Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells / 中华肿瘤杂志

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

Ubiquitinome Profiling Reveals the Landscape of Ubiquitination Regulation in Rice Young Panicles / 基因组蛋白质组与生物信息学报·英文版

Ubiquitination, an essential post-transcriptional modification (PTM), plays a vital role in nearly every biological process, including development and growth. Despite its functions in plant reproductive development, its targets in rice panicles remain unclear. In this study, we used proteome-wide profiling of lysine ubiquitination in rice (O. sativa ssp. indica) young panicles. We created the largest ubiquitinome dataset in rice to date, identifying 1638 lysine ubiquitination sites on 916 unique proteins. We detected three conserved ubiquitination motifs, noting that acidic glutamic acid (E) and aspartic acid (D) were most frequently present around ubiquitinated lysine. Enrichment analysis of Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these ubiquitinated proteins revealed that ubiquitination plays an important role in fundamental cellular processes in rice young panicles. Interestingly, enrichment analysis of protein domains indicated that ubiquitination was enriched on a variety of receptor-like kinases and cytoplasmic tyrosine and serine-threonine kinases. Furthermore, we analyzed the crosstalk between ubiquitination, acetylation, and succinylation, and constructed a potential protein interaction network within our rice ubiquitinome. Moreover, we identified ubiquitinated proteins related to pollen and grain development, indicating that ubiquitination may play a critical role in the physiological functions in young panicles. Taken together, we reported the most comprehensive lysine ubiquitinome in rice so far, and used it to reveal the functional role of lysine ubiquitination in rice young panicles.

The destruction complex of beta-catenin in colorectal carcinoma and colonic adenoma / O complexo destruidor de betacatenina no carcinoma colorretal e no adenoma cólico

ABSTRACT Objective To evaluate the destruction complex of beta-catenin by the expression of the proteins beta-catetenin, adenomatous polyposis coli, GSK3β, axin and ubiquitin in colorectal carcinoma and colonic adenoma. Methods Tissue samples from 64 patients with colorectal carcinoma and 53 patients with colonic adenoma were analyzed. Tissue microarray blocks and slides were prepared and subjected to immunohistochemistry with polyclonal antibodies in carcinoma, adjacent non-neoplastic mucosa, and adenoma tissues. The immunoreactivity was evaluated by the percentage of positive stained cells and by the intensity assessed through of the stained grade of proteins in the cytoplasm and nucleus of cells. In the statistical analysis, the Spearman correlation coefficient, Student’s t, χ2, Mann-Whitney, and McNemar tests, and univariate logistic regression analysis were used. Results In colorectal carcinoma, the expressions of beta-catenin and adenomatous polyposis coli proteins were significantly higher than in colonic adenomas (p<0.001 and p<0.0001, respectively). The immunoreactivity of GSK3β, axin 1 and ubiquitin proteins was significantly higher (p=0.03, p=0.039 and p=0.03, respectively) in colorectal carcinoma than in the colonic adenoma and adjacent non-neoplastic mucosa. The immunohistochemistry staining of these proteins did not show significant differences with the clinical and pathological characteristics of colorectal cancer and colonic adenoma. Conclusions These results suggest that, in adenomas, the lower expression of the beta-catenin, axin 1 and GSK3β proteins indicated that the destruction complex of beta-catenin was maintained, while in colorectal carcinoma, the increased expression of beta-catenin, GSK3β, axin 1, and ubiquitin proteins indicated that the destruction complex of beta-catenin was disrupted.

