N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia.

Recent results have demonstrated that the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) reduces infarct size due to middle cerebral artery occlusion (MCAO), even when given after ischemia. The objective of the present study was to explore whether PBN influences recovery of energy metabolism. MCAO of 2-hr duration was induced in rats by an intraluminal filament technique. Brains were frozen in situ at the end of ischemia and after 1, 2, and 4 hr of recirculation. PBN was given 1 hr after recirculation. Neocortical focal and perifocal ("penumbra") areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 hr of ischemia. Recirculation for 1 hr led to only a partial recovery of energy state, with little further improvement after 2 hr and signs of secondary deterioration after 4 hr, particularly in the focus. After 4 hr of recirculation, PBN-treated animals showed pronounced recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values. Although an effect of PBN on mitochondria cannot be excluded, the results suggest that PBN acts by preventing a gradual compromise of microcirculation. The results justify a reevaluation of current views on the pathophysiology of focal ischemic damage and suggest that a therapeutic window of many hours exists in stroke.

Cardiac arrest in rodents: Maximal duration compatible with a recovery of neuronal activity

We report here that during a permanent cardiac arrest, rodent brain tissue is “physiologically” preserved in situ in a particular quiescent state. This state is characterized by the absence of electrical activity and by a critical period of 5–6 hr during which brain tissue can be reactivated upon restoration of a simple energy (glucose/oxygen) supply. In rat brain slices prepared 1–6 hr after cardiac arrest and maintained in vitro for several hours, cells with normal morphological features, intrinsic membrane properties, and spontaneous synaptic activity were recorded from various brain regions. In addition to functional membrane channels, these neurons expressed mRNA, as revealed by single-cell reverse transcription–PCR, and could synthesize proteins de novo. Slices prepared after longer delays did not recover. In a guinea pig isolated whole-brain preparation that was cannulated and perfused with oxygenated saline 1–2 hr after cardiac arrest, cell activity and functional long-range synaptic connections could be restored although the electroencephalogram remained isoelectric. Perfusion of the isolated brain with the γ-aminobutyric acid A receptor antagonist picrotoxin, however, could induce self-sustained temporal lobe epilepsy. Thus, in rodents, the duration of cardiac arrest compatible with a short-term recovery of neuronal activity is much longer than previously expected. The analysis of the parameters that regulate this duration may bring new insights into the prevention of postischemic damages.

Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny

Nuclear-coded valyl-tRNA synthetase (ValRS) of eukaryotes is regarded of mitochondrial origin. Complete ValRS sequences obtained by us from two amitochondriate protists, the diplomonad, Giardia lamblia and the parabasalid, Trichomonas vaginalis were of the eukaryotic type, strongly suggesting an identical history of ValRS in all eukaryotes studied so far. The findings indicate that diplomonads are secondarily amitochondriate and give further evidence for such conclusion reached recently concerning parabasalids. Together with similar findings on other amitochondriate groups (microsporidia and entamoebids), this work provides critical support for the emerging notion that no representatives of the premitochondrial stage of eukaryotic phylogenesis exist among the species living today.

CM101-mediated recovery of walking ability in adult mice paralyzed by spinal cord injury

CM101, an antiangiogenic polysaccharide derived from group B streptococcus, was administered by i.v. injection 1 hr post-spinal-cord crush injury in an effort to prevent inflammatory angiogenesis and gliosis (scarring) in a mouse model. We postulated that gliosis would sterically prevent the reestablishment of neuronal connectivity; thus, treatment with CM101 was repeated every other day for five more infusions for the purpose of facilitating regeneration of neuronal function. Twenty-five of 26 mice treated with CM101 survived 28 days after surgery, and 24 of 26 recovered walking ability within 2–12 days. Only 6 of 14 mice in the control groups survived 24 hr after spinal cord injury, and none recovered function in paralyzed limbs. MRI analysis of injured untreated and treated animals showed that CM101 reduced the area of damage at the site of spinal cord compression, which was corroborated by histological analysis of spinal cord sections from treated and control animals. Electrophysiologic measurements on isolated central nervous system and neurons in culture showed that CM101 protected axons from Wallerian degeneration; reversed γ-aminobutyrate-mediated depolarization occurring in traumatized neurons; and improved recovery of neuronal conductivity of isolated central nervous system in culture.

