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.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Interleukin 1β-converting Enzyme Related Proteases/Caspases Are Involved in TRAIL-induced Apoptosis of Myeloma and Leukemia Cells

The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNFfamily and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1β-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor AcYVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO.

These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

     

Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5′ to 3′ direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.     

On the multiplicity of platelet prostaglandin receptors: II. The use of N-0164 for distinguishing the loct of action for PGI2, PGD2, PGE2 and hydantoin analogs

The ω-chain variant analogs of prostacyclin (PGI₂) and PGD₂ in which the n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1)(1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI₂, but converts PGD₂ to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD₂ by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD₂, cyclohexyl-PGD₂, and BW-245C; with essentially no effect on PGI₂, cyclohexyl-PGI₂ and PGE₂ at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre (2,3), but supports the view that the anti-aggregatory effect of high doses of PGE₂ ($\text{EC}{}_{50}\text{=50μ}\text{M}$) is mediated by the PGI₂ receptor (4). The hydantoin acts at the platelet PGD₂ receptor.     

Extracellular enzymes of some additional fungi associated with mushroom culture

Twenty-four fungus isolates from the compost utilized in commercially growing Agaricus brunnescens were tested for their ability to produce extracellular enzymes involved in the degradation of cellulose, lignin and xylan, the major components of the straw of the compost. All 24 isolates were able to degrade carboxymethyl cellulose. Most were classified as weak or moderate producers of exo--glucanase. Twenty of the 24 were also able to hydrolyze filter paper, a crystalline cellulose. Nineteen of the 24 were able to hydrolyze xylan, a hemicellulose. The production of extracellular polyphenol oxidases was detected utilizing two tests; the blueing of alcoholic gum guaiacol, which indicates tyrosinase production, and the browning of malt extract-gallic acid agar, which indicates laccase production. Twenty produced tyrosinase, but only eight produced laccase. Agaricus brunnescens was also included in all of the tests. It produced exo--glucanase, hemicellulase, tyrosinase and lactase.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Social behavior of captive,group-living Orangutans

The behavior of orang-utans (Pongo pygmaeus)was observed in two captive groups (one adult group, one juvenile group). Activity profiles,animal interactions, and compartmental spatial use for both adult-group and juvenile-group individuals were recorded over a 9-month period. Behavioral repertoires for both groups included large amounts of social activity. Equivalent amounts of social activity were found for each group. The social behavior of juvenile animals involved more active behavior such as play. The social behavior of the adult animals was more subtle, involving social monitoring and allogrooming. These results indicate that orangutans, at least when group-living in captivity, exhibit the potential to display social behavior which is apparently of greater frequency and complexity than that which has been observed in the wild. These findings suggest that the solitary behavior of wild orangutans is not a necessary characteristic of orangutan behavior. Under different environmental conditions orangutans appear to readily adapt socially, and, like other nonhuman primates,they have the capacity to exhibit complex and subtle social behavior. This report is based on part of a senior thesis submitted by Sara D. Edwards     

Polyamines and protein synthesis: Studies in various polyamine-requiring mutants ofEscherichia coli

Summary DifferentEscherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. Thein vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutants MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.Dedicated to ProfessorLuis F. Leloir on the occasion of ths 70^th birthday.     

Characterization of glycerol nonutilizing and protoperithecial mutants ofNeurospora

Summary Mutants defective in polyol metabolism and/or in protoperithecial development were selected inNeurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed intoN. crassa to facilitate genetic analysis. One,glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, betweenad-1 andrib-1; the other,glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal toad-9. Another mutant,gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal toad-9.