Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes.

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels.

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.

Lentil lectin enriched microsomes from the plasma membrane of the human B-lymphocyte cell line H2LCL carry a heavy load of type-1 porin.

Using an established biochemical approach, five subcellular fractions of human B lymphocytes were prepared by differential centrifugation. Crude membranes were passed over a lentil lectin column to enrich carbohydrate-coated cell surface microsomes. The lectin-bound fraction contained a high amount of plasma membrane-derived microsomes as indicated by cell surface markers. All subcellular fractions in Western blots proved to contain distinct but variable amounts of porin. There was a strong increase in porin content from crude membranes to plasma membrane-derived vesicles. The porin content of this fraction appeared to be higher than that of mitochondria. In the final step the plasma membrane-derived microsome fraction proved to be devoid of contamination by outer mitochondrial membranes, as revealed by antibodies against the established markers MAO B and Tom20 applied in Western blots. These data prove the extramitochondrial expression of human type-1 porin/ type-1 VDAC.

Purification and characterization of two putative HLA class II associated proteins: PHAPI and PHAPII.

In addition to their well defined role in presentation of processed antigen on the cell surface, class II molecules are able to transduce signals into the cell after binding of ligands. The cytoplasmic regions of class II molecules might function as docking sites for as yet unidentified proteins that are components of this signalling pathway. Here we report on two putative HLA class II associated proteins (PHAPI and PHAPII) which have been purified from the cytosolic fraction of the human lymphoblastoid B-cell line H2LCL using an affinity matrix composed of the synthetic biotinylated cytoplasmic region of the DR2 alpha chain immobilized on avidin agarose. The sequence obtained for PHAPI revealed a novel primary structure with a leucine/isoleucine rich N-terminal region. Protein data and the cDNA sequence obtained for PHAPII agree with the cDNA sequence of SET that has been described recently. Both PHAPI and PHAPII have an extended highly acidic C-terminal region. Based on their primary structure we speculate that PHAPI and PHAPII are involved in the generation of intracellular signalling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules.

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro.

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibacterial and antiproliferative activities of vulpinic acids in vitro.

The antimicrobial and antiproliferative activities of vulpinic acids (1 a, b, c) have been assayed in vitro. Activity was demonstrated by vulpinic acids on Gram-positive bacteria only. The MIC values of these compounds were found to be ranging from 3.8-31.5 micrograms/ml. The significance of these results is discussed.

Synthesis and biological evaluation of N-salicyloyl-N-benzyl thiourea and 2,2-dimethyl-4-oxo-6-methoxy benzo-1,3-dioxin.

The synthesis of N-salicyloyl-N-benzyl-thiourea and 2,2-dimethyl-4-oxo-6-methoxy-benzo-1,3-dioxin are described. They were studied for their antiviral, antiproliferative and antimicrobial activities in vitro. N-salicyloyl-N-benzyl-thiourea exhibited significant activity against Gram-positive bacteria and against influenza viruses types A/Philippine/H3N2, A/Chilli/H1N1 and B/Paraha, as well as against K562 cell proliferation. By contrast, no such activity was demonstrated by 2,2-dimethyl-4-oxo-6-methoxy-benzo-1,3-dioxin.