RESUMEN
Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.
Asunto(s)
Fertilidad/genética , Infertilidad Masculina/genética , Carioferinas/genética , Motilidad Espermática/genética , Espermatozoides/anomalías , alfa Carioferinas/genética , Reacción Acrosómica/genética , Animales , Flagelos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Espermatogénesis/genética , Testículo/anomalíasRESUMEN
Aggregation of 42-residue amyloid ß-protein (Aß42) can be prevented by ß-sheet breaker peptides (BSBps) homologous to LVFFA residues, which are included in a ß-sheet region of Aß42 aggregates. To enhance the affinity of BSBps to the Aß42 aggregates, we designed and synthesized ß-strand-fixed peptides (BSFps) whose side chains were cross-linked by ring closing metathesis. Conformation analysis verified that the designed peptides could be fixed in ß-strand conformation. Among the synthesized pentapeptides, 1 and 12, whose side chains of 2nd and 4th residues were cross-linked, significantly inhibited the aggregation of Aß42. This suggested that ß-strand-fixation of BSBps could enhance their inhibitory activity against the Aß42 aggregation. However, pentapeptides 1 and 12 had little effect on morphology of Aß42 aggregates (fibrils) and neurotoxicity of Aß42 against SH-SY5Y cells.
Asunto(s)
Amiloide/química , Fármacos Neuroprotectores/química , Oligopéptidos/química , Agregado de Proteínas/efectos de los fármacos , Secuencia de Aminoácidos , Amiloide/metabolismo , Diseño de Fármacos , Humanos , Modelos Moleculares , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Unión Proteica , Conformación Proteica , Conformación Proteica en Lámina beta , Relación Estructura-ActividadRESUMEN
Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2 accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2 in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.
Asunto(s)
Membrana Basal Glomerular/metabolismo , Tasa de Filtración Glomerular , Podocitos/metabolismo , Tensinas/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Adhesión Celular , Células Cultivadas , Membrana Basal Glomerular/ultraestructura , Laminina/genética , Laminina/metabolismo , Ratones Noqueados , Podocitos/ultraestructura , Estrés Mecánico , Tensinas/deficiencia , Tensinas/genéticaRESUMEN
Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid ß42 (Aß42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer's disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aß42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aß42 formed a partial ß-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated ß-sheet-rich 8-16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aß42 persulfidation in Alzheimer's disease.
RESUMEN
Here, we report the first synthesis of quasi-stable trimer models of full-length Aß40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.
RESUMEN
The formation of soluble oligomers of amyloid ß42 and 40 (Aß42, Aß40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aß42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aß42, Aß40 dimers can also contribute to AD progression because of the more abundance of Aß40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aß40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel ß-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aß40 dimer (1) and E22P-A30DAZ-Aß40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aß40 dimer (3) formed ß-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel ß-sheet, triggers the formation of toxic Aß oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Línea Celular , Dicroismo Circular , Humanos , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Neuronas/efectos de los fármacos , Neuronas/patologíaRESUMEN
Amyloid ß-protein (Aß42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aß42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aß42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aß42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aß42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aß42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aß42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/líquido cefalorraquídeo , Fragmentos de Péptidos/inmunología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/toxicidad , Animales , Anticuerpos Monoclonales/química , Estudios de Casos y Controles , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Masculino , Conformación Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/toxicidad , Ratas Wistar , Resonancia por Plasmón de SuperficieRESUMEN
Tensin2 (Tns2) is an essential component for the maintenance of glomerular basement membrane (GBM) structures. Tns2-deficient mice were previously shown to develop mild glomerular injury on a DBA/2 background, but not on a C57BL/6J or a 129/SvJ background, suggesting that glomerular injury by the deletion of Tns2 was strongly dependent on the genetic background. To further understand the mechanisms for the onset and the progression of glomerular injury by the deletion of Tns2, we generated Tns2-deficient mice on an FVB/N (FVB) strain, which is highly sensitive to glomerular disease. Tns2-deficient mice on FVB (FVBGN) developed severe nephrotic syndrome, and female FVBGN mice died within 8 weeks. Ultrastructural analysis revealed that FVBGN mice exhibited severe glomerular defects with mesangial process invasion of glomerular capillary tufts, lamination and thickening of the GBM and subsequent podocyte foot process effacement soon after birth. Aberrant laminin components containing α1, α2 and ß1 chains, which are normally expressed in the mesangium, accumulated in the GBM of FVBGN, suggesting that these components originated from mesangial cells that invaded glomerular capillary tufts. Compared to Tns2-deficient mice on the other backgrounds in previous reports, FVBGN mice developed earlier onset of glomerular defects and rapid progression of renal failure. Thus, this study further extended our understanding of the possible genetic background effect on the deterioration of nephrotic syndrome by Tns2 deficiency.
