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The second international meeting on “SMC proteins: Chromosomal Organizers from Bacteria to Human” (SMC2017) was held in Nanyo City, Yamagata, Japan, from 13 to 16 June 2017. The meeting was attended by 134 participants (among them, 76 from outside of Japan) who were interested in one of the highly conserved classes of chromosomal proteins regulating large-scale chromosome structure and function. A keynote lecture was followed by 41 oral presentations and 71 poster presentations in the four-day meeting. Diverse topics surrounding eukaryotic SMC protein complexes (cohesins, condensins and SMC5/6) and prokaryotic SMCs, and a wide range of cutting-edge approaches (from polymer physics through medical genetics) were presented. Dominant themes discussed in the meeting included mechanistically how the SMC protein complexes might form chromatin loops and domains. The participants enjoyed both exciting debate about chromosome organization and warm welcome offered by local people in a small city located in the northern part of Japan.

BACKGROUND AND PURPOSE The free fatty acid FFA1 receptor and GPR120 are GPCRs whose endogenous ligands are medium- and long-chain FFAs, and they are important in regulating insulin and GLP-1 secretion respectively. Given that the ligands of FFA1 receptor and GPR120 have similar properties, selective pharmacological tools are required to study their functions further. EXPERIMENTAL APPROACH We used a docking simulation approach using homology models for each receptor. Biological activity was assessed by phosphorylation of ERK and elevation of intracellular calcium ([Ca(2+) ]i ) in cells transfected with FFA1 receptor or GPR120. Insulin secretion from murine pancreatic beta cells (MIN6) was also measured. KEY RESULTS Calculated hydrogen bonding energies between a series of synthetic carboxylic acid compounds and the homology models of the FFA1 receptor and GPR120, using docking simulations, correlated well with the effects of the compounds on ERK phosphorylation in transfected cells (R(2) = 0.65 for FFA1 receptor and 0.76 for GPR120). NCG75, the compound with the highest predicted selectivity for FFA1 receptors from this structure-activity relationship analysis, activated ERK and increased [Ca(2+) ]i as potently as the known FFA1 receptor-selective agonist, Compound 1. Site-directed mutagenesis analysis based on the docking simulation showed that different amino acid residues were important for the recognition and activation by FFA1 receptor agonists. Moreover, NCG75 strongly induced ERK and [Ca(2+) ]i responses, and promoted insulin secretion from MIN6 cells, which express endogenous FFA1 receptors. CONCLUSION AND IMPLICATIONS A docking simulation approach using FFA1 receptor and GPR120 homology models could be useful in predicting FFA1 receptor-selective agonists.

Metal-particle-assisted hydrofluoric acid etching of silicon (Si) is a unique method of preparing Si nanopores with metal nanoparticles at the bottom of each nanopore. Autocatalytic electroless deposition, which is the conventional method to metalize nonmetallic substrates, requires catalyzation of the substrates before deposition. For Si substrates, obtaining adhesive metal films with conventional catalyzation pretreatments is difficult. We recently developed a new method to produce adhesive metal films on Si substrates using catalytic nanopores that consists of three steps: 1) displacement deposition of metal nanoparticles; 2) Si nanopore formation by metal-particle-assisted hydrofluoric acid etching; and 3) autocatalytic electroless deposition of metal films. In this study, the new method is applied not only to previously reported p-Si (100) 1 Ω cm substrates but also to various Si substrates, such as low and high resistibility, p- and n-types, (100), (111), and (110) planes, and single-, multi- and micro-crystalline substrates. Adhesive and bright metal films were formed on all Si substrates. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The mitotic chromosome, which is composed of a pair of sister chromatids, is a large macromolecular assembly that ensures faithful transmission of genetic information into daughter cells. Despite its fundamental importance, how a nucleosome fiber is folded and assembled into a large-scale chromatid structure remains poorly understood. To address this question, we have established a biochemically tractable system in which mitotic chromatids can be reconstituted in vitro by mixing a simple substrate (sperm nucleus) and a limited number of purified factors. The minimum set of required factors includes core histones, three histone chaperones, topoisomerase II, and condensin I. In this article, we describe a set of protocols for the preparation of key reagents and the setup of reconstitution reactions. We believe that this classical approach of biochemical reconstitution will be of great help to dissect the mechanisms of action of individual factors during mitotic chromatid assembly and to assess the contribution of nucleosome dynamics to this process from a fresh angle. © 2018 by John Wiley & Sons, Inc.

Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge. Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers. This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.

We present a case of fever of unknown origin and life-threatening stomatitis developed about 60 months after renal transplantation. He was 15 yr old at the transplantation. Bacterial, fungal, and viral infections were not evident. Fever and stomatitis were resistant to acyclovir and to any anti-bacterial or anti-fungal treatment. Graft biopsy revealed a small focus of acute vascular rejection, but the findings were not severe enough to be an etiology of the fever in this case. The administration of cyclosporine (CYA) was stopped 19 d before graftectomy, but the clinical picture was unchanged. Fever and stomatitis was resolved immediately after graftectomy and the discontinuation of immunosuppressants such as mizoribine (MZ) and prednisolone. Pathological changes of the graft included chronic transplant glomerulopathy, acute glomerulitis, and lymphocyte infiltration in peritubular capillaries. Thus we suppose that immunosuppressants were the cause of both fever and stomatitis in this case. We speculate that a fever in this case might be due to the immunosuppressant itself, i.e., CYA or MZ, or viral infection probably herpes-simplex virus infection. It is probably the immunosuppressive state per se that may cause the resistance of his muco-cutaneous lesion to anti-viral agent.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1+ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.