Porphyrin-retinamides: synthesis and cellular studies

A series of four porphyrin-retinamides containing either all-trans- or 13-cis-retinoid acid residues, directly linked to the para-phenyl position of meso-tetraphenylporphyrin or via a low-molecular-weight PEG spacer, have been synthesized. The biological properties of these conjugates were evaluated in a model cell line, human HEp2, and in neuroblastoma SK-N-DZ cells, which exhibit moderate expression of retinoic acid receptors and retinoic acid-induced differentiation. The directly linked porphyrin-retinamides were taken up by a greater extent (20-50% more) in SK-N-DZ than in HEp2 cells. However, the PEG-containing conjugates accumulated maximally within both cell lines and approximately by the same amount, probably due to their increased amphiphilicity. Among all conjugates, the porphyrin-PEG-13-cis-retinamide accumulated the most in both cell lines (about 5 times more than the non-pegylated conjugates). None of the porphyrin-retinamide conjugates were toxic toward HEp2 cells at concentrations up to 100 microM, and only the hydrophobic non-pegylated conjugates were moderately toxic to SK-N-DZ cells [IC50 (dark) = 56-92 microM, and IC50 (at 1 J/cm2) = 6-8 microM]. All conjugates preferentially localized within cellular vesicles that correlated well to the lysosomes and, in addition, the PEG-containing porphyrin-retinamides were also found in the ER.     

Regulation and cellular localization of ent-kaurene synthesis

The two-step cyclization reaction of ent -kaurene synthesis from geranylgeranyl diphosphate is the first committed step in the biosynthetic pathway of the plant hormone gibberellin. Recent molecular cloning and characterization of the genes encoding the two corresponding enzymes, copalyl diphosphate synthase (CPS) and ent -kau-rene synthase (KS), have demonstrated that ent -kaurene synthesis is localized in the plastids and is highly regulated in specific tissues and cell types during plant development. In addition to occurring in actively growing tissues, ent -kaurene synthesis also takes place in fully expanded leaves. Therefore mature leaves may produce gibberellin intermediates or bioactive gibberellins for transport to responsive tissues. DNA sequence analyses have revealed a conserved aspartate-rich motif, D(I/V)DDTA among CPS and other protonation-initiated terpene cyclases, while KS contains a highly conserved DDXXD motif which was proposed to function as a divalent metal ion-diphos-phate complex binding site in ionization-initiated terpene cyclases and prenyltrans-ferases.     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

Anti-idiotypic antibody identifies the cellular receptor of reovirus type 3

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.     

Hydrostatic pressure-induced changes in cellular protein synthesis

Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.     

Stimulation of cellular DNA synthesis by human cytomegalovirus

Human cytomegalovirus (CMV) is able to induce cellular DNA synthesis in both permissive (human embryonic lung) and nonpermissive (Vero) cells. The induction of cell DNA synthesis was assayed by the incorporation of [methyl-³H]thymidine into macromolecules having the buoyant density characteristics of cell DNA. The DNA synthesis induced by CMV infection appears to represent normal semiconservative replication as opposed to repair synthesis. Both strains of CMV tested were capable of inducing cell DNA synthesis. Virus exposed to heat or UV light prior to infection lost the ability to induce DNA synthesis, indicating that a virus-coded function expressed after infection is responsible for stimulation of cell DNA synthesis.