Asunto(s)
Glomérulos Renales/patología , Síndrome Nefrótico/etiología , Tensinas/deficiencia , Animales , Femenino , Membrana Basal Glomerular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos , Síndrome Nefrótico/patología , Podocitos/patología , Especificidad de la EspecieRESUMEN
Amyloid fibrils in senile plaque mainly consist of the 40-mer and 42-mer amyloid ß-proteins (Aß40 and Aß42). Although Aß42 plays more important role in the pathogenesis of Alzheimer's disease (AD), Aß40 could be involved in the progression of AD pathology because of its large amount. Recent studies revealed that variable sizes of Aß oligomers contributed to the neuronal death and cognitive dysfunction. However, how large oligomeric species are responsible for AD pathogenesis remains unclear. We previously proposed a toxic dimer model of Aß with turn structure at positions 22 and 23 using solid-state NMR and systematic proline replacement. Based on this model, we herein show the synthesis and biological activities of an E22P-Aß40 dimer at position 30, which was connected to l,l-2,6-diaminopimeric acid. The E22P-Aß40 dimer formed stable 6â¼8-mer oligomers without amyloid fibrils, but was not neurotoxic on human neuroblastoma cells. On the other hand, E22P-Aß40 generated high molecular-weight oligomers into fibrils, and showed the neurotoxicity. These results suggest that such kind of Aß40 dimer with a parallel ß-sheet might not be pathological.
Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/metabolismo , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Modelos Moleculares , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Prolina/química , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Células Madre Mesenquimatosas/metabolismo , Transcripción Genética/genética , Aneuploidia , Animales , Técnicas de Cultivo de Célula , Ciclo Celular/genética , Proliferación Celular/genética , Inestabilidad Cromosómica/genética , Cromosomas Humanos Par 13/genética , Reparación del ADN/genética , Fibroblastos/metabolismo , Glipicanos/genética , Erizos/genética , Papillomavirus Humano 6/genética , Humanos , Ratones , Ratones Desnudos , Oncogenes/genética , Transducción de Señal/genética , Telomerasa/genética , Activación Transcripcional/inmunologíaRESUMEN
Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.
Asunto(s)
Ataxia/genética , Atrofia/genética , Cerebelo/patología , Receptores de Glutamato/genética , Animales , Ataxia/complicaciones , Ataxia/patología , Atrofia/complicaciones , Atrofia/patología , Cerebelo/metabolismo , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/análisis , Precursores de Proteínas/análisis , Células de Purkinje/metabolismo , Células de Purkinje/patología , Receptores de Glutamato/análisisRESUMEN
We report a case of general anesthesia for laparoscopic cholecystectomy at 12 weeks of gestation. A 20-year-old woman weighing 123 kg was admitted with epigastralgia. She was diagnosed as pregnancy of 6 weeks of gestation and acute cholecystitis. Percutaneous trans-gallbladder drainage was performed to delay operation until 12 weeks of gestation. Laparoscopic cholecystectomy was performed uneventfully under general anesthesia combined with epidural anesthesia. There were no clinical signs of fetal distress during the perioperative period.