory genes nef and tat of the human immunodeficiency virus type 1 (HIV-1) were transferred into the rat pheochromocytoma cells (line PC12) under the control of the eukaryotic promoters. Proliferative activity of the PC12 cells transfected with the tat HIV-1 gene was substantially increased as compared to the control. Conversely, the nef gene introduced into the cultivated PC12 cell caused inhibition of their proliferative activity and formation of cell agglomerates resembling in morphology the multinuclear syncytial cells. Thus, our results suggest that the tat gene activates proliferation of the cultivated PC12 cells, whereas the nef gene inhibits proliferation of the same cells. We have obtained for the first time a direct indication for the possible role of the nef gene in formation of multinuclear T-lymphocyte and macrophage syncytium in HIV-1-infected patients. The HIV-1 nef and tat genes had no significant effect on the neuronal differentiation of the PC12 cells induced by the nerve growth factor (NGF).

[Characteristics of mutants induced by the c-Ha-ras1 oncogene and the nature of the oncogene's mutagenic action]. / Kharakteristika mutantov, indutsirovannykh onkogenom c-Ha-ras1, i priroda mutagennogo deistviia onkogena.

Chinese hamster cell clones of independent origin, which were resistant to purine base analogs and induced by the activated c-Ha-ras1 oncogene, were isolated. It was shown that the isolated clones stably retained resistance after cultivation on a medium without an analog, confirming mutational nature of the resistance. Most of the clones are able to grow on the HAT medium, retaining partial activity of the hypoxanthine phosphoribosyltransferase enzyme (HPRT); i.e., they are leaky mutants. Analysis by blot-hybridization did not reveal the presence of human ras-sequences in any of the mutants studied. Evidently, the mutagenic action of the oncogene is not insertional, and resistance is not linked to the stably integrated oncogene. The mutagenic effect of c-Ha-ras1 is likely to be of the "hit-and-run" type.

[Plasmid pSVc-myc-1 induces gene mutations and chromosome aberrations in cultured Chinese hamster cells]. / Plazmidnye pSVc-myc-1 indutsiruet gennye mutatsii i khromosomnye aberratsii v kul'tiviruemykh kletkakh kitaiskogo khomiachka.

The induction of mutations to 6-thioguanine-resistance and chromosome aberrations by the plasmid pSVc-myc-1, carrying the activated cellular oncogene c-myc, isolated from a mouse plasmocytoma was studied in a cultured Chinese hamster cell line. The yield of HPRT- mutants and chromosome aberrations increased 1.6 times on the average after pSVc-myc-1 treatment. The mutagenic activity of pSVc-myc-1 was statistically significant. The role of mutagenic effects of activated cellular oncogenes in malignant transformations is discussed.

[Role of activated cellular c-Ha-ras-1 oncogene in the mutagenic effect of the plasmid pEJ6.6]. / Rol' aktivirovannogo kletochnogo onkogena c-Ha-ras-1 v mutagennom éffekte plazmidy pEJ6.6.

The role of the activated oncogene c-Ha-ras-1 from human bladder carcinoma integrated into the pEJ6.6 plasmid in the mutagenic effect of the plasmid was studied in Chinese hamster cells. The frequency of hypoxanthine-phosphoribosyltransferase defective (HPRT-) mutants after treatment with pEJ6.6 containing an active c-Ha-ras-1 exceeded that in control dishes treated with a derivative of pEJ6.6 plasmid with an inactivated oncogene. The inactivation was achieved by introducing a deletion into the coding region of the oncogene. The mutagenic effect was rather weak but statistically significant. Thus, the data obtained show that the mutagenic activity of pEJ6.6 plasmid is determined by its oncogene. The role of mutagenic effects of activated cellular oncogenes in malignant transformation is discussed.

[The plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene increases the mutation frequency in Chinese hamster cells]. / Plazmida pEJ6.6, nesushchaia aktivirovannyi onkogen c-Ha-ras-1, povyshaet chastotu mutatsii v kletkakh kitaiskogo khomiachka.

The induction of gene mutations and chromosome aberrations by the plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene from human bladder carcinoma was studied in cultured Chinese hamster cells. Both an increase in the frequency of gene mutations to 6-mercaptopurine resistance and of chromosome aberrations was observed after pEJ6.6 treatment as compared to control series (pBR322). Thus the results of experiments carried out show that the pEJ6.6 plasmid possesses a mutagenic activity.