Identification of dystrophin Dp71dΔ71-associated proteins in PC12 cells by quantitative proteomics.

Dystrophin Dp71 is essential for the development of the nervous system. Its alteration is associated with intellectual disability. Different Dp71 isoforms are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand the complex functions. PC12 cells, stably transfected with pTRE2pur-Myc/Dp71dΔ71 or pTRE2pur-Myc empty vector (EV), were analyzed by immunoprecipitation followed with quantitative proteomics with data-independent acquisition and ion mobility separation. We used the Top3 method to quantify absolutely every protein detected. A total of 106 proteins were quantified with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12-Myc/Dp71dΔ71 versus PC12-EV cells. These results revealed new proteins that interact with Dp71dΔ71, including ß-Tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10, which are related to cell migration and/or cell growth. The results lay the foundation for future research on the relationship between these proteins and Dp71 isoforms.

Nuclear transport and subcellular localization of the dystrophin Dp71 and Dp40 isoforms in the PC12 cell line.

The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/ß importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.

Overexpression of the dystrophins Dp40 and Dp40L170P modifies neurite outgrowth and the protein expression profile of PC12 cells.

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown. Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression. Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated. The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

The dystrophin isoform Dp71eΔ71 is involved in neurite outgrowth and neuronal differentiation of PC12 cells.

The Dp71 protein is the most abundant dystrophin in the central nervous system (CNS). Several dystrophin Dp71 isoforms have been described and are classified into three groups, each with a different C-terminal end. However, the functions of Dp71 isoforms remain unknown. In the present study, we analysed the effect of Dp71eΔ71 overexpression on neuronal differentiation of PC12 Tet-On cells. Overexpression of dystrophin Dp71eΔ71 stimulates neuronal differentiation, increasing the percentage of cells with neurites and neurite length. According to 2-DE analysis, Dp71eΔ71 overexpression modified the protein expression profile of rat pheochromocytoma PC12 Tet-On cells that had been treated with neuronal growth factor (NGF) for nine days. Interestingly, all differentially expressed proteins were up-regulated compared to the control. The proteomic analysis showed that Dp71eΔ71 increases the expression of proteins with important roles in the differentiation process, such as HspB1, S100A6, and K8 proteins involved in the cytoskeletal structure and HCNP protein involved in neurotransmitter synthesis. The expression of neuronal marker TH was also up-regulated. Mass spectrometry data are available via ProteomeXchange with identifier PXD009114. SIGNIFICANCE: This study is the first to explore the role of the specific isoform Dp71eΔ71. The results obtained here support the hypothesis that the dystrophin Dp71eΔ71 isoform has an important role in the neurite outgrowth by regulating the levels of proteins involved in the cytoskeletal structure, such as HspB1, S100A6, and K8, and in neurotransmitter synthesis, such as HCNP and TH, biological processes required to stimulate neuronal differentiation.

Overexpression of mutant dystrophin Dp71[INCREMENT]78-79 stimulates cell proliferation.

Dp71 dystrophin is the main DMD gene product expressed in the central nervous system. Experiments using PC12 cells as a neuronal model have shown that Dp71 isoforms are involved in differentiation, adhesion, cell division, and nuclear architecture. To contribute to the knowledge of Dp71 domains function, we previously reported the isolation and partial characterization of the dystrophin Dp71[INCREMENT]78-79 (a mutant that lacks exons 71, 78, and 79), which stimulates the neuronal differentiation of PC12-C11 clone. In this article, we generated a doxycycline (Dox)-inducible expression system in PC12 Tet-On cells (B10 cells) to overexpress and control the transcription of Dp71[INCREMENT]78-79. Western blotting and confocal microscopy showed an increase in the amount of Dp71[INCREMENT]78-79 (217±75-fold) with the addition of Dox to growth medium. Cell proliferation assays and morphometric analyses demonstrated that Dp71[INCREMENT]78-79 increases the growth rate of B10 cells and reduces the nerve growth factor-neuronal differentiation. Western blotting analysis revealed an upregulation in the expression of proliferating cell nuclear antigen, focal adhesion kinase, and ß-dystroglycan in B10 cells compared with control cells. Our results show that the inducible expression of Dp71[INCREMENT]78-79 increases the growth rate of PC12 Tet-On cells, suggesting a role of this protein in cell proliferation.

Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with ß-Dystroglycan and α1-Syntrophin.

Several dystrophin Dp71 messenger RNA (mRNA) alternative splice variants have been described. According to the splicing of exon 78 or intron 77, Dp71 proteins are grouped as Dp71d, Dp71f, and Dp71e, and each group has a specific C-terminal end. In this study, we explored the expression of Dp71 isoforms at the complementary DNA (cDNA) level and the subcellular localization of recombinant Myc-Dp71 proteins in PC12 cells. We determined that PC12 cells express Dp71a, Dp71c, Dp71ab, Dp71e, and Dp71ec mRNA splice variants. In undifferentiated and nerve growth factor-differentiated PC12 Tet-ON cells, Dp71a, Dp71ab, and Dp71e were found to localize and colocalize with ß-dystroglycan and α1-syntrophin in the periphery/cytoplasm, while Dp71c and Dp71ec were mainly localized in the cell periphery and showed less colocalization with ß-dystroglycan and α1-syntrophin. The levels of Dp71a, Dp71e, and Dp71ec were increased in the nucleus of differentiated PC12 Tet-ON cells compared to undifferentiated cells. Dp71 isoforms were also localized in neurite extensions and growth cones.

In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling.

Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids (93)LEQEHNNLV(101) and (168)LLLHDSIQI(176) could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled "EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40" (J. Aragón et al. Neurosci. Lett. 600 (2015) 115-120) [1].

EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40.

Dp40 is the shortest DMD gene product that has been reported to date. It is encoded by exons 63-70, a region required for a ß-dystroglycan interaction. Its expression has been identified in rat, mouse, and human; however, its function remains unknown. To explore the expression of Dp40 transcript and subcellular localization of epitope-tagged Dp40 proteins, RT-PCR and immunofluorescence assays were performed in PC12 cells. The expression of Dp40 mRNA was found in undifferentiated and nerve growth factor-differentiated PC12 cells. According to immunofluorescence analyses, the recombinant protein Dp40 was mainly localized in the cell periphery/cytoplasm of undifferentiated and differentiated PC12 cells, a small amount of this protein is localized to the nucleus of differentiated cells. With the aim to identify the amino acids involved in the nuclear localization of Dp40, an in silico analysis was performed and it predicted that prolines 93 and 170, located within EF1 and EF2-hand domains, are involved in the nuclear localization of this protein. This prediction was confirmed by site-directed mutagenesis, the Dp40-L93P mutant was localized to the nucleus and cell periphery, while Dp40-L170P and Dp40-L93/170P showed mainly a nuclear localization. Dp40 co-localizes with ß-dystroglycan and the co-localization score was statistically reduced in Dp40-L93P, Dp40-L170P and Dp40-L93/170P mutants.

Identification of Dp71e, a new dystrophin with a novel carboxy-terminal end.

Several dystrophin Dp71 isoforms have previously been described and can be grouped into two subfamilies (Dp71d or Dp71f) depending upon the splicing of exon 78. As a consequence of this splicing, each group has a carboxy-terminal end with a unique amino acid composition; this composition imparts specific characteristics with respect to subcellular localization and interactions with particular members of the dystrophin-associated proteins (DAPs) complex. We have discovered a new alternative splicing event at the 3' region of the Dp71 transcript. This spliced region has a unique sequence that codes for 10 amino acids and prevents the translation of exons 78 and 79. This novel Dp71 isoform is called Dp71e and is expressed in undifferentiated cells and during nerve growth factor-induced differentiation of PC12 cells. Interestingly, Dp71e mRNA and protein expression increase during PC12 cell differentiation mediated by NGF. This new Dp71 isoform is also expressed in rat organs and in human cell lines.

A 63 kDa VSP9B10A-like protein expressed in a C-8 Giardia duodenalis Mexican clone.

BACKGROUND: It is well documented that Giardia duodenalis undergoes surface antigenic variation both in vivo and in vitro. Proteins involved have been characterized and referred to as VSP (variable surface protein). METHODS: Two cloned cDNA inserts of 0.45 and 1.95 kb were obtained from G. duodenalis expression library and sequenced. Comparison sequence analyses were made against Genbank. PCR analysis was performed on G. duodenalis isolates to identify isolates bearing genes encoding such a peptide. Specific antiserum was prepared against 450-bp encoded peptide and tested by Western blot, immunofluorescence, and inhibition of adhesion of G. duodenalis to target cells. RESULTS: We cloned and characterized a G. duodenalis 450-bp DNA fragment; its DNA sequence analysis revealed that this fragment displayed 99% identity with vsp9B10A gene. Predicted amino acid sequence for this fragment also had significant (99%) identity to VSP9B10A. A second 1.95-kb insert, which encompassed the 450-bp cDNA fragment, was also isolated; its DNA and amino acid sequence displayed 99.5% identity with vsp9B10A gene and 99.2% with the corresponding inferred protein, respectively. This inferred protein contained 24 Cys-X-X-Cys motifs and long ORF of 642 aminoacids. PCR analysis showed that DNA sequence encoding a fragment of this gene was present in P1, CIEA:0487:2-C-8 clone and in INP:180800-B2 G. duodenalis human isolates, while it was absent in sheep isolate of G. duodenalis INP:150593-J10. CONCLUSIONS: Immunofluorescence analysis using antibodies raised against the peptide encoded by 450-bp fragment showed that expression of this epitope varies on trophozoite surface of the C-8 Mexican clone and is involved in parasite adhesion to target epithelial cells.

Abordaje terapéutico de los aneurismas aórticos asociados a neoplasia / Association between aortic aneurysm and malignant neoplasia therapeutic accesible

La asociación entre un aneurisma aórtico y neoplasia maligna es poco frecuente. Cuando se encuentra, es necesario tomar una decisión en cuanto a cuál deberá ser resecado primero. En el presente artículo, se muestra una serie de nueve pacientes con aneurisma de la aorta y carcinoma en localizaciones intra o extraabdominales, su tratamiento y su evolución postoperatoria. Finalmente, se hace una serie de comentarios en cuanto a las pautas de tratamiento cuando se encuentra esta asociación

Comparative analisis plasmids from nosocomial Klebsiella pneumoniae strains isolated from two hospitals

A total of 46 clinical isolates of Klebsiella pneumoniae were studied. Of these, 33 were from "Hospital Infantil de Mexico" (HIM) and 13 from "Hospital General de Mexico" (HGM). The susceptibility of these strains to five antibiotics, as well as the plasmid DNA profiles, were determined for each group. Antibiotic susceptibility profiles were very similar in strains from both hospitals; however, most of the strains analyzed exhibited heterogeneous plasmid DNA profiles. Results showed that strains isolated in the two hospitals did not differ regarding morphology, biochemical profiles, antibiotic susceptibility or plasmid populations, and these characteristics may not be used as markers to differentiate Klebsiella pneumoniae strains from different hospitals