Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering.

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotBC2) and Vibrio alginolyticus (PomBC5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotBC2, the dimeric conformation of the PomBC5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.

Variables influencing patient satisfaction for hypnotics: difference between zolpidem and brotizolam.

WHAT IS KNOWN AND OBJECTIVE: The pharmacokinetics of zolpidem and brotizolam is affected by gender and age, that is, increased clearance in males taking zolpidem and younger subjects taking brotizolam. The purpose of this study was to determine the variables including gender and age influencing patient satisfaction for hypnotics, zolpidem and brotizolam. METHODS: The study included 329 patients who were treated with zolpidem (n = 172) and brotizolam (n = 157) for insomnia. Patients were interviewed to evaluate individual satisfaction and drug efficacy. The factors associated with dissatisfaction of zolpidem and brotizolam were identified using multiple logistic analysis. RESULTS AND DISCUSSION: Of the participating patients, 40 (23%) and 41 (26%) complained of dissatisfaction with zolpidem and brotizolam, respectively. An insufficient amount of sleep (<6 h) and the number of awakenings were common factors cited for dissatisfaction for both drugs. Males were found to report a higher rate of dissatisfaction for zolpidem, whereas patients younger than 65 years and those receiving corticosteroid therapy reported a higher rate of dissatisfaction with brotizolam. WHAT IS NEW AND CONCLUSION: These results suggested that patient satisfaction was different between zolpidem and brotizolam in terms of gender for zolpidem and age and corticosteroid co-administration for brotizolam, which could be used to help choose a better drug among the two in patients with insomnia.

TKI Treatment Sequencing in Advanced Gastrointestinal Stromal Tumors.

Prior to the early 2000s, patients with advanced gastrointestinal stromal tumors (GIST) had very poor prognoses owing to a lack of effective therapies. The development of tyrosine kinase inhibitors at the turn of the century significantly improved the overall survival for patients with GIST. The resounding success of imatinib in the first clinical trial of a tyrosine kinase inhibitor to treat GIST led to its approval for first-line therapy for advanced GIST; this study was open to all comers and not restricted to any GIST subtype(s). The trials that led to the approvals of second-, third-, and fourth-line therapy for advanced GIST were also open to all patients with advanced/metastatic GIST. Only in retrospect do we realize the role that the molecular subtypes played in the results observed in these studies. In this review, we discuss the studies that led to the US Food and Drug Administration approval of imatinib (first line), sunitinib (second line), regorafenib (third line), and ripretinib (fourth line) for advanced KIT-mutant GIST. In addition, we review how information about GIST molecular subtypes has been used to accelerate the approval of other targeted therapies for non-KIT mutant GIST, leading to the approval of five additional drugs indicated for the treatment of specific GIST molecular subtypes. We also discuss how our understanding of the molecular subtypes will play a role in the next generation of therapeutic approaches for treating advanced GIST.

New treatment strategies for advanced-stage gastrointestinal stromal tumours.

When gastrointestinal stromal tumour (GIST), the most common form of sarcoma, was first recognized as a distinct pathological entity in the 1990s, patients with advanced-stage disease had a very poor prognosis owing to a lack of effective medical therapies. The discovery of KIT mutations as the first and most prevalent drivers of GIST and the subsequent development of the first KIT tyrosine kinase inhibitor (TKI), imatinib, revolutionized the treatment of patients with this disease. We can now identify the driver mutation in 99% of patients with GIST via molecular diagnostic testing, and therapies have been developed to treat many, but not all, molecular subtypes of the disease. At present, seven drugs are approved by the FDA for the treatment of advanced-stage GIST (imatinib, sunitinib, regorafenib, ripretinib, avapritinib, larotrectinib and entrectinib), all of which are TKIs. Although these agents can be very effective for treating certain GIST subtypes, challenges remain and new therapeutic approaches are needed. In this Review, we discuss the molecular subtypes of GIST and the evolution of current treatments, as well as their therapeutic limitations. We also highlight emerging therapeutic approaches that might overcome clinical challenges through novel strategies predicated on the biological features of the distinct GIST molecular subtypes.

Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer.

Many transmembrane receptors are oligomeric proteins. Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both. The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands. Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA. In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate. These results suggest that heterodimers with only one intact cytoplasmic domain are functional. The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.

Assessment of hypnotic effects and patient satisfaction in empirical use of sleep medicines.

