Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

Biodiversity among haptophyte algae

The division Haptophyta is represented only by about 300 extant species showing wide diversity in morphology, biochemistry and ecology. They have a world-wide distribution and are numerically important in phytoplankton populations in nearly all marine environments. Evidence from the geological record shows that they have been the major constituent of calcareous deposits since the Late Triassic and, as they have evolved quickly through time, their coccoliths have always shown wide morphological diversity. In today's oceans they occasionally produce extensive blooms, visible by satellite imagery, which have ecological impact. As a consequence of these blooms the haptophyte algae are now receiving greater attention, as their role in the global sulphur and carbon cycles may influence the world's climate, and their potential as nuisance bloom algae have implications for commercial fishing and the marine ecosystem. As it is likely that these organisms have always produced such blooms, these effects may have been in operation for the last 200 million years.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3–7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate β-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with β-carotene reduced the amount of cross-linking. We conclude that the high levels of β-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.     

Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3)

The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Structure and processing of precursor 5 S RNA in Drosophila melanogaster.

The 135-nucleotide-long “5 + S” RNA molecule found in Drosophila tissue culture cells after labelling at 37 °C has been identified as a precursor to 5 S RNA by pulse-chase experiments. The structure of the 15-nucleotide-long 3′-terminal sequence which differentiates this molecule from mature 5 S RNA has been determined. This ends in a stretch of U residues, suggestive of a polymerase termination signal.