Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

Minor lamin polypeptides from rat liver nuclei can be cross-linked into heteropolymers by disulfide bridges

The peripheral lamina of rat liver nuclei is characterized by the presence of three major polypeptides called lamins A, B, and C. Recent studies have identified in rat liver lamina two quantitatively minor polypeptides that have some of the biochemical and immunological properties of the lamins and were tentatively called minor lamin species. We have further characterized these minor lamin polypeptides. Both minor lamin species copurified quantitatively with the major lamins in dissociation-reassociation experiments and shared epitopes with all three major lamins as well as with intermediate filament proteins, including an epitope involved in coiled-coil interactions in lamina and filaments. Minor lamins generated partial peptide maps very similar to each other but completely different from those of lamins A, B, and C. The two minor lamin species could be cross-linked into heteropolymers containing a constant ratio of both polypeptides by exposure to O-phenanthroline - cupric ion complexes, although they did not appear to be cross-linked by disulfide bonds in the native envelope. Preliminary results suggest that the cross-linked minor lamins could be preferentially associated with lamin B. It therefore appears that in addition to the network of lamins A, B, and C, the peripheral lamina is characterized by the presence of two closely juxtaposed minor lamin polypeptides. The molecular interactions between these various polypeptides and their respective roles remain to be identified.     

CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (>99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.     

Nuclear processing of the 3''-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes.

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.     

Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomnnose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B withA. saitoi (1–2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.     

Detection of the gangliosideN-glycolyl-neuraminyl-lactosyl-ceramide by biotinylatedEscherichia coli K99 lectin

K99 lectin fromEscherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay. Abbreviations: NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid; PBS, phosphate buffered saline (0.9% NaCl containing 150mm sodium phosphate, pH 7.2); LPS, lipopolysaccharide; BCHZ, biocytin hydrazide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle medium. For the gangliosides, trivial names and structures are given according to the recommendations in [43]. NeuAc2-3Gal1-4Glc1-1Cer (NeuAc-GM₃); NeuGc2-3Gal1-4Glc1-1Cer (NeuGc-GM₃); GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₂); NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer (GD3); Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₁); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GD_1a); Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Gle1-1Cer (GD_1b); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3) Gal1.-4Glc1-1Cer (GT_1b). NeuGc2-3Gal1-4GleNAc1-4Gal1-4Glc1-1Cer (NeuGc-SPG).     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.