RESUMO
Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit proton pump. The proton pump is essential for the regulation of pH in various eukaryotic cellular processes. Among the 14 subunits that constitute V-ATPase, d subunit mediates coupling between cytosolic and membrane domains. Whereas d1 is expressed ubiquitously in various types of cells, its isoform d2 is only expressed in specific cells or tissues. To characterize these isoforms, we expressed and purified the isoforms of human V-ATPase d subunits using Escherichia coli over-expression system. Subunit d1 and d2 were purified as homogeneous monomers as demonstrated by dynamic light scattering (DLS) analysis. Secondary structures of d subunits were estimated to be composed of 73% α-helix and 2% ß-sheet, as analyzed using circular dichroism (CD) analysis. Although sequence identity and secondary structures of d subunits were highly similar, the relative stability against thermal stress was higher for d1 than d2. Efficient expression and purification of d subunits, together with biophysical and biochemical characterization, presented in this study is expected to facilitate further structural analysis to clarify specific inter-molecular interactions involved in multi-subunit assembly and regulation of H(+) transporters.
Assuntos
Escherichia coli/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/isolamento & purificação , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
PURPOSE: Mesenchymal stem cells (MSCs) have demonstrated great promises for the treatment of ischemic stroke. Previously, we identified a new source of MSCs located in the inferior turbinate. We investigated therapeutic potentials of human turbinate- derived mesenchymal stem cells (hTMSCs) in ischemic stroke. METHODS: Ischemic stroke was induced by the intraluminal occlusion of middle cerebral artery (MCAo) for 50 minutes in rats. At one day after MCAo, hTMSCs, adipose tissue-derived MSCs (AdMSCs), or phosphate buffered saline (PBS) were transplanted into the striatum. Functional recovery was assessed by repeating behavioral tests including modified neurologic severity score and corner test. At 14 days after MCAo, brains were stained with hematoxylin and eosin (H&E) for measuring infarct volume. The survival of grafted MSCs was evaluated by immunohistochemistry to human nuclei (hNU). Immunohistochemistry with anti-doublecortin (anti-DCX) was performed to assess hippocampal neurogenesis. RESULTS: Transplantation of hTMSCs following MCAo showed improvements of neurologic function, which was comparable with that of AdMSCs. H&E staining showed no difference in infarct volume among 3 groups. Regarding the survival of grafted MSCs, the number of hNU-expressing cells was not different between hTMSCs- and AdMSCs-treated groups. Finally, hTMSCs increased the number of subgranular DCX-positive cells compared to PBS-treated controls, without affecting hilar ectopic migration of newborn neurons. CONCLUSION: hTMSCs could improve functional recovery following ischemic stroke, of which efficacy was similar to AdMSCs. Although hTMSCs showed comparable infarct size and survival of grafted MSCs, transplantation of hTMSCs could upregulate subgranular neurogenesis with no impact on ectopically migrating newborn neurons.
RESUMO
The anti-melanogenic effects of the extract of Angelica tenuissima (AT) root and the extract of AT root fermented by Aspergillus oryzae (FAT) were investigated. These effects were determined by measuring the inhibitory activity of AT and FAT on melanin production in B16F10 melanocytes and with in vitro tyrosinase activity assays. The AT extract inhibited melanin production at concentrations above 250 µg/ml, and this inhibitory effect was significantly enhanced by the fermentation process with A. oryzae. HPLC analysis resulted in the isolation of two active compounds from both the AT and FAT extracts. Their chemical structures were identified as decursin and Z-ligustilide through comparison with previously reported NMR data. The decursin and Z-ligustilide contents were increased in the FAT extract and could be responsible for its enhanced inhibitory effects on melanin production and tyrosinase activity compared with that of the AT extract.
Assuntos
4-Butirolactona/análogos & derivados , Angelica/química , Aspergillus oryzae/metabolismo , Benzopiranos/farmacologia , Butiratos/farmacologia , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Plantas Medicinais/química , 4-Butirolactona/química , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/metabolismo , 4-Butirolactona/farmacologia , Angelica/microbiologia , Animais , Benzopiranos/química , Benzopiranos/isolamento & purificação , Benzopiranos/metabolismo , Butiratos/química , Butiratos/isolamento & purificação , Butiratos/metabolismo , Técnicas de Cultura de Células , Linhagem Celular/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fermentação , Alimentos Fermentados , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Camundongos , Monofenol Mono-Oxigenase/análise , Extratos Vegetais/química , Raízes de Plantas/microbiologia , Plantas Medicinais/microbiologiaRESUMO
The hypothetical protein TTC0263 of Thermus thermophilus HB27 is a thermophilic tetratricopeptide repeat (TPR)-containing protein. In the present study, the TPR region (residues 26-230) was resolved at 2.5 A with R-factors of R/Rfree = 23.6%/28.6%. TTC0263 consists of 11 helices that form five TPR units. Uniquely, it contains one atypical "extended" TPR (eTPR) unit. This comprises extended helical residues near the loop region of TTC0263, such that the helical length of eTPR is longer than that of the canonical TPR sequence. In addition, the hybrid TPR domain of TTC0263 possesses oligomer-forming characteristics. TPR domains are generally involved in forming multi-subunit complexes by interacting with each other or with other subunit proteins. The dynamic structure of TTC0263 described here goes some way to explaining how TPR domains mediate the formation of multi-subunit complexes.
Assuntos
Proteínas de Bactérias/química , Thermus thermophilus/química , Motivos de Aminoácidos , Aminoácidos/análise , Cristalização , Cristalografia por Raios X , Modelos MolecularesRESUMO
YrrB is a hypothetical protein containing a tetratricopeptide repeat (TPR) domain from a Gram-positive bacterium, Bacillus subtilis. We determined YrrB structure in the C2 space group to 2.5A resolution, which is the first TPR structure of the Gram-positive bacterium B. subtilis. In contrast to other known TPR structures, the concave surface of the YrrB TPR domain is composed of the putative peptide-binding pocket lined with positively-charged residues. This unique charge distribution reveals that YrrB can interact with partner proteins via an unusual TPR-mediated interaction mode, compared to that of other TPR-containing structures. Functional annotation using genomics analysis suggested that YrrB may be an interacting mediator in the complex formation among RNA sulfuration components. No proteins containing a TPR domain have been identified in the biosynthesis of sulfur-containing biomolecules. Thus, YrrB could play a new role as a connecting module among those proteins in the conserved gene cluster for RNA sulfuration.