RESUMO
The axon initial segment (AIS) is essential for action potential generation. Recently, the AIS was identified as a site of neuronal plasticity. A subpopulation of AIS in cortical principal neurons contains stacks of endoplasmic reticulum (ER) forming the cisternal organelle (CO). The function of this organelle is poorly understood, but roles in local Ca2+-trafficking and AIS plasticity are discussed. To investigate whether the presence and/or the size of COs are linked to the development and maturation of AIS of cortical neurons, we analyzed the relationship between COs and the AIS during visual cortex development under control and visual deprivation conditions. In wildtype mice, immunolabeling for synaptopodin, ankyrin-G, and ßIV-spectrin were employed to label COs and the AIS, respectively. Dark rearing resulted in an increase in synaptopodin cluster sizes, suggesting a homeostatic function of the CO in this cellular compartment. In line with this observation, synaptopodin-deficient mice lacking the CO showed AIS shortening in the dark. Collectively, these data demonstrate that the CO is an essential part of the AIS machinery required for AIS plasticity during a critical developmental period of the visual cortex.
Assuntos
Segmento Inicial do Axônio/metabolismo , Axônios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Plasticidade Neuronal/fisiologia , Córtex Visual/crescimento & desenvolvimento , Potenciais de Ação/fisiologia , Animais , Retículo Endoplasmático/metabolismo , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Córtex Visual/metabolismoRESUMO
INTRODUCTION: Orthodontic patients usually complain about masticatory limitations associated with the activation of fixed appliances. The aim of this investigation was to evaluate whether orthodontic pain reflects differences in the objective evaluation of mastication and in the levels of proinflammatory cytokines in the crevicular fluid of patients undergoing orthodontic treatment. METHODS: Twenty patients with malocclusions requiring orthodontic treatment were included in this prospective study. Their pain experience, masticatory performance, and levels of interleukin 1-beta and prostaglandin E2 in crevicular fluid were evaluated at 3 times: before bracket placement, 24 hours after archwire placement, and 30 days after the initial appointment. All variables were compared with those of a control group of 25 subjects with normal occlusion. RESULTS: The masticatory performance of the patients was significantly reduced at 24 hours after bracket placement, the period in which they reported higher values of pain and had higher levels of interleukin 1-beta. The levels of prostaglandin E2 did not change in the periods evaluated, and there were no correlations between the levels of cytokines and the functional limitations observed. The only significant correlation was between pain and decreased masticatory performance. CONCLUSIONS: The masticatory performance of orthodontic patients is significantly reduced only during the period of greatest pain. However, these alterations did not correlate with any measurement of interleukin 1-beta or prostaglandin E2 in the crevicular fluid, suggesting that these solitary measurements are inadequate to predict the temporary pain and masticatory limitations experienced by patients undergoing orthodontic treatment.
Assuntos
Dinoprostona/análise , Líquido do Sulco Gengival/imunologia , Mediadores da Inflamação/análise , Interleucina-1beta/análise , Mastigação/fisiologia , Braquetes Ortodônticos , Fios Ortodônticos , Dor/fisiopatologia , Adolescente , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Má Oclusão/classificação , Má Oclusão/terapia , Dor/imunologia , Medição da Dor/métodos , Tamanho da Partícula , Estudos Prospectivos , Silicones/química , Fatores de Tempo , Adulto JovemRESUMO
Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.
Assuntos
Comportamento Animal/fisiologia , Endocitose/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Dopamina D2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Agonistas de Dopamina/metabolismo , Antagonistas de Dopamina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/genética , Receptores de Dopamina D2/genéticaRESUMO
Soft tissue sarcomas are mesenchymal tumors that are fatal in approximately one-third of patients. To explore mechanisms of sarcoma pathogenesis, we have generated a mouse model of soft tissue sarcoma. Intramuscular delivery of an adenovirus expressing Cre recombinase in mice with conditional mutations in Kras and Trp53 was sufficient to initiate high-grade sarcomas with myofibroblastic differentiation. Like human sarcomas, these tumors show a predilection for lung rather than lymph node metastasis. Using this model, we showed that a prototype handheld imaging device can identify residual tumor during intraoperative molecular imaging. Deletion of the Ink4a-Arf locus (Cdkn2a), but not Bak1 and Bax, could substitute for mutation of Trp53 in this model. Deletion of Bak1 and Bax, however, was able to substitute for mutation of Trp53 in the development of sinonasal adenocarcinoma. Therefore, the intrinsic pathway of apoptosis seems sufficient to mediate p53 tumor suppression in an epithelial cancer, but not in this model of soft tissue sarcoma.
Assuntos
Modelos Animais de Doenças , Sarcoma/patologia , Animais , Transformação Celular Neoplásica , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Knockout , Sarcoma/genética , Sarcoma/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genéticaRESUMO
The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.
RESUMO
The inhibitor of NF-κB alpha (IκBα) protein is an important regulator of the transcription factor NF-κB. In neurons, IκBα has been shown to play a role in neurite outgrowth and cell survival. Recently, a phosphorylated form of IκBα (pIκBα Ser32/36) was reported to be highly enriched at the axon initial segment (AIS) and was proposed to function upstream of ankyrinG in AIS assembly, including ion channel recruitment. However, we report here that the AIS clustering of ankyrinG and Na(+) channels in the brains of IκBα knockout (Nfkbia(-/-)) mice is comparable to that in wild-type littermates. Furthermore, we found that multiple phospho-specific antibodies against pIκBα Ser32/36 non-specifically label AIS in Nfkbia(-/-) cortex and AIS in dissociated Nfkbia(-/-) hippocampal neurons. With the exception of ankyrinG, shRNA-mediated knockdown of known AIS proteins in cultured hippocampal neurons did not eliminate the AIS labeling with pIκBα antibodies. Instead, the pIκBα antibodies cross-react with a phosphorylated epitope of a protein associated with the microtubule-based AIS cytoskeleton that is not integrated into the AIS membrane complex organized by ankyrinG. Our results indicate that pIκBα is neither enriched at the AIS nor required for AIS assembly.
Assuntos
Axônios/metabolismo , Proteínas I-kappa B/metabolismo , Animais , Anquirinas/antagonistas & inibidores , Anquirinas/metabolismo , Células Cultivadas , Proteínas I-kappa B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , RNA Interferente Pequeno , Ratos , Ratos Sprague-DawleyRESUMO
Cholic acid (1, CD), deoxycholic (3, DCA), chenodeoxycholic acid (5, CDCA), ursodeoxycholic acid (7, UDCA), and lithocholic acid (9, LCA) were acetylated and converted into their piperazinyl spacered rhodamine B conjugates 16-20. While the parent bile acids showed almost no cytotoxic effects for several human tumor cell lines, the piperazinyl amides were cytostatic but an even superior effect was observed for the rhodamine B conjugates. Extra staining experiments showed these compounds as mitocans; they led to a cell arrest in the G1 phase.
Assuntos
Ácidos e Sais Biliares , Ácido Ursodesoxicólico , Humanos , Ácidos e Sais Biliares/farmacologia , Ácido Cólico/farmacologia , Ácido Ursodesoxicólico/farmacologia , Ácido Quenodesoxicólico , Linhagem Celular Tumoral , Ácido Desoxicólico/farmacologia , Ácidos Cólicos/farmacologiaRESUMO
Here we investigated the regulation of NF-κB activity by post-translational modifications upon reconstitution of NF-κB p65-deficient cells with the wild-type protein or phosphorylation-defect mutants. Analysis of NF-κB target gene expression showed that p65 phosphorylations alone or in combination function to direct transcription in a highly target gene-specific fashion, a finding discussed here as the NF-κB barcode hypothesis. High-resolution microscopy and surface rendering revealed serine 536 phosphorylated p65 predominantly in the cytosol, while serine 468 phosphorylated p65 mainly localized in nuclear speckles. TNF stimulation resulted in the translocation of the cytosolic p65 kinase IKKε to the nucleus and also to promyelocytic leukemia (PML) nuclear bodies. This inducible IKKε translocation was dependent on p65 phosphorylation and was prevented by the oncogenic PML-RARα fusion protein. Chromatin immunoprecipitation experiments revealed the inducible association of IKKε to the control regions of several NF-κB target genes. In the nucleus, the kinase contributes to the expression of a subset of NF-κB-regulated genes, thus revealing a novel role of IKKε for the control of nuclear NF-κB activity.
Assuntos
Núcleo Celular/enzimologia , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/genética , Células Cultivadas , Cromatina/enzimologia , Células HeLa , Humanos , Quinase I-kappa B/genética , Camundongos , Mutação , Fosforilação , Fator de Transcrição RelA/análise , Transcrição GênicaRESUMO
Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG(-/-) mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG(-/-) mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.
Assuntos
Anquirinas/fisiologia , Axônios/fisiologia , Polaridade Celular/fisiologia , Dendritos/fisiologia , Sinapses/fisiologia , Animais , Anquirinas/deficiência , Anquirinas/genética , Genes Reporter , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Regiões Promotoras Genéticas , Células de Purkinje/fisiologia , Potenciais Sinápticos/fisiologiaRESUMO
The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D: -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.
Assuntos
Proteínas de Bactérias/química , Desidrogenase do Álcool de Açúcar/química , Thermotoga maritima/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Expressão Gênica , Cinética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Desidrogenase do Álcool de Açúcar/metabolismo , Thermotoga maritima/química , Thermotoga maritima/genéticaRESUMO
The axon initial segment (AIS) is a critical microdomain for action potential initiation and implicated in the regulation of neuronal excitability during activity-dependent plasticity. While structural AIS plasticity has been suggested to fine-tune neuronal activity when network states change, whether it acts in vivo as a homeostatic regulatory mechanism in behaviorally relevant contexts remains poorly understood. Using the mouse whisker-to-barrel pathway as a model system in combination with immunofluorescence, confocal analysis and electrophysiological recordings, we observed bidirectional AIS plasticity in cortical pyramidal neurons. Furthermore, we find that structural and functional AIS remodeling occurs in distinct temporal domains: Long-term sensory deprivation elicits an AIS length increase, accompanied with an increase in neuronal excitability, while sensory enrichment results in a rapid AIS shortening, accompanied by a decrease in action potential generation. Our findings highlight a central role of the AIS in the homeostatic regulation of neuronal input-output relations.
Assuntos
Segmento Inicial do Axônio/metabolismo , Córtex Cerebral/metabolismo , Homeostase , Envelhecimento/fisiologia , Animais , Comportamento Exploratório , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Privação Sensorial , Fatores de Tempo , Vibrissas/fisiologiaRESUMO
Inhibitor kappaB kinase (IKK) regulates the activity of the transcription factor nuclear factor-kappa B that normally protects neurons against excitotoxicity. Constitutively active IKK is enriched at axon initial segments and nodes of Ranvier (NR). We used mice with a Cre-loxP-mediated specific deletion of IKKbeta in sensory neurons of the dorsal root ganglion (SNS-IKKbeta(-/-)) to evaluate whether IKK plays a role in sensory neuron excitability and nociception. We observed increased sensitivity to mechanical, cold, noxious heat and chemical stimulation in SNS-IKKbeta(-/-) mice, with normal proprioceptive and motor functions as revealed by gait analysis. This was associated with increased calcium influx and increased inward currents in small- and medium-sized primary sensory neurons of SNS-IKKbeta(-/-) mice during stimulation with capsaicin or Formalin, specific activators of transient receptor potentials TRPV1 and TRPA1 calcium channels, respectively. In vitro stimulation of saphenous nerve preparations of SNS-IKKbeta(-/-) mice showed increased neuronal excitability of A- and C-fibers but unchanged A- and C-fiber conduction velocities, normal voltage-gated sodium channel currents, and normal accumulation of ankyrin G and the sodium channels Nav1.6 at NR. The results suggest that IKKbeta functions as a negative modulator of sensory neuron excitability, mediated at least in part by modulation of TRP channel sensitivity.
Assuntos
Gânglios Espinais/citologia , Quinase I-kappa B/deficiência , Nociceptores/fisiologia , Limiar da Dor/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Anquirinas/metabolismo , Área Sob a Curva , Comportamento Animal , Cálcio/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Regulação da Expressão Gênica/genética , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Atividade Motora/genética , Canal de Sódio Disparado por Voltagem NAV1.8 , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Condução Nervosa/genética , Condução Nervosa/fisiologia , Nociceptores/efeitos dos fármacos , Medição da Dor/métodos , Técnicas de Patch-Clamp/métodos , Estimulação Física/efeitos adversos , Tempo de Reação/genética , Nervo Isquiático , Fármacos do Sistema Sensorial/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/genética , Tetrodotoxina/farmacologiaRESUMO
We previously showed that the 2-oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (DeltapknA, DeltapknB, DeltapknL), five double (DeltapknAG, DeltapknAL, DeltapknBG, DeltapknBL, DeltapknLG) and two triple deletion mutants (DeltapknALG, DeltapknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C. glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho-Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Proteínas do Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Divisão Celular , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Especificidade por Substrato/genética , Treonina/genética , Treonina/metabolismoRESUMO
Protein aggregation is a major pathological hallmark of many neurodegenerative disorders including polyglutamine diseases. Aggregation of the mutated form of the disease protein ataxin-3 into neuronal nuclear inclusions is well described in the polyglutamine disorder spinocerebellar ataxia type 3 (SCA3 or Machado-Joseph disease), although these inclusions are not thought to be directly pathogenic. Neuropil aggregates have not yet been described in SCA3. We performed a systematic immunohistochemical study of serial thick sections through brains of seven clinically diagnosed and genetically confirmed SCA3 patients. Using antibodies against ataxin-3, p62, ubiquitin, the polyglutamine marker 1C2 as well as TDP-43, we analyzed neuronal localization, composition and distribution of aggregates within SCA3 brains. The analysis revealed widespread axonal aggregates in fiber tracts known to undergo neurodegeneration in SCA3. Similar to neuronal nuclear inclusions, the axonal aggregates were ubiquitinated and immunopositive for the proteasome and autophagy associated shuttle protein p62, indicating involvement of neuronal protein quality control mechanisms. Rare TDP-43 positive axonal inclusions were also observed. Based on the correlation between affected fiber tracts and degenerating neuronal nuclei, we hypothesize that these novel axonal inclusions may be detrimental to axonal transport mechanisms and thereby contribute to degeneration of nerve cells in SCA3.
