EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis.

Bacterial translation elongation factor G (EF-G) catalyzes translocation during peptide elongation and mediates ribosomal disassembly during ribosome recycling in concert with the ribosomal recycling factor (RRF). Two homologs of EF-G have been identified in mitochondria from yeast to man, EF-G1mt and EF-G2mt. Here, we demonstrate that the dual function of bacterial EF-G is divided between EF-G1mt and EF-G2mt in human mitochondria (RRFmt). EF-G1mt specifically catalyzes translocation, whereas EF-G2mt mediates ribosome recycling with human mitochondrial RRF but lacks translocation activity. Domain swapping experiments suggest that the functional specificity for EF-G2mt resides in domains III and IV. Furthermore, GTP hydrolysis by EF-G2mt is not necessary for ribosomal splitting, in contrast to the bacterial-recycling mode. Because EF-G2mt represents a class of translational GTPase that is involved in ribosome recycling, we propose to rename this factor mitochondrial ribosome recycling factor 2 (RRF2mt).

PPARγ ligand attenuates portal inflammation in the MRL-lpr mouse: a new strategy to restrain cholangiopathy in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by chronic destructive cholangitis, which is associated with the reduced expression of an anti-inflammatory molecule, peroxisome proliferator-activated receptor-γ (PPARγ), in intrahepatic bile ducts. We previously demonstrated the anti-inflammatory effects of PPARγ ligands using cultured human biliary epithelial cells. In this study, we evaluated the effectiveness of PPARγ ligand against peribiliary inflammation in vivo. As an animal model of PBC, we used MRL/lpr mice in which a PBC-like cholangitis occurs naturally. Anti-inflammatory effects of the intraperitoneal administration of a PPARγ ligand, the prostaglandin D metabolite 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2), were evaluated. In untreated mice, portal inflammation including cholangitis was found to some degree in the majority of portal tracts. In mice given a high-dose group, the degree of portal inflammation was significantly reduced and mice mostly lacking portal inflammation and cholangitis were also found. T cell numbers in portal tracts were markedly decreased in the high-dose group, compared with controls, whereas there was no significant difference in terms of B cells and macrophages. This study is the first to assess the therapeutic potential of a PPARγ ligand against portal inflammation including cholangitis. Anti-inflammatory effects of PPARγ ligands may prevent the progression of cholangiopathy in PBC patients.

Novel approach to evaluating granulation and segregation level considering the contribution of hydroxypropyl cellulose to the surface property change of granules.

The granulation process is critical to the uniformity of not only the active ingredient (API) but also other excipients in granules. Insufficient granulation results in unexpected product quality, e.g. delayed dissolution and lack of uniformity of API. Therefore, evaluating the granulation and segregation level of granules helps secure the uniformity of drug product quality. Here, we found that the polar surface free energy (SFE) of studied granules increased as granulation by a high shear granulator proceeded. Among the excipients formulated in the studied granules, only hydroxypropyl cellulose (HPC) showed a higher specific free energy of adsorption (ΔGsp) of chloroform, which is a parameter used to calculate polar SFE. This indicates that the ΔGsp of chloroform in granules helps detect the level of contribution of HPC to the granulation progress by inverse gas chromatography (IGC). We concluded that the ΔGsp of chloroform in a granulated sample is a novel critical material attribute (CMA) in relation to granulation level. In addition, we propose a novel approach to evaluating the quantitative granulation and segregation level based on the ΔGsp of chloroform in a granulated sample by focusing on the distribution of HPC in the granulated sample.

HMRF1L is a human mitochondrial translation release factor involved in the decoding of the termination codons UAA and UAG.

While all essential mammalian mitochondrial factors involved in the initiation and elongation phases of translation have been cloned and well characterized, little is known about the factors involved in the termination process. In the present work, we report the functional analysis of human mitochondrial translation release factors (RF). Here, we show that HMRF1, which had been previously denoted as a human mitochondrial RF, was inactive in in vitro translation system, although it is a mitochondrial protein. Instead, we identified another human mitochondrial RF candidate, HMRF1L, and demonstrated that HMRF1L is indeed a mitochondrial protein that functions specifically as an RF for the decoding of mitochondrial UAA and UAG termination codons in vitro. The identification of the functional mitochondrial RF brings us much closer to a detailed understanding of the translational termination process in mammalian mitochondria as well as to the unraveling of the molecular mechanism of diseases caused by the dys-regulation of translational termination in human mitochondria.

The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC.

