Pirarubicin Combination Low-Dose Chemotherapy for Early Infantile Stage MS Neuroblastoma: Case Report.

Neuroblastoma (NB) is a neural crest-derived malignant tumor which is diagnosed during infancy in approximately 40% of cases; spontaneous regressions are observed, but there are varying degrees of severity. Treatment is indicated if an infant's condition is at risk of deterioration. Herein, we report the case of a 42-day-old boy who presented with hepatomegaly and was diagnosed with stage MS NB. A pathological diagnosis of "poorly differentiated neuroblastoma with low mitosis-karyorrhexis index, favorable histology" was made; his tumor cells were hyperdiploid and MYCN was not amplified. Because he had respiratory distress caused by the rapidly evolving hepatomegaly, two cycles of chemotherapy containing vincristine and cyclophosphamide were administered in the second and fourth weeks of admission; however, his abdominal tumor did not shrink. In the sixth week of admission, chemotherapy was revised to pirarubicin and cyclophosphamide, and the tumor began to shrink. After discharge, there was no re-elevation of tumor markers; after 1 year, the hepatomegaly and liver metastases disappeared. During the 5-year follow-up, his growth and development were normal and he progressed without sequelae. A regimen that includes pirarubicin could merit further study in the treatment of early infants with stage MS low-risk NB who are at risk of complications.

The potentially therapeutic role of tonsillectomy in the alleviation of several renal diseases apart from IgA nephropathy.

Tonsils are located mainly at the gateway of the respiratory tract, and are reportedly one of the secondary lymphatic organs of the immune system. The development of several diseases including IgA nephropathy (IgAN) is associated with inflammatory stimulation and an aberrant immune response of the tonsils. Several studies have reported an improvement in and/or an increase in the stability of the clinicopathological findings of patients with IgAN post tonsillectomy. However, the efficacy in and precise mechanism of the alleviation of symptoms of other renal diseases by tonsillectomy remain unknown. We hypothesize that tonsillectomy may play a potentially therapeutic role in renal diseases apart from IgAN, which are thought to be caused by an impaired regulation of the immune system.

Structure of Vibrio FliL, a New Stomatin-like Protein That Assists the Bacterial Flagellar Motor Function.

Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella.

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliGC). We have identified point mutations in FliGC from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliGC from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliGC are observed in the Cα1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliGC at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix α1 in the Cα1-6 domain of FliGC contribute to ensure the precise regulation of the motor switching.

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus.

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na+-driven flagellar motor.

Many bacteria move using their flagellar motor, which generates torque through the interaction between the stator and rotor. The most important component of the rotor for torque generation is FliG. FliG consists of three domains: FliGN, FliGM, and FliGC. FliGC contains a site(s) that interacts with the stator. In this study, we examined the physical properties of three FliG constructs, FliGFull, FliGMC, and FliGC, derived from sodium-driven polar flagella of marine Vibrio. Size exclusion chromatography revealed that FliG changes conformational states under two different pH conditions. Circular dichroism spectroscopy also revealed that the contents of α-helices in FliG slightly changed under these pH conditions. Furthermore, we examined the thermal stability of the FliG constructs using differential scanning calorimetry. Based on the results, we speculate that each domain of FliG denatures independently. This study provides basic information on the biophysical characteristics of FliG, a component of the flagellar motor.

Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli.

The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB91 , PotB129, and PotB138 , with various chimeric junctions. When those chimeric proteins were produced with PomA in a ΔmotAB strain of E. coli or in ΔpomAB and ΔpomAB ΔmotX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the T ring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in ΔpomAB, PotB138 had the highest motility. The difference between the origin of the α1 and α2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor.

The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomBC) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121-153) and following the N-terminal two-thirds of α1(154-164) in PomBC changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.