Assuntos
Axônios/ultraestrutura , Encéfalo/patologia , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Doença de Machado-Joseph/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Axônios/patologia , Encéfalo/metabolismo , Feminino , Humanos , Doença de Machado-Joseph/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Triptofano Hidroxilase/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas tau/metabolismoRESUMO
In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33 degrees C to 39 degrees C. A dephosphorylation of OdhI in C. glutamicum 2262 was observed during the biotin-limited as well as the temperature-induced glutamate-producing process. Deletion of pknG in C. glutamicum 2262 did not affect the phosphorylation status of OdhI during growth and glutamate production phases triggered by a temperature upshock, though a 40% increase in the specific glutamate production rate was measured. These results suggest that, under the conditions analyzed, PknG is not the kinase responsible for the phosphorylation of OdhI in C. glutamicum 2262. The phosphorylation status of OdhI alone is, as expected, not the only parameter that determines the performance of a specific strain, as no clear relation between the specific glutamate production rate and OdhI phosphorylation level was demonstrated.
Assuntos
Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Deleção de Genes , Cinética , Mutagênese Insercional , Fosfoproteínas Fosfatases/genética , FosforilaçãoRESUMO
Non-neuronal cells may be pivotal in neurodegenerative disease, but the mechanistic basis of this effect remains ill-defined. In the polyglutamine disease spinocerebellar ataxia type 7 (SCA7), Purkinje cells undergo non-cell-autonomous degeneration in transgenic mice. We considered the possibility that glial dysfunction leads to Purkinje cell degeneration, and generated mice that express ataxin-7 in Bergmann glia of the cerebellum with the Gfa2 promoter. Bergmann glia-specific expression of mutant ataxin-7 was sufficient to produce ataxia and neurodegeneration. Expression of the Bergmann glia-specific glutamate transporter GLAST was reduced in Gfa2-SCA7 mice and was associated with impaired glutamate transport in cultured Bergmann glia, cerebellar slices and cerebellar synaptosomes. Ultrastructural analysis of Purkinje cells revealed findings of dark cell degeneration consistent with excitotoxic injury. Our studies indicate that impairment of glutamate transport secondary to glial dysfunction contributes to SCA7 neurodegeneration, and suggest a similar role for glial dysfunction in other polyglutamine diseases and SCAs.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Fatores Etários , Idoso , Animais , Animais Recém-Nascidos , Ataxina-7 , Comportamento Animal , Western Blotting/métodos , Encéfalo/patologia , Células Cultivadas , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neuroglia/ultraestrutura , Transfecção/métodosRESUMO
STUDY DESIGN: A literature review. OBJECTIVE: To summarize the implant removal rate, common bacterial organisms found, time of onset, ratio of superficial to deep infection, and regurgitating the prevalence among all the retrospective and prospective studies on management and characterization of surgical site infections (SSIs). METHODS: PubMed was searched for articles published between 2000 and 2018 on the management or characterization of SSIs after spinal surgery. Only prospective and retrospective studies were included. RESULTS: A total of 49 articles were found relevant to the objective. These studies highlighted the importance of implant removal to avoid recurrence of SSI. The common organisms detected were methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermis, Staphylococcus epidermis, Staphylococcus aureus, and Propionibacterium acnes, with prevalence of 1% to 15%. A major proportion of all were deep SSI, with minority reporting on late-onset SSI. CONCLUSION: Long-term antibiotics administration, and continuous irrigation and debridement were common suggestion among the authors; however, the key measure undertaken or implied by most authors to avoid risk of recurrence was removal or replacement of implants for late-onset SSI.
