Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.     

Demonstration of T-cell-dependent and T-cell-independent components of 8-mercaptoguanosine-mediated adjuvanticity

The contribution of T cells to potentiation of humoral immunity by the C8-substituted guanine ribonucleosides and the origin of the increased numbers of antigen-responsive B cells generated consequent to their action have been investigated. Augmentation of the antigen-specific antibody response by these nucleosides, exemplified by 8-mercaptoguanosine (8MGuo), can be separated into T-cell-dependent and T-cell-independent components, both by use of the T-cell tropic immunosuppressive agent, cyclosporin A, and by experiments using separated populations of T and B cells. Augmentation of adjuvanticity by T cells is hypothesized to involve a B-cell subpopulation not otherwise subject to the action of 8MGuo. This subpopulation could potentially arise by either of two mechanisms, one whereby preexisting antigen-specific B cells undergo clonal expansion, and one in which cells not normally participating in the response are recruited in the absence of clonal expansion. Although the former mechanism makes a minor contribution to adjuvanticity, the latter mechanism appears to be the dominant one, insofar as models in which 8MGuo-induced proliferation fails to occur (such as after irradiation, or in the SJL mouse) nonetheless exhibit strong adjuvant effects. Analysis of precursor frequency of antigen-specific B cells indicates that for each mature, antigen-responsive B cell present in adult murine spleen, an average of four additional cells can be recruited by the conjoint actions of antigen and 8MGuo. One group subject to such recruitment is the immature antigen-specific B cell, whose degree of functional maturity is accelerated in the presence of antigen and 8MGuo.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Effects of pentobarbital anesthesia on tonic luteinizing hormone secretion in the ewe: evidence for active inhibition of luteinizing hormone in anestrus

In the ewe, seasonal anestrus appears to result from two effects of inhibitory photoperiod: 1) estradiol gains the capacity to suppress luteinizing hormone (LH) pulse frequency and hence becomes a potent inhibitor of tonic LH secretion and 2) a steroid-independent decrease in LH pulse frequency occurs in ovariectomized ewes. In this study, we have obtained evidence, using pentobarbital anesthesia, that both these actions of photoperiod reflect the activation, in anestrus, of an inhibitory neural system. Administration of pentobarbital to intact anestrous ewes produced a dramatic, 3-fold increase in LH pulse frequency during the 6 h of anesthesia. In contrast, during the breeding season, pentobarbital inhibited LH pulse frequency in luteal phase animals. There was also a seasonal variation in the effects of pentobarbital in ovariectomized ewes. During the breeding season this drug again suppressed LH secretion, inhibiting both LH pulse amplitude and frequency. In anestrus, pentobarbital also suppressed pulse amplitude, but it produced a transitory increase (lasting 3 h) in pulse frequency. To account for the stimulatory actions of pentobarbital, we propose that in anestrus, but not the breeding season, LH pulse frequency is held in check by a set of estradiol-sensitive inhibitory neurons. Further, we suggest that these neurons are activated by inhibitory photoperiod and account for both the steroid-dependent and steroid-independent actions of photoperiod.     

Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii

Candida yeasts rapidly form peroxisomes of simple function and composition when grown on methanol. Because the induction is both massive and rapid, this system may be useful for a detailed dissection of peroxisomal biogenesis. We report procedures to express peroxisomal proteins in cells and spheroplasts of Candida boidinii to stabilize peroxisomes in a lysate of spheroplasts and to obtain an enriched peroxisomal fraction. With these techniques we have been able to study the assembly of alcohol oxidase, a homo-octomeric flavoprotein, into the organelle in vivo. The primary translation product of alcohol oxidase comigrates on sodium dodecyl sulfate-polyacrylamide gels with the mature subunit. Pulse-chase experiments indicate that the newly synthesized monomer of alcohol oxidase has a half-life of about 20 min in intact cells and 13 min in spheroplasts before conversion to octomer. The monomer first appears in a high speed supernatant of a lysate of spheroplasts and then chases into a purified peroxisomal fraction before or during its octomerization. There is no detectable intermediary organelle involved in this process.     

Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

The present study is one component of a comprehensive investigation of oxygen tolerance of tissues and organs in normal human subjects. The focus of this study was the acylation of membrane phospholipid in situ by erythrocytes. Activation of exogenous [9,10-3H]oleic acid to acyl thioester and transesterification of the acyl thioester into phospholipid by intact human erythrocytes incubated in vitro decreased 30% after exposure of 10 human subjects to hyperbaric hyperoxia (100% O2, 3 ATA, 3.5 h). Partial recovery of activity could be detected when additional cells were obtained from these subjects and assayed in vitro 24 h after cessation of exposure. No significant change in membrane phospholipid fatty acid composition was detected under these conditions. The reduced glutathione content of intact erythrocytes increased by 15% after hyperbaric hyperoxia and remained elevated 24 h after exposure. In isolated membranes prepared from the same cells activation of [9,10-3H]oleic acid to acyl thioester and its transesterification into phospholipid did not change after hyperoxia. Since the ability of intact cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be necessary for the maintenance of membrane integrity during exposure to oxidative stress, the decrease in [9,10-3H]oleic acid incorporation by human erythrocytes detected in vitro after hyperbaric hyperoxia in vivo may reflect an early event in the pathogenesis of oxygen-induced cellular injury and may be a useful index for assessment of the tolerance of tissues to hyperoxia.     

Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes

Summary The genetic control of hexokinase isozymes (ATP: d-hexose-6-phosphotransferase, E.C. 2.7.7.1, HEX) in maize (Zea mays L.) was studied by starch gel electrophoresis. Genetic analysis of a large number of inbred lines and crosses indicates that the major isozymes observed are encoded by two nuclear loci, designated Hex1 and Hex2. Five active allozymes and one null variant are associated with Hex1, while Hex2 has nine active alleles in addition to a null variant. Alleles at both loci govern the presence of single bands, with no intragenic or intergenic heteromers visible, suggesting that maize HEX's are active as monomers. Organelle preparations demonstrate that the products of both loci are cytosolic. All alleles, including the nulls, segregate normally in crosses. Vigorous and fertile plants were synthesized that were homozygous for null alleles at both loci, suggesting that other hexosephosphorylating enzymes exist in maize that are undetected with our assay conditions. Linkage analyses and crosses with B-A translocation stocks place Hex1 on the short arm of chromosome 3, 27 centimorgans from Pgd2 (phosphogluconate dehydrogenase) and Hex2 on the long arm of chromosome 6, approximately 45 centimorgans from Pgd1. It is suggested that the parallel linkages among these two pairs of duplicated genes reflects an evolutionary history involving chromosome segment duplication or polyploidy.Paper No. 10170 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.     

Isolation of Mycobacterium avium-intracellulare-scrofulaceum complex from faeces of patients with AIDS.

Thirty faecal specimens from 22 patients with the acquired immune deficiency syndrome were examined by microscopy after Ziehl-Neelsen staining and by culture after decontamination with sodium hydroxide. Thirteen specimens (from 11 patients) were positive for Mycobacterium avium-intracellulare-scrofulaceum on culture, and only five of these on Ziehl-Neelsen staining. Five of the 11 patients had evidence of disseminated infection. Lipid analysis showed six of the nine strains tested to be indistinguishable. These findings support the theory that the gastrointestinal tract is a portal of entry for the organism.