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Recovery of hearing and vocal behavior after hair-cell regeneration

Postmitotic hair-cell regeneration in the inner ear of birds provides an opportunity to study the effect of renewed auditory input on auditory perception, vocal production, and vocal learning in a vertebrate. We used behavioral conditioning to test both perception and vocal production in a small Australian parrot, the budgerigar. Results show that both auditory perception and vocal production are disrupted when hair cells are damaged or lost but that these behaviors return to near normal over time. Precision in vocal production completely recovers well before recovery of full auditory function. These results may have particular relevance for understanding the relation between hearing loss and human speech production especially where there is consideration of an auditory prosthetic device. The present results show, at least for a bird, that even limited recovery of auditory input soon after deafening can support full recovery of vocal precision.

Use of health services by children and young people according to ethnicity and social class: secondary analysis of a national survey

Objective To assess whether equity is achieved in use of general practitioner, outpatient, and inpatient services by children and young people according to their ethnic group and socioeconomic background.

Recovery of Diadema antillarum reduces macroalgal cover and increases abundance of juvenile corals on a Caribbean reef

The transition of many Caribbean reefs from coral to macroalgal dominance has been a prominent issue in coral reef ecology for more than 20 years. Alternative stable state theory predicts that these changes are reversible but, to date, there is little indication of this having occurred. Here we present evidence of the initiation of such a reversal in Jamaica, where shallow reefs at five sites along 8 km of coastline now are characterized by a sea urchin-grazed zone with a mean width of 60 m. In comparison to the seaward algal zone, macroalgae are rare in the urchin zone, where the density of Diadema antillarum is 10 times higher and the density of juvenile corals is up to 11 times higher. These densities are close to those recorded in the late 1970s and early 1980s and are in striking contrast to the decade-long recruitment failure for both Diadema and scleractinians. If these trends continue and expand spatially, reefs throughout the Caribbean may again become dominated by corals and algal turf.

Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase.

Agents that damage DNA in Escherichia coli or interfere with its replication induce DNA repair and mutagenesis via the SOS response. This well-known activity is regulated by the RecA protein and the LexA repressor. Following repair or bypass of the DNA lesion, the cell returns to its resting state by a largely unknown process. We found that 2-keto-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase; EC 4.1.3.16) is necessary for the recovery of respiration and that it is regulated by the SOS response. This protein was induced by DNA-damaging agents. Induction required RecA activation. When the LexA regulon was repressed, activation of RecA was not sufficient for induction, indicating the requirement for an additional protein under LexA control. Finally, a mutant in the corresponding hga gene was UV sensitive. 2-Keto-4-hydroxyglutarate aldolase also plays a role in respiratory metabolic pathways, which suggests a mechanism for respiration resumption during the termination of the SOS response.

Lyophilization-induced reversible changes in the secondary structure of proteins.

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.

Direct evidence for secondary loss of mitochondria in Entamoeba histolytica.

Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.

Efficient induction of point mutations allowing recovery of specific locus mutations in zebrafish.

A technique is described that greatly increases the efficiency of recovering specific locus point mutations in zebrafish (Danio rerio). Founder individuals that were mosaic for point mutations were produced by mutagenizing postmeiotic gametes with the alkylating agent N-ethyl-N-nitrosourea. Under optimal conditions, each founder carried an average of 10 mutations affecting genes required for embryogenesis. Moreover, approximately 2% of these founders transmitted new mutations at any prespecified pigmentation locus. Analyses of new pigmentation mutations confirmed that most were likely to be point mutations. Thus, mutagenesis of postmeiotic gametes with N-ethyl-N-nitrosourea yielded frequencies of point mutations at specific loci that were 10- to 15-fold higher than previously achieved in zebrafish. Our procedure should, therefore, greatly facilitate recovery of multiple mutant alleles at any locus of interest.