MiR-379-5p improved locomotor function recovery after spinal cord injury in rats by reducing endothelin 1 and inhibiting astrocytes expression.

OBJECTIVE: The aim of this study was to investigate the effect of microRNA-214-5p (miR-214-5p) on spinal cord injury (SCI) and to explore the mechanism of action in pathophysiological relevance. MATERIALS AND METHODS: The model of SCI was successfully established in rats aged 6-8 weeks. The levels of the locomotor function recovery in rats of the miR-379-5p group and SCI group were detected one month later by Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Biochemical indexes were measured by Western blotting and real time-PCR, respectively. In addition, rat astrocytes were cultured to verify the effect of miR-379-5p on activated astrocytes in vitro. RESULTS: Compared with the SCI group, the rats in the miR-379-5p group showed prominent improvement in the locomotor function in vivo. MiR-379-5p attenuated the activation of astrocytes and significantly suppressed the expressions of the nerve growth inhibitors. Furthermore, the down-regulation of endothelin-1 (ET-1) ameliorated the spinal cord ischemia, thereby reducing apoptosis and oxidative stress. Compared with the pentylenetetrazol (PTZ) group, ET-1 and chondroitin sulfate poly-glycoprotein (CSPG) in miR-379-5p group decreased significantly in the astrocytes transfected with miR-214-5p in vitro. CONCLUSIONS: MiR-379-5p retarded the neurofilament regeneration block effect by inhibiting endothelin 1 and the expression of the astrocytes after SCI. Furthermore, it might relieve nerve structure destruction, resist oxidative stress, and inhibit apoptosis, eventually promoting functional recovery.

Resonance Raman investigation of nickel microperoxidase-11.

Resonance Raman and UV-visible absorption spectra show that nickel(II) microperoxidase-11 (NiMP-11) is four-coordinate in aqueous solution in the pH range from 1.0 to 13.0. In aqueous solutions of NiMP-11 in the absence of cetyltrimethylammonium bromide (CTAB), NiMP-11 is aggregated. In CTAB micellar solutions, where aggregation of NiMP-11 does not occur, the Raman spectra of NiMP-11 are similar to that of nickel(II) cytochrome c (NiCyt-c). The presence of the peptide segment shifts the equilibrium heavily in favor of the nonplanar form, just as does the entire protein component in the case of NiCyt-c. This further elucidates the structural mechanism by which the protein segment ruffles the heme, most likely modulating the redox potential as indicated for the cytochromes c3 [Ma, J.-G., et al. (1998) Biochemistry 37, 12431-12442]. Furthermore, the hydrophobic environment that is provided by the CTAB micelle is found to be crucial to the native folding of the pentapeptide and formation of two hydrogen bonds in the peptide backbone. These two H-bonds act to contract the peptide segment exerting the force on the macrocycle that causes the ruffling and makes the redox potential more negative than if the heme were to remain planar. The structure of the heme and pentapeptide may also be associated with redox-linked triggering of the formation and release of cytochrome-protein complexes.

The structural origin of nonplanar heme distortions in tetraheme ferricytochromes c3.

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

Protein-induced changes in nonplanarity of the porphyrin in nickel cytochrome c probed by resonance Raman spectroscopy.

The influence of the protein on the nonplanarity of the macrocycle for nickel(II)-reconstituted cytochrome c (NiCyt-c) has been investigated with pH-dependent resonance Raman and UV-visible absorption spectroscopy and molecular mechanics calculations. The spectra reveal that NiCyt-c near neutral pH has axially coordinated Ni, but below pH 3 and above pH 12, four-coordinate species predominate. The shape of the structure-sensitive Raman line nu10 of NiCyt-c is asymmetric and broad and it changes with pH. This broad line can be decomposed well into at least two sublines, a low-frequency line that results from a nonplanar conformer and a high-frequency line that arises from a nearly planar conformer. Upon lowering the pH from 3.0 to 1.0, the amount of the nonplanar conformer decreases relative to that of the planar conformer. The decreased nonplanarity can be accounted for in terms of the disruption of a hydrogen-bonding network in the peptide backbone upon lowering the pH. Molecular mechanics (MM) calculations on iron(III) and nickel(II) microperoxidase 5 (MP-5) as well as some model heme derivatives have been carried out in order to locate the part of the protein that causes the heme distortion observed in the X-ray crystal structures of cytochromes c. The energy-optimized structures of MP-5 and the model compounds were analyzed using the normal-coordinate structural decomposition method to specify and quantify the out-of-plane macrocyclic distortions. MM calculations for MP-5 show that two hydrogen bonds formed between the amide groups in the peptide backbone are important in maintaining the ruffled deformation of the macrocycle. All evidence presented supports the hypothesis that the nonplanar distortion of the porphyrin of cytochromes c is largely maintained by a relatively small protein segment including the cysteines, the amino acids between the cysteines, and the adjacent histidine ligand. Hydrogen bonding within the backbone of this segment is important in maintaining the conformation of the peptide that induces the porphyrin distortion.

[Effects of m-nifedipine and nifedipine given intraduodenally on coronary flow, myocardial oxygen, and carbon dioxide metabolism in dogs].

In anesthetized dogs intraduodenal administration of m-nifedipine (m-Nif) 0.4 mg.kg-1, coronary flow increased from 708 +/- 72 to 880 +/- 150 ml.min-1.kg-1 (+24%) at 5 min, to 1976 +/- 350 ml.min-1.kg-1 (+179%) at 30 min, and to 998 +/- 250 ml.min-1.kg-1 (+41%) at 4 h. At the dose of nifedipine (Nif), coronary flow increased from 778 +/- 91 to 1080 +/- 90 ml.min-1.kg-1 (+39%) at 3 min and to 1836 +/- 280 ml.min-1.kg-1 (+136%) at 90 min. m-Nif was more 43% potent than Nif and the duration of action of m-Nif was longer. m-Nif reduced heart rate by -39%, while Nif reduced only -11%. m-Nif increased coronary sinus O2 content and reduced MVO2, and no difference was seen between these 2 drugs. However, the rate of CO2 production with m-Nif was reduced to a greater degree than that of Nif. The results indicated that m-Nif was more beneficial in alleviating myocardial ischemia than that of Nif.