RESUMO
BACKGROUND: Recently, the management of musculoskeletal disorders with the patients' own stem cells, isolated from the walls of small blood vessels, which can be found in great numbers in the adipose tissue, has received considerable attention. On the other hand, there are still misconceptions about these adipose-derived regenerative cells (ADRCs) that contain vascular-associated pluripotent stem cells (vaPS cells) in regenerative medicine. METHODS: Based on our previous publications on this topic, we have developed a concept to describe the significance of the ADRCs/vaPS cells in the field of orthobiologics as briefly as possible and at the same time as precisely as possible. RESULTS: The ADRCs/vaPS cells belong to the group of orthobiologics that are based on autologous cells. Because the latter can both stimulate a patient's body's localized self-healing power and provide new cells that can integrate into the host tissue during the healing response when the localized self-healing power is exhausted, this group of orthobiologics appears more advantageous than cell-free orthobiologics and orthobiologics that are based on allogeneic cells. Within the group of orthobiologics that are based on autologous cells, enzymatically isolated, uncultured ADRCs/vaPS cells have several advantages over non-enzymatically isolated cells/microfragmented fat as well as over uncultured bone marrow aspirate concentrate and cultured cells (adipose-derived stem cells, bone marrow-derived mesenchymal stem cells). CONCLUSIONS: The use of ADRCs/vaPS cells can be seamlessly integrated into modern orthopedic treatment concepts, which can be understood as the optimization of a process which-albeit less efficiently-also takes place physiologically. Accordingly, this new safe and effective type of treatment is attractive in terms of holistic thinking and personalized medicine.
Assuntos
Tecido Adiposo , Regeneração Óssea , Doenças Musculoesqueléticas/terapia , Transplante de Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Células-Tronco Pluripotentes , Medicina Regenerativa , Transplante AutólogoRESUMO
BACKGROUND: N-acylhomoserine lactones (AHLs) are well-studied signalling molecules produced by some Gram-negative Proteobacteria for bacterial cell-to-cell communication or quorum sensing. We have previously demonstrated the degradation of AHLs by an Antarctic bacterium, Planococcus versutus L10.15T, at low temperature through the production of an AHL lactonase. In this study, we cloned the AHL lactonase gene and characterized the purified novel enzyme. RESULTS: Rapid resolution liquid chromatography analysis indicated that purified AidP possesses high AHL-degrading activity on unsubstituted, and 3-oxo substituted homoserine lactones. Liquid chromatography-mass spectrometry analysis confirmed that AidP functions as an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Multiple sequence alignment analysis and phylogenetic analysis suggested that the aidP gene encodes a novel AHL lactonase enzyme. The amino acid composition analysis of aidP and the homologous genes suggested that it might be a cold-adapted enzyme, however, the optimum temperature is 28 °C, even though the thermal stability is low (reduced drastically above 32 °C). Branch-site analysis of several aidP genes of Planococcus sp. branch on the phylogenetic trees also showed evidence of episodic positive selection of the gene in cold environments. Furthermore, we demonstrated the effects of covalent and ionic bonding, showing that Zn2+ is important for activity of AidP in vivo. The pectinolytic inhibition assay confirmed that this enzyme attenuated the pathogenicity of the plant pathogen Pectobacterium carotovorum in Chinese cabbage. CONCLUSION: We demonstrated that AidP is effective in attenuating the pathogenicity of P. carotovorum, a plant pathogen that causes soft-rot disease. This anti-quorum sensing agent is an enzyme with low thermal stability that degrades the bacterial signalling molecules (AHLs) that are produced by many pathogens. Since the enzyme is most active below human body temperature (below 28 °C), and lose its activity drastically above 32 °C, the results of a pectinolytic inhibition assay using Chinese cabbage indicated the potential of this anti-quorum sensing agent to be safely applied in the field trials.
Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Planococcus (Bactéria)/enzimologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Modelos Moleculares , Pectinas/metabolismo , Pectobacterium carotovorum/metabolismo , Percepção de Quorum , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are collectively the most frequent autosomal-recessive neurodegenerative disease of childhood, but the underlying cellular and molecular mechanisms remain unclear. Several lines of evidence have highlighted the important role that non-somatic compartments of neurons (axons and synapses) play in the instigation and progression of NCL pathogenesis. Here, we report a progressive breakdown of axons and synapses in the brains of two different mouse models of NCL: Ppt1(-/-) model of infantile NCL and Cln6(nclf) model of variant late-infantile NCL. Synaptic pathology was evident in the thalamus and cortex of these mice, but occurred much earlier within the thalamus. Quantitative comparisons of expression levels for a subset of proteins previously implicated in regulation of axonal and synaptic vulnerability revealed changes in proteins involved with synaptic function/stability and cell-cycle regulation in both strains of NCL mice. Protein expression changes were present at pre/early-symptomatic stages, occurring in advance of morphologically detectable synaptic or axonal pathology and again displayed regional selectivity, occurring first within the thalamus and only later in the cortex. Although significant differences in individual protein expression profiles existed between the two NCL models studied, 2 of the 15 proteins examined (VDAC1 and Pttg1) displayed robust and significant changes at pre/early-symptomatic time-points in both models. Our study demonstrates that synapses and axons are important early pathological targets in the NCLs and has identified two proteins, VDAC1 and Pttg1, with the potential for use as in vivo biomarkers of pre/early-symptomatic axonal and synaptic vulnerability in the NCLs.
Assuntos
Axônios/metabolismo , Modelos Animais de Doenças , Lipofuscinoses Ceroides Neuronais/genética , Sinapses/metabolismo , Animais , Axônios/patologia , Western Blotting , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Humanos , Imuno-Histoquímica , Lactente , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Securina , Sinapses/patologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Tálamo/metabolismo , Tálamo/patologia , Tioléster Hidrolases/deficiência , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Fatores de Tempo , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P(5B) ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.
Assuntos
Cádmio/toxicidade , Manganês/toxicidade , Níquel/toxicidade , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Selênio/toxicidade , Cádmio/metabolismo , Cátions Bivalentes/metabolismo , Cátions Bivalentes/toxicidade , Histidina/metabolismo , Humanos , Manganês/metabolismo , Níquel/metabolismo , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Selênio/metabolismo , Vacúolos/enzimologiaRESUMO
Nutrient enrichment is known to increase bacterioplankton population density in a variety of Antarctic freshwater lakes. However, relatively little is known about the associated changes in species composition. In this study, the bacterioplankton community composition of one such lake was studied following natural nutrient enrichment to investigate the resistance of the system to environmental change. Heywood Lake is an enriched freshwater maritime Antarctic lake, with nitrogen and phosphorus concentrations significantly higher than its more oligotrophic neighbours (by at least an order of magnitude). This major change in lake chemistry has occurred following large increases in the fur seal population over the last 30 years. Using analysis of 16S rRNA gene fragments, fatty acid methyl ester analysis, denaturing gradient gel electrophoresis and fluorescence in situ hybridization, significant changes are reported in lake microbiology which have resulted in a distinct bacterioplankton community. In comparison to its more oligotrophic neighbours, nutrient-enriched Heywood Lake has a high bacterioplankton population density, reduced species richness and an increasing evenness among key groups. Only 42.3 % of the clones found with > or =97 % similarity to a named genus were also present in adjacent oligotrophic lakes, including three of the dominant groups. Critically, there was an apparent shift in dominance with trophic status (from the beta-Proteobacteria to the Actinobacteria). Other key observations included the absence of a dominant group of Cyanobacteria and the presence of marine bacteria. The significant impact of natural nutrient enrichment on the microbiology of Heywood Lake, therefore, suggests that low-temperature oligotrophic freshwater lake systems might have low resistance to environmental change.
Assuntos
Bactérias/crescimento & desenvolvimento , Ecossistema , Água Doce/microbiologia , Nitrogênio/metabolismo , Fósforo/metabolismo , Plâncton/crescimento & desenvolvimento , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/crescimento & desenvolvimento , Animais , Regiões Antárticas , Bactérias/classificação , Bactérias/genética , Betaproteobacteria/classificação , Betaproteobacteria/genética , Betaproteobacteria/crescimento & desenvolvimento , Meios de Cultura/química , DNA Bacteriano , Eletroforese/métodos , Ácidos Graxos/análise , Água Doce/química , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Plâncton/classificação , Plâncton/genética , RNA Ribossômico 16S , Análise de Sequência de DNARESUMO
Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marked rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC-(ATP-binding cassette) superfamily, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it is also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome C550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome C550-alkaline phosphatase fusion protein and apocytochrome cd1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.