Cellular compartments of GABA in brain and their relationship to anticonvulsant activity

Summary The effects of GABA-elevating agents were examined with respect to the cellular compartments in which GABA increases occurred and the brain region(s) that mediate the anticonvulsant activity of these compounds. Changes in GABA occurring in the presence and absence of GABAergic nerve terminals were estimated in vivo using rats in which the GABA projection to the substantia nigra (SN) was destroyed on one side of the brain. One week post-operatively, the GABA concentration in the denervated SN was 10–20% of control. The net increase in GABA content of the denervated SN was compared to that of the intact SN after intraperitoneal injection of amino-oxacetic acid (AOAA), di-n-propylacetate (DPA) and -vinyl GABA (GVG). In the intact SN, all drugs produced significant increases in GABA. In the denervated SN, both AOAA and GVG produced marked increases in GABA (nearly equivalent to those obtained in the intact SN) whereas DPA was without effect. It therefore appears that the DPA-induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA and GVG primarily elevate GABA in non-nerve terminal compartments. An increase in GABA associated with nerve terminals was obtained with GVG only after a latency of more than 12 h following a single injection. The time course of elevation of nerve terminal-associated GABA coincided with the time course of anticonvulsant action of GVG; both effects were maximal at 60 h after a single injection. Taken together, our results indicate that the ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures is directly correlated with increases in nerve terminal GABA and not related to increases in other GABA compartments.Localization of the anatomical site that mediates anticonvulsant activity was examined using intracerebral injections of GVG into fore-, mid-and hindbrain areas. Blockade of tonic hindlimb extension in the maximal electroshock test and blockade of tonic and clonic seizures produced by pentylenetetrazol and bicuculline was obtained by microinjection of GVG (10 µg) into the ventral tegmental area of the midbrain. Injections of GVG (10–40 µg) into forebrain areas (striatum, thalamus) or into hindbrain (pontine tegmentum) were without anticonvulsant activity. Anticonvulsant effects of midbrain GVG were correlated with GABA elevation (3–4 fold) within a 1.5 mm radius of the injection site; these effects were obtained within 6 h and lasted three to four days after a single treatment. After four days seizure activity returned to control. No changes in spontaneous motor activity or reflexes accompanied the GVG injections. Similar but shorter lasting anticonvulsant effects were obtained with the direct GABA receptor agonist muscimol (50 ng) injected into the midbrain site. On the other hand, doses of muscimol up to 500 ng placed in the rostral pontine tegmentum were without anticonvulsant effect, despite the appearance of marked sedation.The time to peak anticonvulsant activity after midbrain microinjection of GVG (6 h) was considerably more rapid than that after intraperitoneal injection (60 h). Compartmental analysis revealed that nerve terminal associated GABA was elevated by 6 h after GVG when the direct microinjection route was used. These results suggest that GABAergic synapses in the midbrain may be critically involved in the control of seizure propagation.     

Lack of effect on the chromosomal non-disjunction in aged female mice after low dose X-irradiation.

Karyotypes were determined in 1064 embryos of aged C57/BL mothers. The virgin female mice were irradiated with 0, 4, 8 or 16 R of X-rays, respectively, and placed with young untreated males 5 days after irradiation. 10.5-days old embryos were recovered from the uterus. Aneuploid embryos classified as alive (heart beats observed at the dissection) were 1 monosomic in the control group (496 embryos) and 2 trisomics in the irradiated group (568 embryos). The number of aneuploid embryos classified as dead was 4 trisomic cases in the control group and 3 trisomics in the irradiated group. The data indicate that trisomic embryos are not uncommon in the mouse but are eliminated in post-implantation death. In contrast to the results of Yamamoto et al. the present data do not demonstrate an increased frequency of chromosome abnormalities in embryos of aged mice X-irradiated before mating as compared to non-irradiated ones.     

Biogenesis of mitochondria 49 identification and mapping of a new mitochondrial locus (tsr1) which maps within polar region of yeast mitochondrial genome.

