Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.     

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.     

Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.     

The expression of plasmalemma transcobalamin-II receptors on human blood lymphocytes stimulated by mitogens]

The influence of mitogens on the expression of surface membrane TC-II receptors of human blood lymphocytes and internalization of (TS-II+57Co-CNCbl) complex into cytoplasm were investigated. Mature lymphocytes have a very small number of surface receptors to plasma TC-II but their expression is increased significantly by PHA or Con-A stimulation. CBl transport to cytoplasma is activated in definite sequence by two different mechanisms. Stimulated cells take free CBl without participation of TC-II in early hours of mitogen action (12-42 hrs) before maximal 3H-thymidine incorporation into DNA. On day 3 of cultivation, specific mechanism of CBl transport triggers and the number of lymphoblast receptors is increased manifold. Radioactive CBl enters cytoplasma due to interaction of TC-II-CN [57Co] CBl of the medium with surface membrane receptor of the cells. Thus, the definition of TC-II receptors as an important functional parameter may serve a marker of proliferating cells.     

Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.     

Studies on rheomelanins. V. Hemolysis associated with the transformation of catechol into rheomelanin in human blood.

Incubation of 2 mg amounts of catechol in 5 ml samples of heparinated blood plasma from four subjects at 38 degrees C for 24 h produced plasma-soluble rheomelanins. These solutions had the brown color and the yellow-green fluorescence in ultraviolet light of 366 nm of other rheomelanins. Their differential ultraviolet and visible spectra showed a rheomelanin absorption maximum at 344 nm. Paper chromatograms of the rheomelanin-plasma solutions in 5% methanol-95% water showed elongated spots of rheomelanins with RF values of 0.82, on Whatman No. 1 paper. Using heparinated distilled water adjusted to pH 7.4 with sodium bicarbonate instead of human blood plasma gave markedly different findings from those obtained with the plasma rheomelanin solutions. Incubation of 4 mg amounts of catechol in 10 ml samples of heparinated whole blood from four subjects for 24, 32 and 48 at 38 degrees C produced rheomelanins as found in the plasma separated from the blood after incubation. The differential ultraviolet and visible spectra of these solutions revealed hemolysis caused by the catechol rheomelanins; this was more marked with longer incubations. The hemolysis was manifested by two absorption peaks at about 270 and 400 nm. Paper chromatography revealed the brown elongated spots of catechol rheomelanins with an RF value of 0.82. Other spots owing to the products of hemolysis were also present.     

The regulation of expression of the porin gene ompC by acid pH.

The regulation of expression of the porin genes of Escherichia coli by acid pH was investigated using reporter gene fusions. The ompC-lacZ gene fusion was expressed in response to acidification of the external medium. The kinetics of beta-galactosidase synthesis under acid-induction differed significantly from those obtained under conditions of osmotic stress. The latter led to rapid induction without a lag, followed by establishment of a rate that was equal to the growth rate; acid-induction was frequently preceded by a short lag period, was relatively slow and did not achieve a rate that was in balance with the growth rate. Further, induction of the ompC gene at acid external pH was dependent upon the presence of glucose as sole carbon source; growth with either glycerol or succinate as sole carbon source reduced induction of ompC at acid pH. Osmotic induction was independent of carbon source. The induction of the ompC gene at acid pH was also reduced by addition of cAMP to the growth medium. The porins are known to be subject to catabolite repression and our data are consistent with the exposure to acidic pH resulting in progressive changes in the state of catabolite repression. Acidification of the cytoplasm also provoked a rapid induction of the ompC-lacZ gene fusion. The kinetics of induction resembled the response to osmotic upshock. This response was independent of the identity of the carbon source supplied for growth. The contribution of changes in cytoplasmic pH to the induction of ompC at acid pH is discussed.     

Studies on the immunoglobulin V kappa locus in human lymphoid cell lines

DNA digests of 16 human lymphoid cell lines were studied in blot hybridization experiments with probes from V kappa genes and their immediate neighborhood as well as with single or low-copy probes from intergenic regions. The patterns were compared with those of placenta DNA digests in which the kappa genes are in the germline configuration. The differences of patterns which were detected with the first type of hybridization probes can be attributed to V kappa--J kappa rearrangements or to restriction site polymorphisms between individuals. Some of the pattern differences observed with the second type of probes can be interpreted best as arising from deletions of parts of the kappa locus. Such deletions may be individual variations but they may also be caused by the V kappa-J kappa rearrangement process. The results obtained with one particular probe which was derived from a nonduplicated part of the kappa locus allow some conclusions as to the mechanism of the V kappa--J kappa rearrangement: the genomic situation in some lymphoid cell lines can be explained by an inversion while in other cell lines clearly deletions have occurred. The observations are in agreement with the inversion-deletion mechanism of V kappa--J kappa rearrangement as proposed by Lewis et al. (1982, 1984).     

Synchronization of the human promyelocytic cell line HL 60 by thymidine

Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G1, S and G2 + M, respectively. The doubling time was 26 hr and the growth fraction was estimated as 1.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector.

We constructed a human cDNA expression vector by combining an episomal Epstein-Barr virus (EBV) vector with the expression cassette from the transient-expression vector, pCDM8. This new vector, designated pEBS7, exhibited high-level expression of reporter genes in normal and repair-deficient xeroderma pigmentosum cell lines. Reconstruction experiments indicated that marker genes diluted to a frequency of 10(-5) can be rescued on a single transfection dish. Moreover, derivative cell lines that constitutively express the gene encoding EBV nuclear antigen 1 exhibited a tenfold enhancement in the frequency of rescue of marker genes. The feasibility of preparing large-scale directional or nondirectional cDNA libraries in pEBS7 was demonstrated and reconstruction experiments indicated that marker genes could be rescued from either library with equal efficiency. These results establish a high-efficiency system for the isolation of genes by direct phenotypic selection in human mutant cell lines.     

The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation

The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1–3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1–3 displays an unusually low propensity to form a stem–loop structure, an effect potentiated by miR-BHRF1–3''s proximity to the BHRF1 polyA site. Cloning miR-BHRF1–2 or a cellular microRNA, but not a ribozyme, 5′ of miR-BHRF1–3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1–2 seed regions expressed miR-BHRF1–3 at normal levels and was fully transforming. Therefore, miR-BHRF1–2''s role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1–2 and miR-BHRF1–3 in EBV enhanced miR-BHRF1–3''s expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1–3 under the control of miR-BHRF1–2.