Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.     

Structure–activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure–activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.     

Effects of salmon calcitonin and anti-salmon calcitonin antibody on plasma calcium concentrations in the goldfish,Carassius auratus

The role of calcitonin (CT) in plasma calcium regulation was studied by the administration of exogenous CT and anti-salmon(s) CT antibody using goldfish,Carasius auratus, loaded or otherwise with calcium. CT elicited a decrease in plasma calcium concentrations at a dose of 10 ng/g body weight 1 h after administration. However, no effects were observed following doses of 30 ng and 50 ng/g 1 h, nor for the three doses 3 h after administration. In calcium-loaded fish, the effect of CT was different depending on the dosage of CT. Ten ng and 50 ng/g induced a decrease and an increase in plasma calcium concentrations, respectively, 3 h after administration. Anti-sCT antibody (0.02 μg or 0.1 μg/g) did not affect plasma calcium concentrations. In calcium-loaded fish, neither dose of anti-sCT antibody changed plasma calcium concentrations 1 h after administration. However, following a dose of 0.1 μg/g, plasma calcium concentrations decreased after 3 h. A positive correlation between plasma calcium concentrations and the gonad somatic index (GSI) in females was no longer apparent after administration of anti-sCT antibody. There was no relationship between plasma calcium concentrations and GSI in control and anti-sCT antibody-treated males. These results suggested that CT regulates plasma calcium concentrations in different ways depending on the dosage with CT having a role in calcium physiology during vitellogenesis.     

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.     

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser⁶⁵ by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.     

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.     

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

<Emphasis Type="Italic">Coprinus cinereus</Emphasis> Mer3 is required for synaptonemal complex formation during meiosis

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25°C but were similar to the wild type at 37°C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.