RESUMO Objetivo Avaliar o complexo de destruição da betacatenina no carcinoma colorretal e no adenoma do colo pela expressão das proteínas betacatenina, adenomatous polyposis coli, GSK3β, axina e ubiquitina. Métodos Amostras de tecidos de 64 doentes com carcinoma colorretal e de 53 pacientes com adenoma do colo foram analisadas. Blocos de tecidos foram submetidos ao estudo imuno-histoquímico com anticorpos policlonais nos tecidos do carcinoma, mucosa não neoplásica adjacente e adenoma. A imunorreatividade foi avaliada pela porcentagem de positividade de células coradas e pela intensidade do grau de coloração das proteínas no citoplasma e no núcleo das células. Na análise estatística, foram utilizados o coeficiente de correlação de Spearman, os testes t de Student, χ2, Mann-Whitney e de McNemar, e a análise de regressão logística univariada. Resultados No carcinoma colorretal, as expressões da betacatenina e da adenomatous polyposis coli foram significativamente maiores do que em adenomas do colo (p<0,001 e p<0,0001, respectivamente). A imunorreatividade das proteínas GSK3β, axina 1 e ubiquitina foi significativamente maior (p=0,03, p=0,039 e p=0,03, respectivamente) no carcinoma colorretal do que no adenoma e na mucosa não neoplásica adjacente. A coloração imuno-histoquímica dessas proteínas não apresentou diferenças significantes em relação às características clinicopatológicas do câncer colorretal e do adenoma. Conclusões Em adenomas, as menores expressões de betacatenina, axina 1 e GSK3β indicaram que o complexo de destruição da betacatenina estava conservado, enquanto que, no carcinoma colorretal, o aumento das expressões da betacatenina, GSK3β, 1 axina, e ubiquitina indicaram que o complexo de destruição de betacatenina estava alterado.

Six-week anaerobic training improves proteolytic profile of diabetic rats

Objective To evaluate the effect of six-week anaerobic training on the mRNA expression of genes related to proteolysis Ubb (Ubiquitin), E2-14kDa, Trim63 (MuRF1 protein) and Nfkb1 in the skeletal muscle of diabetic rats.Materials and methods Four groups were established: DE (DiabetesExercised), DS (Diabetes Sedentary), CE (Control Exercised) and CS (Control Sedentary). The training consisted of 3 sets of 12 jumps in the liquid mean with load equivalent to 50% of BW for 6 weeks. Euthanasia occurred under ip anesthesia, and blood, adipose tissue and skeletal muscles were collected. Gene expression was quantified by RT–PCR in the gastrocnemius muscle. ANOVA one-way was used for comparison among groups, with post-hoc (Tukey) when necessary, considering p < 0.05.Results We observed reduction in the body weight and adipose tissue in the diabetic groups. The muscle mass was reduced in DS, which could be reversed by training (DE). Although DS and DE have presented similar body weight, the training protocol in DE promoted reduction in the adipose tissue, and increase of muscle mass. Anaerobic training was efficient to reduce glycaemia only in the diabetic animals until 6 hours after the end of training. The Trim63 gene expression was increased in DS; decreased Ubb gene level was observed in trained rats (CE and DE) compared to sedentary (CS and DS), and DE presented the lowest level of E2-14kDa gene expression.Conclusion Six-week anaerobic training promoted muscle mass gain, improved glycemic control, and exerted inhibitory effect on the proteolysis of gastrocnemius muscle of diabetic rats.

HIF-1alpha Upregulation due to Depletion of the Free Ubiquitin Pool

Hypoxia-inducible factor 1alpha (HIF-1alpha), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1alpha is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1alpha. The poly-ubiquitination of HIF-1alpha was resumed by restoration of free ubiquitin, which suggests that the HIF-1alpha stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1alpha with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1alpha.

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into beta-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

Necessidades proteicas, morbidade e mortalidade no paciente grave: fundamentos e atualidades / Protein requirements, morbidity and mortality in critically ill patients: fundamentals and applications

Evidências recentes sugerem que o balanço proteico negativo secundário à doença grave se associa ao aumento de morbidade. A perda da proteína corporal total é inevitável nesse cenário, mesmo com uma abordagem nutricional agressiva, e resulta, principalmente, do catabolismo da fibra muscular esquelética. O principal mecanismo bioquímico e metabólico envolvido nesse processo é o sistema ubiquitina-proteassoma, que, paradoxalmente, consome a adenosina trifosfatocomo fonte energética e motriz. É possível que a neutralidade do balanço proteico nessas instâncias clínicas, seja tão importante na melhora dos desfechos quanto atingir a meta calórica estimada ou medida pela calorimetria indireta. Estudos recentes apontam a utilização de concentrações mais elevadas de proteínas na terapia nutricional do paciente grave como importante para um impacto positivo na mortalidade. A proposta deste trabalho foi revisar alguns princípios da terapia nutricional relativos ao metabolismo proteico, sinalizar para as principais assertivas das diretrizes das sociedades especializadas e comentar estudos recentes, que abordam a questão em tela, sob a visão crítica da experiência clínica dos autores.

