Free amino acids from pollens

Pollens from Pinus canariensis, P. nigra, P. pinaster, P. pinea, Castanea sativa, Magnolia grandiflora, Olea sativa cv frantoio, cv itrana, cv pisciottana were examined for their free amino acid composition. A large amount of proline was found in all species; pollens of Olea also contain a large amount of serine.     

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.     

Synthesis of the marine epoxy sterol 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol

The synthesis of 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol (1), a highly oxygenated marine sterol containing a 9,11-epoxide moiety in the nucleus, is described. Epoxy sterol 1 was synthesized from cholesta-5,7-dien-3 beta-ol. Oxidation of this sterol with m-chloroperbenzoic acid followed by hydrolysis and acetylation furnished 5 alpha-cholest-7-ene-3 beta,5,6 alpha-triol 3,6-diacetate (2). Mercuric acetate dehydrogenation of diacetate 2, followed by oxidation with manganese dioxide and epoxidation with m-chloroper-benzoic acid, afforded 9 alpha,11 alpha-epoxy-3 beta,5-dihydroxy-5 alpha-cholest-7-en-6-one (5). Reduction of 5 with lithium aluminum hydride gave the desired compound 1. The structures of all synthetic intermediates were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A reassignment of resonances for carbons 1, 8, and 15 in the 13C NMR spectrum of 1, based on 2D-NMR correlation spectroscopy, has been accomplished.     

Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation     

THE CALCAREOUS RESTING CYST OF PENTAPHARSODINIUM TYRRHENICUM COMB. NOV. (DINOPHYCEAE)1

While investigating dinoflagellate cyst assemblages in surface sediments of the Gulfs of Naples and Salerno (Mediterranean Sea), we found a new calcareous resting cyst. This cyst has a round to oval body surrounded by a thick mineral layer, which gives it the shape of a Napoleon hat, with a flat, oval face bearing the archeopyle and a convex keel on the opposite side. The cyst shape is variable in both natural samples and clonal cultures. The organic membrane underlying the calcareous covering is resistant to acetolysis, thus demonstrating the presence of sporopolleninlike material. The cyst germinated into a motile stage having the same morphological features and thecal plate pattern as Peridinium tyrrhenicum Balech. We believe the validity of the genus Pentapharsodinium Indelicato & Loeblich should be accepted. Based on the comparative examination of the species we collected and of a similar species, Pentapharsodinium trachodium Indelicato & Loeblich, we propose Pentapharsodinium tyrrhenicum as a new combination for Peridinium tyrrhenicum. The genus Pentapharsodinium also includes P. dalei Indelicato & Loeblich (= Peridinium faeroense Dale), which produces spiny, organic-walled cysts. The presence of species forming calcareous cysts and species producing noncalcareous cysts in the same genus raises questions about maintaining the family Calciodinellaceae. This family should only include calcareous cyst-forming peridinioids, in order to maintain a unified system of classification of fossil and recent dinoflagellates.     

A specific microassay for evaluating hepatic LDH activity in co-cultures of hepatocytes with other cells

This study describes the development of a simple, rapid and reproducible microassay for determining the intracellular LDH activity of rat hepatocytes present in a co-culture system with other cells. The procedure involves treatment of cellular homogenates with an anti-LDH antiserum that specifically inhibits the LDH activity of rat hepatocytes. The assay is performed in 96-well plates and LDH activity can be measured directly in the same wells using a colorimetric method. The difference in LDH activity values measured before and after antiserum incubation reflects the LDH content of the hepatocytes in the sample. The advantages of this method are the small number of cells required, a reduction in sample handling and the possibility of differentiating LDH activity in hepatic and non-hepatic cells. The possible applications of this technique as a parameter for biochemical data and as a test for cytotoxicity studies in co-cultures are also discussed.     

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.     

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.     

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V)

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 microM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.