We have recently identified the human mitochondrial release factor, HMRF1L, which is responsible for decoding of UAA/UAG termination codons. Here, we identified human mitochondrial methyltransferase, HMPrmC, which methylates the glutamine residue in the GGQ tripeptide motif of HMRF1L. We demonstrate that HMPrmC is targeted to mitochondria and the glutamine residue in the GGQ motif of HMRF1L is methylated in vivo. HMPrmC depletion in HeLa cells leads to decreased mitochondrial translation activity in the presence of the translation fidelity antibiotic streptomycin in galactose containing medium. These results suggest that the methylation of HMRF1L by HMPrmC in human mitochondria is involved in the control of the translation termination process, probably by preventing the undesired suppression of termination codons and/or abortive termination events at sense codons under such conditions, as observed in prokaryotes and eukaryotes systems.

Molecular targeting of inosine-5'-monophosphate dehydrogenase by FF-10501 promotes erythropoiesis via ROS/MAPK pathway.

One of the major symptoms of myelodysplastic syndromes (MDS) is severe cytopenia. Despite cytokine therapies, such as erythropoiesis-stimulating agents, many patients still require blood transfusions, and the development of new therapeutic approaches is needed. In this work, we studied the effects of the inosine-5'-monophosphate (IMP) dehydrogenase (IMPDH) inhibitor FF-10501 on erythropoiesis of human hematopoietic cells. Differentiation of K562 chronic myeloid leukemia cells to an erythroid lineage was promoted by FF-10501 in a dose-dependent manner. Interestingly, we found that metabolic conversion of IMP to hypoxanthine leads to elevation of reactive oxygen species (ROS). The differentiative effects of FF-10501 were abolished by the ROS scavenger dimethylthiourea or the p38 MAPK inhibitor SB203580. Furthermore, FF-10501 promoted erythropoiesis from CD34+ hematopoietic stem/progenitor cells, accompanied with ROS accumulation, while high-dose FF-10501 mainly showed cytotoxic effects. These findings denote the potential of IMPDH inhibition therapy with FF-10501 in amelioration of anemia in MDS patients.

Therapeutic effects of garenoxacin in murine experimental secondary pneumonia by Streptococcus pneumoniae after influenza virus infection.

In a pneumococcal pneumonia murine model following influenza virus infection, garenoxacin was more effective than other fluoroquinolones and demonstrated high levels of bacterial eradication in the lung, low mortality, and potent histopathological improvements. Garenoxacin could potentially be used for the treatment of secondary pneumococcal pneumonia following influenza.

Collaborative work on evaluation of ovarian toxicity. 6) Two- or four-week repeated-dose studies and fertility study of cisplatin in female rats.

The main aim of the present study is to determine the optimal administration period of cisplatin with regards to its toxic effects on ovarian morphology in the repeated-dose toxicity study. Cisplatin was administered to female SD rats intraperitoneally once daily at dose levels of 0.25, 0.5, 1.0 and 2.0 mg/kg for 2 weeks, or at dose levels of 0.125, 0.25 and 0.5 mg/kg for 4 weeks in the repeated-dose toxicity study. In the female fertility study, 0.25, 0.5 and 1.0 mg/kg of cisplatin were administered in the same manner from 14 days prior to mating to Day 7 of gestation. In the repeated-dose toxicity study, a decrease in large follicle, an increase in atresia of medium and large follicles, and/or a decrease in currently formed corpus luteum were observed in animals receiving 1.0 and 2.0 mg/kg for 2 weeks, and decreases in small and/or large follicles and an increase in atresia of large follicle were observed in animals receiving 0.25 and 0.5 mg/kg for 4 weeks on the histopathological examination of the ovaries. In the female fertility study, the copulation and fertility indices in the animals receiving 1.0 mg/kg tended to be lower than those in the control animals. In conclusion, histopathological changes in the ovary that were attributable to cisplatin dosing were detected by detailed observation of the ovary in the 2-week study; and therefore, a 2-week administration period is sufficient to evaluate the ovarian toxicity of cisplatin.

Changes in density of calcium-binding-protein-immunoreactive GABAergic neurons in prefrontal cortex in schizophrenia and bipolar disorder.

There is evidence that GABAergic neurotransmission is altered in mental disorders such as schizophrenia (SCZ) and bipolar disorder (BPD). The calcium-binding proteins (CBPs) calbindin (CB), calretinin (CR), and parvalbumin (PV) are used as markers of specific subpopulations of cortical GABAergic interneurons. We examined the postmortem prefrontal cortical region (Brodmann's area 9) of patients with SCZ and BPD, and of age-matched control subjects, excluding suicide cases. The laminar density of neurons immunoreactive (IR) for three CBPs, namely CB, CR, and PV, was quantified. The densities of CB-IR neurons in layer 2 and PV-IR neurons in layer 4 in the SCZ subjects decreased compared with those in the control subjects. When CBP-IR neurons were classified according to their size, a reduction in the density of medium CB-IR neurons in layer 2 in SCZ subjects and an increase in the density of large CR-IR neurons in layer 2 in BPD subjects were observed. These results suggest that alterations in specific GABAergic neurons are present in mental disorders, and that such alterations may reflect the vulnerability toward the disorders.