Recent evidence suggests that a negative protein balance secondary to severe disease is associated with increased morbidity. A loss of total body protein is inevitable in this scenario, even with an aggressive nutritional approach, primarily due to the catabolism of skeletal muscle fibers. The ubiquitin-proteasome system is the primary metabolic and biochemical mechanism involved in this process; paradoxically, this system consumes adenosine triphosphate as its energy source. It is possible that a neutral protein balance in these clinical situations is important for improving outcomes and achieving the caloric goals estimated or measured by indirect calorimetry. Recent studies have suggested that the use of higher protein concentrations in nutritional therapy for critically ill patients may help to reduce mortality. The purpose of this study was to review some of the nutrition therapy principles related to protein metabolism, evaluate the main assertions of the guidelines of specialty societies and review the recent studies that address these issues using critical insights from the authors' clinical experience.

Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting / La degradación intracelular de proteínas: Desde una vaga idea, a través del lisosoma y el sistema ubiquitina-proteosoma a las enfermedades humanas y el blanco de las drogas

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely non-lysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.

Entre los años 1950 y 1980 los científicos focalizaron sus estudios sobre la forma en que el código genético es transcripto al ARN y traducido a las proteínas, dejando de lado la forma en que éstas se degradan. Con el descubrimiento de los lisosomas por Christian de Duve se asumió que las proteínas se degradaban en el interior de esa organela. Sin embargo, varias líneas de trabajo independientes sugerían fuertemente que la proteólisis intracelular era en su mayor parte no lisosómica, aunque se desconocían sus mecanismos. El descubrimiento del sistema ubiquitina-proteosoma resolvió el enigma. Ahora sabemos que la degradación intracelular de proteínas participa en la regulación de un amplio espectro de procesos celulares como la división y el ciclo celular, la regulación de los factores de transcripción y el control de la calidad celular. No es sorpresa entonces que las aberraciones del sistema estén relacionadas con la patogénesis de enfermedades humanas como tumores y desórdenes neurodegenerativos, lo que llevó luego a un esfuerzo para desarrollar drogas basadas en este mecanismo.

The quantitative analysis of Ub and UbE1 in focus of traumatic epilepsy / 法医学杂志

OBJECTIVE@#To study the expression of ubiquitin proteasome system (UPS) in the traumatic epilepsy pathogenesis and its value in traumatic epilepsy by quantitative analysis.@*METHODS@#Fifteen specimens from human epileptic temporal cortex from PTE were collected as the PTE group. Fifteen specimens from non-PTE were collected as the non-PTE group. Fifteen normal cerebral cortex specimens died from acute traffic accident were collected as the control group. The expression of mRNA and protein of Ub and UbE1 were detected by RT-PCR and Western blot. Statistical analysis was used to compare the data between three groups.@*RESULTS@#The expression of mRNA and protein of Ub and UbE1 were the following order: PTE group(high), non-PTE group(middle) and control group(low).@*CONCLUSION@#The study confirms that UPS is up-regulated in the epilepsy's focus, especially in traumatic epilepsy. The activation of UPS may be an important pathological change in neurons in pathogenesis of traumatic epilepsy.

Morphology changes of ubiquitin-proteasome system in traumatic epilepsy / 法医学杂志

OBJECTIVE@#To investigate the value of ubiquitin(Ub) and ubiquitin-activating enzymel(UbE1) for the appraisement of post traumatic epilepsy (PTE).@*METHODS@#Fifteen specimens from human epileptic temporal cortex originating from PTE were collected as the PTE group. Fifteen specimens from non-PTE were collected as the non-PTE group. Meanwhile, 15 normal cerebral cortex specimens from people dead from acute traffic accident were collected as the control groups. Observe morphology changes of each group with HE, then with immunohistochemistry of Ub and UbE1.@*RESULTS@#Compared to the control group, morphology changes of neuron quantity reduction, neuron denaturation and so on were observed both in the PTE group and the non-PTE group under HE, especially in the PTE group. Ub and UbE1 mainly expressed in the nucleus and cytoplasm of the neurons in epilepsy spot without extracellular expression. The expression of Ub and UbE1 is PTE group > non-PTE group > control group (P < 0.05).@*CONCLUSIONS@#The neuron denaturation are one of the main pathology changes of epilepsy, and it is more obvious in the PTE group. Immunohistochemistry of Ub and UbE1 may be more helpful to distinguish PTE and non-PTE than HE staining.

Selectively Decreased Expression of Peroxiredoxins Induced by Silica in Pulmonary Epithelial Cells

BACKGROUND/AIMS: Peroxiredoxin (Prx) belongs to a ubiquitous family of antioxidant enzymes that regulates many cellular processes through intracellular oxidative signal transduction pathways. Silica-induced lung damage involves reactive oxygen species (ROS) that trigger subsequent toxic effects and inflammatory responses in alveolar epithelial cells resulting in fibrosis. Therefore, we investigated the role of Prx in the development of lung oxidant injury caused by silicosis, and determined the implication of ROS in that process. METHODS: Lung epithelial cell lines A549 and WI26 were treated with 1% silica for 0, 24, or 48 hours, following pretreatment of the A549 cells with N-acetyl-L-cysteine and diphenylene iodonium and no pretreatment of the WI26 cells. We transfected an HA-ubiquitin construct into the A549 cell line and then analyzed the cells via Western blotting and co-immunoprecipitation. RESULTS: Silica treatment induced cell death in the A549 lung epithelial cell line and selectively degraded Prx I without impairing protein synthesis in the A549 cells, even when the ROS effect was blocked chemically by N-acetyl-L-cysteine. A co-immunoprecipitation study revealed that Prx I did not undergo ubiquitination. CONCLUSIONS: Silica treatment induces a decrease of Prx I expression in lung epithelial cell lines regardless of the presence of ROS. The silica-induced degradation of Prx does not involve the ubiquitin-proteasomal pathway.

Suppression of prostate cancer growth by resveratrol in the transgenic rat for adenocarcinoma of prostate (TRAP) model.

Research into actions of resveratrol, abundantly present in red grape skin, has been greatly stimulated by its reported beneficial health influence. Since it was recently proposed as a potential prostate cancer chemopreventive agent, we here performed an in vivo experiment to explore its effect in the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model, featuring the rat probasin promoter/SV 40 T antigen. Resveratrol suppressed prostate cancer growth and induction of apoptosis through androgen receptor (AR) down-regulation, without any sign of toxicity. Resveratrol not only downregulated androgen receptor (AR) expression but also suppressed the androgen responsive glandular kallikrein 11 (Gk11), known to be an ortholog of the human prostate specific antigen (PSA), at the mRNA level. The data provide a mechanistic basis for resveratrol chemopreventive efficacy against prostate cancer.

Inmunohistoquímica de los cambios degenerativos del sistema nervioso central en paraparesias esp sticas asociadas al virus linfotrópico humanoTtipol(HTLV-l) / Immunohistochemistry of degenerative changes in the central nervous system in spastic paraparesis associated to human T lymphotropic virus type I (HTLV-I)

Background: Human T lymphotropic virus type I is associated with tropical spastic paraparesis, that is a chronic and progressive disease which damages specially the cortiespinal tracts. The pathogenesis of this degenerative process remains unknown. Aim: To identify histopathological aspects that could suggest a pathogenic hypothesis we studied immunohistochemical features in spinal cords obtained from patients that died due to progressive spastic paraparesis. Patients and Methods: Five males and five females, who died between 1990 and 2000, with a mean age of 52 years and mean disease duration of 8.6, were studied. All had a complete clinical and virological diagnosis. Samples were obtained from the frontal motor cortex and spinal cord (cervical, dorsal and lumbar segments), were fixed in formol (10 percent), included in paraffin, and stained with Haematoxylin and Luxol-fast-blue. Immunohistochemical study was made with anti-neurofilament antibodies 1:100 (M0762, DAKO), anti-APP 1:20 (Rabbit Pre Amyloid protein 51-2700 ZYMED), anti-tau 1:100 (A0024DAKO) and anti-ubiquitine 1:50 (NCL UBIQm Novocastra). Results: All cases had demyelinization and axonal loss in the cortico-spinal tracts; distal and segmental demyelinization of Goll tract; axonal thickening, amyloid precursor protein deposits in the white matter; tau protein aggregation in the spinal cord oligodendrocytes; axonal ubiquitination of sensitive and motor tracts, and subcortical white matter. Neurona! injury was absent. Conclusions: The systematic damage of motor and sensitive tracts of the spinal-cord and the absence of neurona! damage, defines a degenerative process limited to axons. This central axonopathie could be caused by a disturbance of axoplasmic transport.

