RESUMEN
Atomic motion through excitation of extended surface electronic states on Ge(001) is studied using extraction of electrons by scanning tunneling microscopy and density functional theory. Single-electron excitation into the surface states nonlocally alters the tilting orientation of the surface Ge dimer, and the change rate depends on the excitation energy. Theoretical investigations identify the excited electronic states for the dimer motion, and clarify the strong coupling between the surface state electrons and a local vibrational mode of the dimer for changing the tilting orientation.
RESUMEN
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The aims of this study were to establish Chinese hamster ovary (CHO) cells overexpressing recombinant human GALNS (rhGALNS) and to assess pharmacokinetics and tissue distribution of purified enzymes by using MPS IVA knock-out mouse (Galns(-/-)). The CHO-cell derived rhGALNS was purified from the media by a two-step affinity chromatography procedure. The rhGALNS was administered intravenously to 3-month-old Galns(-/-) mice at a single dose of 250U/g of body weight. The treated mice were examined by assaying the GALNS activity at baseline and up to 240min to assess clearance of the enzyme from blood circulation. The mice were sacrificed 4h after infusion of the enzyme to study the enzyme distribution in tissues. The rhGALNS was purified 1317-fold with 71% yield. The enzyme was taken up by Galns(-/-) chondrocytes (150U/mg/15h). The uptake was inhibited by mannose-6-phosphate. The enzyme activity disappeared from circulation with a half-life of 2.9min. After enzyme infusion, the enzyme was taken up and detected in multiple tissues (40.7% of total infused enzymes in liver). Twenty-four hours after a single infusion of the fluorescence-labeled enzymes into MPS IVA mice, biodistribution pattern showed the amount of tagged enzyme retained in bone, bone marrow, liver, spleen, kidney, and heart. In conclusion, we have shown that the phosphorylated rhGALNS is delivered to multiple tissues, including bone, and that it functions bioactively in Galns(-/-) chondrocytes implying a potential enzyme replacement treatment.
Asunto(s)
Condroitinsulfatasas/farmacocinética , Proteínas Recombinantes/farmacocinética , Animales , Células CHO , Condroitinsulfatasas/genética , Condroitinsulfatasas/aislamiento & purificación , Condroitinsulfatasas/metabolismo , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Estabilidad de Enzimas , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Mucopolisacaridosis IV/tratamiento farmacológico , Mucopolisacaridosis IV/enzimología , Proteínas Recombinantes/aislamiento & purificación , Factores de Tiempo , Distribución TisularRESUMEN
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. In recent studies of enzyme replacement therapy for animal models with lysosomal storage diseases, cellular and humoral immune responses to the injected enzymes have been recognized as major impediments to effective treatment. To study the long-term effectiveness and side effects of therapies in the absence of immune responses, we have developed an MPS IVA mouse model, which has many similarities to human MPS IVA and is tolerant to human GALNS protein. We used a construct containing both a transgene (cDNA) expressing inactive human GALNS in intron 1 and an active site mutation (C76S) in adjacent exon 2 and thereby introduced both the inactive cDNA and the C76S mutation into the murine Galns by targeted mutagenesis. Affected homozygous mice have no detectable GALNS enzyme activity and accumulate glycosaminoglycans in multiple tissues including visceral organs, brain, cornea, bone, ligament and bone marrow. At 3 months, lysosomal storage is marked within hepatocytes, reticuloendothelial Kupffer cells, and cells of the sinusoidal lining of the spleen, neurons and meningeal cells. The bone storage is also obvious, with lysosomal distention in osteoblasts and osteocytes lining the cortical bone, in chondrocytes and in the sinus lining cells in bone marrow. Ubiquitous expression of the inactive human GALNS was also confirmed by western blot using the anti-GALNS monoclonal antibodies newly produced, which resulted in tolerance to immune challenge with human enzyme. The newly generated MPS IVA mouse model should provide a good model to evaluate long-term administration of enzyme replacement.
Asunto(s)
Condroitinsulfatasas/genética , Mucopolisacaridosis IV/enzimología , Mucopolisacaridosis IV/genética , Animales , Condroitinsulfatasas/administración & dosificación , Condroitinsulfatasas/biosíntesis , Condroitinsulfatasas/deficiencia , Condroitinsulfatasas/inmunología , Modelos Animales de Enfermedad , Femenino , Válvulas Cardíacas/patología , Humanos , Tolerancia Inmunológica/genética , Hígado/patología , Masculino , Meninges/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucopolisacaridosis IV/patología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , ARN MensajeroRESUMEN
High mobility group box protein 1, HMGB1, is a major nonhistone chromatin component in higher eukaryotic cells. HMGB1 is thought to be involved in the processes of global nuclear events such as transcription, recombination and repair, but the mechanism of these processes is unclear. Here, we show a concrete example of chromatin structural modulation by HMGB1 in HeLa S3 cells. A co-immunopurification experiment with Flag-tagged HMGB1 revealed that a portion of HMGB1 in HeLa S3 cells is included in a large-molecular-weight multiprotein complex. The multiprotein complex including HMGB1 showed ATP hydrolysis and ATP-dependent chromatin structural modulation activities that increased the susceptibility of chromatin to MNase digestion, while HMGB1 alone had no such activity. Thus, HMGB1 in the multiprotein complex is critical for expressing the chromatin structural modulation activity. These results suggest that HMGB1 is involved in chromatin structural modulation in global nuclear events through its interaction with a multiprotein complex in mammalian cells.
Asunto(s)
Adenosina Trifosfato/fisiología , Cromatina/química , Cromatina/metabolismo , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Cromatina/enzimología , Células HeLa , Humanos , Hidrólisis , Nucleasa Microcócica/química , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismoRESUMEN
We identified several low-abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two-dimensional electrophoresis (2-DE) followed by microsequencing and mass spectrometry. Two major milk proteins, beta-casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2-DE. Protein identification of the spots on 2-DE was performed by either gel comparison, microsequencing, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF-MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight-MS (Q-TOF). Significant differences in protein patterns were observed between the low-abundance proteins of colostrum and mature milk. In addition, several low-abundance proteins including fibrinogen beta-chain, chitinase 3-like 1, alpha-antitrypsin, complement C3 alpha-chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of beta-casein fragments increased significantly during this lactation period. alpha-Lactalbumin and beta-lactoglobulin as well as some low-abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low-abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation.