To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.

c Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.T oxoplasma gondii, an obligate intracellular parasite, invades a wide variety of cells in its vertebrate hosts. The disease caused by T. gondii is usually asymptomatic but can be severe in immunosuppressed individuals, and cyst reactivation causes toxoplasmic encephalitis in AIDS patients (1). In addition, infection of nonimmune women during pregnancy can lead to congenital infection, with hydrocephaly, microcephaly, or intracerebral calcifications occurring in the fetus (2).Systemic infection by the proliferating stage of the parasite, the tachyzoite, is efficiently controlled by the cellular immune response. However, the pathogen persists in its slowly replicating stage, the bradyzoite, in tissue cysts mainly within the muscle and brain. In chronic infections, parasites within neurons can directly cause neuronal death and atrophy of neuronal processes, while inflammation via production of nitric oxide (NO) and inflammatory cytokines from microglia or immune cells may contribute to the death of neighboring neurons (3). However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear.In mice and rats, the specificity of behavioral modifications induced by T. gondii has been examined across a broad range of behaviors that primarily concern anxiety and learned fear in these animals (4). Recently, it has been suggested that chronic infection with T...

cChronic infection with Toxoplasma gondii becomes established in tissues of the central nervous system, where parasites may directly or indirectly modulate neuronal function. Epidemiological studies have revealed that chronic infection in humans is a risk factor for developing mental diseases. However, the mechanisms underlying parasite-induced neuronal dysfunction in the brain remain unclear. Here, we examined memory associated with conditioned fear in mice and found that T. gondii infection impairs consolidation of conditioned fear memory. To examine the brain pathology induced by T. gondii infection, we analyzed the parasite load and histopathological changes. T. gondii infects all brain areas, yet the cortex exhibits more severe tissue damage than other regions. We measured neurotransmitter levels in the cortex and amygdala because these regions are involved in fear memory expression. The levels of dopamine metabolites but not those of dopamine were increased in the cortex of infected mice compared with those in the cortex of uninfected mice. In contrast, serotonin levels were decreased in the amygdala and norepinephrine levels were decreased in the cortex and amygdala of infected mice. The levels of cortical dopamine metabolites were associated with the time spent freezing in the fear-conditioning test. These results suggest that T. gondii infection affects fear memory through dysfunction of the cortex and amygdala. Our findings provide insight into the mechanisms underlying the neurological changes seen during T. gondii infection.T oxoplasma gondii is one of the most successful brain parasites, infecting approximately one-third of the human population (1). T. gondii can persist in brain and muscle throughout the host's life, and chronic infection is asymptomatic in immunocompetent humans (2). However, recent studies have suggested that T. gondii infection is a risk factor for developing mental diseases, such as schizophrenia and depression, as well as human behavior and personality changes and suicide (3, 4). Interestingly, T. gondii infection increases the risk of schizophrenia roughly 2.7 times, which is higher than that for genes associated with schizophrenia (5). Several studies have also suggested that rodents infected with T. gondii exhibit decreased avoidance behavior in response to cat odors, indicating manipulation of the host's behavior by T. gondii to facilitate the parasite's transmission and complete sexual replication in the definitive host (6-11).To date, research on the mechanism(s) underlying behavioral changes following T. gondii infection has been conducted primarily from two points of view. First, the relationship between parasite localization in the brain and behavioral changes has been investigated, with a previous study reporting that T. gondii has no obvious tropism in the brain (12-15). However, another study found that tissue cyst density in amygdalar areas (the medial and basolateral amygdala) is 2-fold higher than that in nonamygdalar areas (9), whereas the presence of tissue cy...

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