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BackgroundMild brain hypothermia (32°-34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C]glucose and [1,2-C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase.MethodsNeonatal rat cerebrocortical slices receiving a C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N=3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen-glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator.ResultsThe most significant metabolite difference (P

Human clinical trials using 72 hours of mild hypothermia (32°C-34°C) after neonatal asphyxia have found substantially improved neurologic outcomes. As temperature changes differently modulate numerous metabolite fluxes and concentrations, we hypothesized that (1)H/(31)P nuclear magnetic resonance (NMR) spectroscopy of intracellular metabolites can distinguish different insults, treatments, and recovery stages. Three groups of superfused neonatal rat brain slices underwent 45 minutes oxygen-glucose deprivation (OGD) and then were: treated for 3 hours with mild hypothermia (32°C) that began with OGD, or similarly treated with hypothermia after a 15-minute delay, or not treated (normothermic control group, 37°C). Hypothermia was followed by 3 hours of normothermic recovery. Slices collected at different predetermined times were processed, respectively, for 14.1 Tesla NMR analysis, enzyme-linked immunosorbent assay (ELISA) cell-death quantification, and superoxide production. Forty-nine NMR-observable metabolites underwent a multivariate analysis. Separated clustering in scores plots was found for treatment and outcome groups. Final ATP (adenosine triphosphate) levels, severely decreased at normothermia, were restored equally by immediate and delayed hypothermia. Cell death was decreased by immediate hypothermia, but was equally substantially greater with normothermia and delayed hypothermia. Potentially important biomarkers in the (1)H spectra included PCr-(1)H (phosphocreatine in the (1)H spectrum), ATP-(1)H (adenosine triphosphate in the (1)H spectrum), and ADP-(1)H (adenosine diphosphate in the (1)H spectrum). The findings suggest a potential role for metabolomic monitoring during therapeutic hypothermia.

Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3'-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases.

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7 at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA-protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.

KH (hnRNP K homology) domains, consisting of approximately 70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 A resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a betaalphaalphabetabetaalpha configuration. The protein-DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein-protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.

The NMR solution structure of a 38-nucleotide RNA representing the entire stem-loop D domain with the consensus sequence for enteroviruses and rhinoviruses is investigated. The results also indicate that although the primary sequences of the apical loops in these three RNAs are different, they adopt very similar three-dimensional structures that conform to the previously established UNCG-type tetraloop motif.

Research has been conducted on prion protein. The transformation of the prion protein normal cellular isoform to the infectious prion protein has entailed a metastable locally disordered prion protein conformer, the stability and structural features of this conformer have been characterized via NMR spectroscopy and the results indicate that the conformer coexists in solution.

Nuclear magnetic resonance spectroscopic analysis of the Okazaki fragment containing a single gemcitabine substitution shows alteration in electrostatic properties in terms of the electron density enhancement at the germinal difluoro group. Furthermore, it alters the structural and thermodynamic properties of DNA.

Results demonstrate that in the CAA-TTG sequence region of duplex DNAs, exhibit a slow motion in the microsecond-to millisecond range. The CAA-TTG sequence has a flexible to unstable pyrimidine-purine dinucleotide step in which the purine base is adenine.

A study was conducted to characterize the structure of a homodimer determined by a synthetic 25 residue DNA oligonucleotide which supports single-stranded nucleic acids called a pseudosquare knot (PSQ). Nuclear magnetic resonance was carried out to represent sequences, structures and numbering schemes. Results indicated the frequency of sequences compatibility with PSQ formation in single-stranded genomes.

Research was conducted to determine the three-dimensional structure of d(GCATATGATAG)*d(CTATCATATGC) using two-dimensional nuclear Overhauser effect and double-quantum-filtered COSY spectra. the complete relaxation matrix program MARDIGRAS was ran several times utilizing 2D NOE spectra for exchangeable and nonexchangeable protons at various mixing times. R factors suggest that the structures were in good agreement with the experimental NOE data.

A study was conducted to characterize DNA duplex structures in solution utilizing nuclear magnetic resonance. Motional models have been utilized to determine data for three DNA duplexes while H8-sugar proton distances were correlated with glycosidic torsion angle for adenosine 5. Results indicated that short base-H1' bounds correlated with glycosyl bond motion. Finding also showed that DNAminiCarlo plots supported MD-tar simulation analyses.