Isolation of peroxisomes from frozen human liver samples.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.     

Substance P affects the GABAergic system in the hypothalamo-pituitary axis

In the present work we examined the effect of the neutralization of endogenous substance P by the administration of an anti-substance P serum (ASPS) on GABA concentration in the anterior pituitary in hyperprolactinemic conditions induced by 5-hydroxytryptophan or by grafting anterior pituitaries. ASPS reduced the increase in the anterior pituitary GABA concentration induced by hyperprolactinemia. In vitro experiments showed that substance P inhibited K⁺-evoked GABA efflux from hypothalamic fragments and decreased GABA concentration in the anterior pituitary but ASPS increased it. Our results demonstrate that substance P modifies hypothalamic GABA release and anterior pituitary GABA concentration and suggest that an interaction exists between substance P and GABA.     

Increased Neostriatal Tyrosine Hydroxylation During Stress: Role of Extracellular Dopamine and Excitatory Amino Acids

Abstract: We examined the regulation of neostriatal tyrosine hydroxylation during acute stress, testing the hypothesis that excitatory amino acids (EAAs) contribute to the stress-evoked increase in dopamine (DA) synthesis. Dialysis probes implanted into neostriatum permitted delivery of drugs and sampling of extracellular fluid. Rats were exposed to 30 min of intermittent tail shock during infusion of an inhibitor of aromatic amino acid decarboxylase (AAAD), NSD-1015 (100 µM), and DOPA was measured in the dialysate. Tail shock was applied beginning either 15 min after the onset of NSD-1015 treatment (the initial rate of DOPA accumulation) or 75 min after the onset of treatment (when DOPA had approached steady state). Tail shock increased the steady-state levels of extracellular DOPA in neostriatum (+40%). However, there was no change in the initial rate of DOPA accumulation unless animals also received the D₂ receptor antagonist eticlopride (50 nM), in which case an increase was observed (+228%). The impact of tail shock on the steady-state level of DOPA was attenuated by the D₂ agonist quinpirole (100 µM), or by 2-amino-5-phosphonovalerate (APV) (100 µM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100 µM), EAA antagonists acting at NMDA or d ,l -α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, respectively. These data suggest that acute stress normally has little effect on tyrosine hydroxylation in neostriatum due to the inhibitory influence of DA in the extracellular fluid. However, when that influence is absent (e.g., during extended inhibition of DOPA decarboxylation or blockade of DA receptors), stress increases tyrosine hydroxylation via EAAs acting on NMDA and AMPA receptors. Thus, EAAs released from corticostriatal projections may stimulate DA synthesis and thereby restore dopaminergic activity under conditions in which the availability of DA for release has been compromised.     

Geographic differences in the allele frequencies of the human Y-linked tetranucleotide polymorphism DYS19

We have studied the allele frequency distribution of the microsatellite locus DYS 19 in several populations with different geographical origins worldwide. Three new alleles were found. In addition, remarkable geographic and ethnic differences were observed in the allele frequency profiles and DNA marker (gene) diversity among populations and major ethnic groups. Amerindians showed an overwhelming predominance of the A allele, while in Caucasians the B allele was modal, and in Greater Asians and Africans allele C became predominant. Even within these geographic regions there were significant gradients, as exemplified by the decreasing frequency profile of the B allele from Great Britain over Germany to Slovakia. Thus, DYS 19 emerges as a useful tool for studying the structure and dynamics of human populations.     

The magic-bead concept: an integrated approach to nitrogen removal with co-immobilized micro-organisms

This paper describes both qualitative and quantitative aspects of simultaneous autotrophic nitrification and heterotrophic denitrification by, respectively, the nitrifierNitrisomonas europaea and either of the denitrifiersPseudomonas denitrificans orParacoccus denitrificans co-immobilized in double-layer gel beads. The system is based on the establishment of well-defined oxic and anoxic zones within the cell supports and on physical separation of the nitrifying and denitrifying populations. Nitrification and denitrification rates were obtained from measured bulk concentrations and head-space analysis. The latter analyses showed that ammonia was primarily converted into molecular nitrogen. Nitrous oxide was not detected. High nitrogen removal rates (up to 5.1 mmol N m^–3 gel s^–1) were achieved in continuous reactors under aerobic conditions. The overall rate of nitrogen removal was controlled by the nitrifying step. The approach followed is, in principle, also suitable to the coupling of other oxidative and reductive bioprocesses having complementary metabolic routes. Two-stage bioconversion processes can be thus conducted as if single-staged, which results in more compact reactor systems.     

Elongation factor 1α genes of the red alga Porphyra purpurea include a novel, developmentally specialized variant

The life cycle of the red alga Porphyra purpurea alternates between two morphologically distinct phases: a shell-boring, filamentous sporophyte and a free-living, foliose gametophyte. From a subtracted cDNA library enriched for sporophyte-specific sequences, we isolated a cDNA encoding an unusual elongation factor 1 (EF-1) that is expressed only in the sporophyte. A second EF-1 gene that is expressed equally in the sporophyte and the gametophyte was isolated from a genomic library. These are the only EF-1 genes detectable in P. purpurea. The constitutively expressed gene encodes and EF-1 very similar to those of most eukaryotes. However, the sporophyte-specific EF-1 is one of the most divergent yet described, with nine insertions or deletions ranging in size from 1 to 26 amino acids. This is the first report of a developmental stage-specific EF-1 outside of the animal kingdom and suggests a fundamental role for EF-1 in the developmental process.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

Enrichment of mixed cultures capable of aerobic degradation of 1,2-dibromoethane.

1,2-dibromoethane (DBE) is a common environmental contaminant; it is potentially carcinogenic and has been detected in soil and groundwater supplies. Most of the biodegradation studies to date have been performed under anaerobic conditions or in the context of soil remediation, where the pollutant concentration was in the parts per billion range. In this work a mixed bacterial culture capable of complete aerobic mineralization of concentrations of DBE up to 1 g liter(-1) under well-controlled laboratory conditions was enriched. In order to verify biodegradation, formation of biodegradation products as well as the disappearance of DBE from the biological medium were measured. Complete mineralization was verified by measuring stoichiometric release of the biodegradation products. This mixed culture was found to be capable of degrading other halogenated compounds, including bromoethanol, the degradation of which has not been reported previously.