1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Analysis of Chlamydomonas reinhardtii histones and chromatin

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.     

Biochemical changes of rat brain membranes with aging

Modification of membrane composition and enzymatic activities both in total brain homogenate and purified synaptic plasma membrane of 3 and 24 month old rats has been investigated. Protein, cholesterol and phospholipid content and (Na+, K+)ATPase and 2',3' cyclic nucleotide phosphohydrolase activities were determined. The major changes occurred in the whole homogenate where a general increase in total protein and cholesterol content with age and a significant increase of the cholesterol/phospholipids molar ratio has been detected. In S.P.M. aging process induced a decrease of protein, cholesterol and phospholipids content associated with an increased membrane viscosity and a decrease of delta E. These data are consistent with a change in the structural organization and in the distribution pattern of different cell population in the aging brain. A possible artifactual effect of freezing on the reported parameter is also discussed.     

Constitutive expression of FosB and its short form, FosB/SF, induces malignant cell transformation in rat-1A cells

We have established Rat-1A cell lines constitutively expressing c-Fos and the two products of the fosB gene, FosB and its short form, FosB/SF. The expressed proteins in the different stable transfectants have been characterized by immunofluorescence and immunoprecipitation analysis. Our results demonstrate that constitutive expression of FosB, like the constitutive expression of c-Fos and, to a lesser extent, FosB/SF, results in cells that grow to increased saturation densities and have the ability to grow in an anchorage-independent manner. Most important is the finding that expression of these proteins augments the tumorigenic potential of Rat-1A cells. These results show that both forms of FosB have a stimulatory effect on cell proliferation.     

DNA repair investigations in nine Italian patients affected by trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients. Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents. Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5-15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells. The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.     

Co-oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co-oligopeptides of tryptophan and glycine

The ¹H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0–2) and those of several di- and tripeptides have been recorded at 360 MHz with CD₃OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(αL,βS)(α,β-d₂)Trp-OH, H-Trp-(αL,βS)(α,β-d₂)Trp-OH, and H-Trp-(δ¹,ε²,ζ²,ζ³,η²-d₅)Trp-OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ₁ and (in two cases) χ₂ torsion angles of the side chains has been obtained from the vicinal coupling constants ³J and from the long-range coupling constants ⁴J. These data and an analysis of the chemical shifts of the Gly-C^α protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N- and C-side: -NH₂ > –NHCO– > –CONH–> –COO^?. This rule does not hold for the second Trp residue of di- and tripeptides containing the -Trp-Trp- sequence, which has tentatively been attributed to steric effects.