RESUMO
The current communication seeks to provide an updated narrative review on latest methods of reducing implant contaminations used during spine surgery. Recent literature review has shown that both preoperative reprocessing and intraoperative handling of implants seem to contaminate implants. In brief, during preoperative phase, the implants undergo repeated bulk cleaning with dirty instruments from the OR, leading to residue buildup at the interfaces and possibly on the surfaces too. This, due to its concealed nature, remains unnoticed by the SPD (sterile processing department) or other hospital staff. Nevertheless, these can be avoided by using individually prepackaged presterilized implants. In the intraoperative phase, the implants (in the sterile field) are directly touched by the scrub tech with soiled (assisting the surgeon dispose the tissues from the instruments in use) gloves for loading onto an insertion device. It is then kept exposed on the working table (either separately or next to the used instruments as the pedicles hole are being prepared). Latest investigation has shown that by the time it is implanted in the patient, it can harbor up to 10e7 bacterial colony-forming units. The same implants were devoid of such colony-forming units, when sheathed by an impermeable sterile sheath around the sterile implant.
RESUMO
STUDY DESIGN: A prospective multicenter study. OBJECTIVE: The objective of this study was to assess bacterial contamination in current practices of pedicle screw handling and comparing it to a novel method of using an intraoperative, sterile implant guard for screws. SUMMARY OF BACKGROUND DATA: Postoperative infections occur at the higher end of 2%-13%, as cited in the literature, and are underestimated due to various reasons in such publications. Despite concerns associated with vancomycin application immediately before closure, it is theoretically impossible to irrigate the screw-bone interface postimplantation. Consequently, any contamination of pedicle screw before implantation is permanent, and has the potential to cause deep-bone infection, or hardware loosening due to encapsulation of biofilm between the bone and the screw. Therefore, continued vigilance and effective preventive measures should be undertaken if available. MATERIALS AND METHODS: Two groups of presterile individually-packaged pedicle screws, one incased in a sterile, protective guard (group 1: G) and the other without such a guard (group 2: NG), 31 samples in each group were distributed over 28 spinal fusion surgeries at 5 independent hospitals groups. Each were loaded onto the insertion device by the scrub tech and left on the sterile table. Twenty minutes later, the lead surgeon who had just finished preparing the surgical site, handles the pedicle screw, to check the fit with the insertion device. Then, instead of implantation, it was transferred to a sterile container using fresh sterile gloves for bacterial analysis. RESULTS: The standard unguarded pedicle screws presented bioburden in the range of 10 to 10 colonies forming units per screw, whereas the guarded pedicle screws showed no bioburden. CONCLUSION: Standard, current, handling of pedicle screws leads to bacterial contamination, which can be avoided if the screws are sterilely prepackaged with an intraoperative guard (preinstalled).
Assuntos
Parafusos Pediculares , Doenças da Coluna Vertebral/cirurgia , Fusão Vertebral , California , Contaminação de Equipamentos , Humanos , Índia , Ohio , Estudos Prospectivos , Infecção da Ferida Cirúrgica/prevenção & controleRESUMO
ACBD5 deficiency is a novel peroxisome disorder with a largely uncharacterized pathology. ACBD5 was recently identified in a tethering complex mediating membrane contacts between peroxisomes and the endoplasmic reticulum (ER). An ACBD5-deficient mouse was analyzed to correlate ACBD5 tethering functions with the disease phenotype. ACBD5-deficient mice exhibit elevated very long-chain fatty acid levels and a progressive cerebellar pathology. Liver did not exhibit pathologic changes but increased peroxisome abundance and drastically reduced peroxisome-ER contacts. Lipidomics of liver and cerebellum revealed tissue-specific alterations in distinct lipid classes and subspecies. In line with the neurological pathology, unusual ultra-long chain fatty acids (C > 32) were elevated in phosphocholines from cerebelli but not liver indicating an organ-specific imbalance in fatty acid degradation and elongation pathways. By contrast, ether lipid formation was perturbed in liver towards an accumulation of alkyldiacylglycerols. The alterations in several lipid classes suggest that ACBD5, in addition to its acyl-CoA binding function, might maintain peroxisome-ER contacts in order to contribute to the regulation of anabolic and catabolic cellular lipid pathways.