Summary An enrichment procedure which facilitates the isolation of conditional respiratory-deficient mutants of Saccharomyces cerevisiae is reported. Detailed genetic analysis of one mutant which exhibits a respiratory deficient phenotype at low temperature (18°C) is also presented. The phenotype is due to a single lesion at a new locus, tsr1, located on the mitochondrial DNA. By analysis of locus retention patterns in a set of physically characterized petite strains, the tsr1 mutation has been mapped within the segment 0–5 map units on the physical map of the yeast mitochondrial genome. This segment of the mitochondrial DNA also contains the cap1 and ery1 loci and the cistron for the mitochondrial 21S rRNA. Studies of the frequencies of co-retention of markers in petite populations, and of the frequencies of recombination of markers in non-polar crosses (⁺ × ⁺), demonstrate linkage of the tsr1 locus to both the cap1 and ery1 loci. The degree of linkage indicates that tsr1 is closer to the ery1 locus. Comparison of pairwise recombination frequencies for these three markers indicate the order cap1-tsr1-ery1. The tsr1 locus lies within the segment of the mitochondrial genome which is influenced by the polarity locus , and analysis of transmission and recombination frequencies and polarities in a polar (⁺ × ^-) cross show that the behaviour of the tsr1 locus is similar to that of ery1. However striking features of this cross are that the recombination frequency between tsr1 and ery1 is comparable to that observed in non-polar crosses, and that the polarity for recombination between tsr1 and cap1 or ery1 is extremely low.     

Relationship of F9 antigen and genes of theT/t complex

Serological studies relating F9 antigen of embryonal carcinoma cells to at the murineT/t complex have been extended and confirm that only the lethal haplotype t¹²- and none of the other five lethal haplotypes-affects the quantitative expression of F9 antigen on sperm. Cytotoxicity tests on preimplantation embryos show that t¹² homozygotes are less susceptible to antiF9 serum than t^w5 homozygotes, and that using specific antimutant haplotype antisera prepared against sperm, t¹² antigen is detectable on morulae, whereas t^w5 antigen is not.     

The cell-surface distribution of alloantigens on the bovine erythrocyte

The topographical distribution of genetically defined alloantigens of the bovine erythrocyte was investigated by a serological absorption procedure known as the blocking test. Blocking was observed between the E′₁ and Y₂ antigens in theB system when these occurred in the same phenogroup. E′₁ was also blocked by G and Q′ in the same phenogroup and Y₂ was blocked by G. No blocking was observed between specificities residing either in different phenogroups or in different systems. We conclude that the several specificities in a givenB-system phenogroup are carried on a single molecule having multiple antigenic determinants, and that this arrangement is entirely consistent with the hypothesis that these factors are controlled by multiple alleles at a single locus.     

A note on the microbial spoilage of undercooked chub-packed luncheon meat

Contrary to expectations slight undercooking (68.5°C instead of 70°C for 90 min) dramatically increased the shelf-life of chub-packed luncheon meat stored at 25°C. The pH of undercooked chubs fell rapidly to below 5.0 as a result of the growth of enterococci. The accumulated acid prevented the growth of Bacillus spores and gave the luncheon meat a not unpleasant tangy flavour. Degradative changes associated with the spoilage of commercially cooked chub-packed luncheon meat did not occur, even after 42 d storage. Apparently, post-cooking fermentation by enterococci can effectively convert a perishable product into a 'shelf stable' one by lowering the pH below 5.0.     

Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

The interaction between eukaryotic elongation factor eEF-2 and reconstituted 80 S ribosomes was investigated by analyzing the accessibility of 5 S ribosomal RNA for chemical and enzymatic modification. Ribosomes reconstituted from derived subunits were modified, and the positions of the modified sites were identified by primer extension using a 5 S rRNA-specific probe. All reactive sites were located between nucleotides 38 and 99, and most of them were found in putative single-stranded regions of the 5 S rRNA. Conversion of the ribosomes to the post-translocation type of particles by treatment with the translational inhibitor ricin resulted in the exposure of 3 additional bases for chemical modification, suggesting that the 5 S rRNA was more exposed in this type of ribosome. After binding of eEF-2 in complex with the non-hydrolyzable GTP analogue guanosine 5'-(beta, gamma-methylene)-triphosphate, most of the exposed bases in the 5 S rRNA were protected against both chemical and enzymatic modification.     

Effects of chemical composition on nitrogen mineralization from green manures of seven tropical leguminous trees

Green manures from seven tropical leguminous trees were incubated with soil to determine the rates and controls of net nitrogen release. Fresh green manure (leaves and succulent twigs) was mixed with moist soil and incubated in polyethylene bags. Net N mineralization from green manures was estimated by the accumulation of extractable ammonium and nitrate minus the accumulation in soil alone. Patterns of N mineralization were complex, differed among species, and at 12 weeks ranged from 10 to 65 percent of original green-manure N. Cumulative net N mineralization was negatively correlated with initial soluble polyphenol content in the early phases of decomposition (1 through 8 weeks) and with initial lignin content in later phases (4 through 12 weeks). Neither initial percent N nor lignin: N ratio were strongly correlated with N mineralization. The best chemical index of N release was the initial polyphenol: N ratio. This study confirms previous findings that N mineralization from tropical legumes is controlled more by soluble polyphenols than by lignin or N content.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.