13C- and 31P-NMR studies of the conformation of carcinogen-modified nucleic acid dimers

The effect of the carcinogen acetylaminofluorene (AAF) on nucleic acid structure was examined using ¹³C- and ³¹P-NMR spectroscopies. Conformational effects were compared in two AAF-modified dinucleoside monophosphates (ApG and GpA) and two AAF-modified deoxydinucleotides (dpApG and dpGpA). Changes in adenine ¹³C chemical shifts on formation of the AAF-adduct and as a function of temperature provided evidence of base stacking. Differences in fluorene ¹³C chemical shifts between the AAF-modified dimer and AAF-modified monomer provided evidence of fluorene stacking. The effect of forming the adduct on the phosphate backbone was examined using ³¹P-NMR. A correlation was demonstrated between the degree of adenine-fluorene stacking on one hand and the change in conformation of the backbone conformation on the other.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

The case for a polyphyletic origin of mitochondria: Morphological and molecular comparisons

The comparative morphology and pigmentation of protists suggest that those with tubular mitochondrial cristae belong to a different lineage than those with lamellar cristae and that the evolutionary divergence might have been very early. We propose that the difference in cristal morphology is the result of separate origins of the mitochondria from endosymbionts related to the Rhodospirillaceae (purple nonsulfur bacteria) but differing in the morphology of their internal membranes. Comparisons of the cytochromes c of protists and the Rhodospirillaceae and of 16s rRNA T1 oligonucleotide catalogs in the Rhodospirillaceae do not contradict, and in fact provide support for, the idea. More extensive evidence may be lacking simply because cytochromes c have been studied in very few protists with tubular mitochondrial cristae.     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

The use of embryo culture for the recovery of plants from cassava (Manihot esculenta Crantz) seeds

Cassava fertility and seed viability are frequently low, which can be a disadvantage in a breeding programme. An embryo culture method is described whereby embryonic axes are excised from mature seeds and placed on a culture medium containing 1.23 M indolebutyric acid (IBA) at 30°C under continuous light. The number of plants recovered by embryo culture was much greater than the number recovered from conventional seed germination procedures.     

Differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture

To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of ₁-protease inhibitor, ceruloplasmin, and ₂-macroglobulin, but had no effect on the export of fibronectin, -fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized ₁-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized ₁-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on ₁-protease inhibitor, ceruloplasmin and ₂-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.