The expression of ubiquitin in the healing process of incised wounds of rat's skin and its significance to forensic medicine / 法医学杂志

OBJECTIVE@#To evaluate the changes of ubiquitin expression in incised wounds of the rat skin.@*METHODS@#The testing rat groups were subjected to incised skin wounds, with normal rat skin used as control. The expression level of ubiquitin was assessed using immunohistochemistry and imaging analysis technique on skin samples taken at 1, 3, 6, 12h, and on day 1, 3, 6, 10, and 14d after injury.@*RESULTS@#The expression level of ubiquitin was low in the skin of normal control group. Increased level of ubiquitin expression could be observed 1 h after injury. The expression level of ubiquitin reached its peak on day 6 and started to decline on day 10, and then returned to its normal level on day 14 d after injury.@*CONCLUSION@#Ubiquitin may serve as a potentially useful marker for forensic determination of the skin wound age.

Conjugation and deconjugation of ubiquitin regulating the destiny of proteins

The homeostasis for a number of cellular proteins is regulated by not only phosphorylation and dephosphorylation, but also ubiquitination and deubiquitination. A number of proteins involved in the degradation of polypeptides have been isolated in various eukaryotic organisms from Saccharomyces cerevisiae to human. Recently, several deubiquitinating enzymes, classified into either the Ub C-terminal hydrolase (UCH) or the Ub-specific processing protease (UBP), have been reported. It has been shown that they contain conserved domains including Cys, His, and Asp residues throughout the enzyme. These proteins have been demonstrated that Cys and His domains are critical for deubiquitinating enzymatic activity. Recently, we have shown that the Asp domain localized between Cys and His domains is also essential for cleaving the ubiquitin from protein substrates. Mouse deubiquitinating enzymes including DUB-1, DUB-2, and DUB-2A have been isolated and they showed the expression specificity. Of these, DUB- 1 and DUB-2 are expressed in lymphocytes depending on the presence of cytokines (interleukin-3 in B-lymphocytes and interleukin-2 in T- lymphocytes, respectively), indicating that they are involved in cytokine signaling pathways. Isolation of all putative DUBs will help to identify their substrates and to regulate the homeostasis of cellular proteins, especially in proliferative cells.

Mechanisms of protein degradation in Trypanosoma cruzi

Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50 percent) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5 percent glucose or 10 percent serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins

Presencia de condensaciones pericanaliculares inmunogénicamente semejantes a cuerpos de Mallory en colestasia / Presence of perinacular condensations immunogenicaly similar to Mallory's bodies in cholestasia

El rol del citoesqueleto del hepatocito en secreción biliar y en la génesis de colestasia ha sido estudiado especialmente en relación a microfilamentos y microtúbulos, restándosele importancia los filamentos intermedios (FI). En cambio, estudios recientes han demostrado que la integridad de los FI del hepatocito es fundamental en la secreción biliar. Los FI del hepatocito corresponden a citoqueratinas y en ciertas patologías, especialmente de etiología alcohólica, éstos se alteran y en algunas ocasiones forman agregados como los cuerpos de Mallory (CM) cambiando sus características antigénicas. Se estudiaron 131 biopsias hepáticas con técnicas inmunohistoquímicas, utilizando sueros anti-queratinas epidérmicas y anti-ubiquitina, polipéptido de función proteolítica de proteínas anormales. De las biopsias estudiadas, 47 por ciento presentaban signos de colestasia y de éstas, un 64 por ciento presentó inmunorreacción en condensaciones pericanulares con anti-ubiquitina y un porcentaje algo menor con anti-queratinas; el resto de las biopsias fueron negativas con ambos anticuerpos. Estas observaciones indican que en biopsias hepáticas con signos de colestasia, las condensaciones pericaniculares correspondían a FI alterados, con características inmunogénicas semejantes a CM