Uniqueness of Entamoeba sulfur metabolism: sulfolipid metabolism that plays pleiotropic roles in the parasitic life cycle

Sulfur metabolism is ubiquitous and terminally synthesizes various biomolecules that are crucial for organisms, such as sulfur‐containing amino acids and co‐factors, sulfolipids and sulfated saccharides. Entamoeba histolytica, a protozoan parasite responsible for amoebiasis, possesses the unique sulfur metabolism features of atypical localization and its terminal product being limited to sulfolipids. Here, we present an overall scheme of E. histolytica sulfur metabolism by relating all sulfotransferases and sulfatases to their substrates and products. Furthermore, a novel sulfur metabolite, fatty alcohol disulfates, was identified and shown to play an important role in trophozoite proliferation. Cholesteryl sulfate, another synthesized sulfolipid, was previously demonstrated to play an important role in encystation, a differentiation process from proliferative trophozoite to dormant cyst. Entamoeba survives by alternating between these two distinct forms; therefore, Entamoeba sulfur metabolism contributes to the parasitic life cycle via its terminal products. Interestingly, this unique feature of sulfur metabolism is not conserved in the nonparasitic close relative of Entamoeba, Mastigamoeba, because lateral gene transfer‐mediated acquisition of sulfatases and sulfotransferases, critical enzymes conferring this feature, has only occurred in the Entamoeba lineage. Hence, our findings suggest that sulfolipid metabolism has a causal relationship with parasitism.     

Anti-microtubule ‘plinabulin’ chemical probe KPU-244-B3 labeled both α- and β-tubulin

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine ‘phenylahistin’ with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both α- and β-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between α- and β-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.     

Study of the structure-activity relationship of polymyxin analogues

A structure-activity relationship study on three classes of polymyxin analogues focusing on hydrophobicity was conducted.     

<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.     

DNA topoisomerase II interacts with Lim15/Dmc1 in meiosis

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein–protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.     

Genetic Polymorphisms of Dihydropyrimidinase in a Japanese Patient with Capecitabine-Induced Toxicity

Dihydropyrimidinase (DHP) is the second enzyme in the catabolic pathway of uracil, thymine, and chemotherapeutic fluoropyrimidine agents such as 5-fluorouracil (5-FU). Thus, DHP deficiency might be associated with 5-FU toxicity during fluoropyrimidine chemotherapy. We performed genetic analyses of the family of a patient with advanced colon cancer who underwent radical colectomy followed by treatment with 5-FU prodrug capecitabine and developed severe toxicity attributable to a lack of DHP. We measured urinary uracil and dihydrouracil, and genotyped DPYS in the patient and her family. We also measured the allele frequency of DPYS polymorphisms in 391 unrelated Japanese subjects. The patient had compound heterozygous missense and nonsense polymorphisms comprising c.1001A>G (p.Gln334Arg) in exon 6 and c.1393C>T (p.Arg465Ter) in exon 8, which are known to result in a DHP enzyme with little or no activity. The urinary dihydrouracil/uracil ratio in the patient was 17.08, while the mean ± SD urinary dihydrouracil/uracil ratio in family members who were heterozygous or homozygous for wild-type DPYS was 0.25 ± 0.06. In unrelated subjects, 8 of 391 individuals were heterozygous for the c.1001A>G mutation, while the c.1393C>T mutation was not identified. This is the first report of a DHP-deficient patient with DPYS compound heterozygous polymorphisms who was treated with a fluoropyrimidine, and our findings suggest that polymorphisms in the DPYS gene are pharmacogenomic markers associated with severe 5-FU toxicity in Japanese patients.     

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.