OBJECTIVE: We assessed the hypnotic effects of and patient satisfaction with three types of hypnotics prescribed empirically: ultra-short-acting (US-a), short-acting (S-a), and intermediate- and long-acting (IL-a) agents. METHODS: We studied 310 insomniac patients (age 60.5 +/- 15.0 years) treated with US-a (n = 124), S-a (n = 149) or IL-a (n = 37) agents. Patients were interviewed to evaluate individual satisfaction and drug efficacy. Efficacy, as assessed by total sleep time (TST) and sleep latency time (SLT), was compared between satisfied and dissatisfied patient groups. Nocturnal awaking curve for each hypnotic was used for comparing the effects between satisfied and dissatisfied patient groups in each type of hypnotics. RESULTS: Thirty-two patients (25.8%) were dissatisfied with US-a, 35 (23.5%) with S-a and 11 (29.7%) with IL-a. TST differed significantly between satisfied and dissatisfied groups: 424 +/- 88 vs. 345 +/- 101 min for US-a (P < 0.001), 440 +/- 84 vs. 359 +/- 111 min for S-a (P < 0.001) and 453 +/- 96 vs. 345 +/- 125 min for IL-a (P < 0.01), respectively. With IL-a agents, the SLT of dissatisfied patients was longer than in satisfied ones (81 +/- 52 vs. 33 +/- 22 min, P < 0.05). Twenty (62.5%) dissatisfied patients taking US-a agents awoke before 05:00 hours - a rate significantly higher than satisfied patients (n = 23, 25.0%, P < 0.001). These characteristics of dissatisfied patients were reflected by the patterns of nocturnal awaking curves, although the patterns for satisfied patients were similar among the three types of hypnotics. CONCLUSION: Between 24% and 30% of patients were dissatisfied with their hypnotics. Shorter TST was common in dissatisfied patients receiving any agent, for reasons differing among hypnotics (longer SLT with IL-a agents and early awakening with US-a). Drug efficacy and patient satisfaction in empirical use of hypnotics can be assessed by nocturnal awaking curves for each hypnotic.

Blood ribavirin concentration in high-dose ribavirin for adenovirus-induced haemorrhagic cystitis - a case report.

Blood ribavirin concentration was monitored after the administration of high-dose oral ribavirin in a case of adenovirus-induced haemorrhagic cystitis post-stem-cell transplantation. Combination use of intravenous gamma immunoglobulin (15 g/3 days) and high-dose ribavirin (RBV; 9000 mg/4 days) provided plasma ribavirin concentration of 24.3 microM and achieved virus eradication. High level of erythrocyte ribavirin (1085 microM; mostly as phosphorylated metabolites) with long half-life (15 days) caused severe anaemia, which required several blood transfusions for 2 weeks after the cessation of the ribavirin treatment. It was suggested that blood transfusion and intensive haemoglobin level monitoring is necessary for at least 4 weeks after the RBV, because of the high accumulation of phosphorylated ribavirin in erythrocytes even after stopping ribavirin administration.

Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap.

Autoantibodies in the serum from a patient with connective tissue disease have been used to define a high molecule weight acidic nuclear protein antigen. The antigen tentatively termed Ku, after the first two letters of patient's name, has distinct physicochemical properties and immunological specificities that distinguish it from previously reported antigens. The Ku antigen has an apparent 300,000 mol wt as determined by gel filtration and sucrose density gradient ultracentrifugation techniques. The antigen is destroyed by trypsin, mild heating, and pH variations greater than 10 and less than 5. Treatment with ribonuclease or deoxyribonuclease did not affect the antigenic reactivity. The Ku antigen was demonstrated in the soluble extracts of human, calf, and rabbit, but not of rat tissues. Purified antibody localized the Ku antigen within the nuclei of human liver where a "reticular" pattern of immunofluorescence was seen. Of 330 patients with various connective tissue diseases, 9 had precipitating antibodies to the Ku antigen. Preliminary results of clinical analysis indicated that antibody to the Ku antigen might become a useful marker for a group of patients with clinical characteristics of both polymyositis and scleroderma with a good prognosis.

Autoantibody reactive with three classes of RNA polymerases in sera from patients with systemic sclerosis.

We have identified a novel autoantibody reactive with all three classes of RNA polymerases, well-characterized nuclear enzymes, in sera from patients with systemic sclerosis (SSc). After incubation with [35S]methionine-labeled HeLa cell extracts, 14 of 275 SSc sera immunoprecipitated 12 or 14 proteins with similar molecular weights as those of several subunit proteins of eukaryotic RNA polymerases I, II, and III. Purified IgG from these two types of sera inhibited RNA transcription catalyzed by RNA polymerases I, II, and III in vitro. Immunoblot analysis using RNA polymerase-enriched fraction showed that the majority of these sera reacted with 42- or 25-kD protein. Anti-RNA polymerase antibody was highly specific to SSc, especially to diffuse cutaneous SSc. Clinical features associated with this antibody included a high frequency of heart and kidney involvement and a poor survival rate at 5 yr after first visit. These findings indicate that the autoantibody to three classes of RNA polymerases is a new marker for a unique subset of diffuse cutaneous SSc.

Establishment of an animal model with side effects induced by mycophenolate mofetil and pharmacohistological analysis of them.

UNLABELLED: Mycophenolate mofetil (MMF) is used for immunosuppression after organ transplantation, but gastrointestinal side effects including diarrhea are sometimes observed with this drug. We sought to construct on animal model of diarrhea with MMF in rodents. MATERIALS AND METHODS: BALB/Cj mice, weighing 25 g received 500 mg /kg of MMF, 60 mg/kg of levofloxacin (LVFX), 1000 mg/kg of Hangeshashin-to (HST), which is traditional Kampo medicine. This cocktail was administered orally to MMF, LVFX, HST, MMF+LVFX, and MMF+LVFX+HST groups for 21 days. We measured the water content fecal collected on days 1, 4, 8, 11, 14, 18, and 21. Feces on day 21 were cultured for identification of fecal flora. Mice were sacrificed on day 21, with blood samples collected to measure mycophenolic acid (MPA) concentrations by HPLC. Jejunum, cecum, and colon were taken for histological evaluation. RESULTS: Significant weight loss of mice and increased fecal water content of were observed in MMF and MMF+LVFX but not in MMF+LVFX+HST groups. Serum MPA levels didn't differ in MMF-administered groups. Inflammatory changes in intestinal villi were observed in the cecum in MMF and MMF+LVFX groups. A change in fecal flora was observed in LVFX-administered groups. CONCLUSION: Diarrhea induced by MMF in a rodent model produced inflammatory changes in the cecum. LVFX seemed to change the activity of beta-glucuronidase in the fecal flora. HST suppressed fecal softening induced by MMF in this animal model.

Na(+)-driven flagellar motor of Vibrio.

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the coupling ion: namely the H(+)-driven motor and the Na(+)-driven motor. Analysis of the flagellar motor at the molecular level is far more advanced in the H(+)-driven motor than in the Na(+)-driven motor. Recently, the genes of the Na(+)-driven motor have been cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In this review, we summarize recent studies of the Na(+)-driven flagellar motor.

Molecular cloning of the murine homologue of CD63/ME491 and detection of its strong expression in the kidney and activated macrophages.

The mouse homologue of CD63/ME491 (Mu-CD63) was molecularly cloned and analyzed. Mu-CD63 exhibited a strikingly high similarity to CD63/ME491 and the rat homologue. Northern blot analysis revealed that Mu-CD63 mRNA was expressed strongly in the kidney of adult mice, especially in the glomerulus fraction, implying the possibility that Mu-CD63 plays an important role in maintaining normal renal function. Activated macrophages and splenocytes exhibited strong expression of Mu-CD63 mRNA, whereas cultured thymocytes barely expressed the mRNA irrespective of cell activation. Taken together, the present results suggest that Mu-CD63 expression is associated with differentiation and/or development of certain cell types, but not necessarily with cell proliferation.

Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum.

Within the bacterial flagellum the basal-body rod, the hook, the hook-associated proteins (HAPs), and the helical filament constitute an axial substructure whose elements share structural features and a common export pathway. We present here the amino acid sequences of the hook protein and the three HAPs of Salmonella typhimurium, as deduced from the DNA sequences of their structural genes (flgE, flgK, flgL and fliD, respectively). We compared these sequences with each other and with those for the filament protein (flagellin) and four rod proteins, which have been described previously (Joys, 1985; Homma et al., 1990; Smith & Selander, 1990). Hook protein most strongly resembled the distal rod protein (FlgG) and the proximal HAP (HAP1), which are thought to be attached to the proximal and distal ends of the hook, respectively; the similarities were most pronounced near the N and C termini. Hook protein and flagellin, which occupy virtually identical helical lattices, did not resemble each other strongly but showed some limited similarities near their termini. HAP3 and HAP2, which form the proximal and distal boundaries of the filament, showed few similarities to flagellin, each other, or the other axial proteins. With the exceptions of the N-terminal region of HAP2, and the C-terminal region of flagellin, proline residues were absent from the terminal regions of the axial proteins. Moreover, with the exception of the N-terminal region of HAP2, the terminal regions contained hydrophobic residues at intervals of seven residues. Together, these observations suggest that the axial proteins may have amphipathic alpha-helical structure at their N and C termini. In the case of the filament and the hook, the terminal regions are believed to be responsible for the quaternary interactions between subunits. We suggest that this is likely to be true of the other axial structures as well, and specifically that interaction between N-terminal and C-terminal alpha-helices may be important in the formation of the axial structures of the flagellum. Although consensus sequences were noted among some of the proteins, such as the rod, hook and HAP1, no consensus extended to the entire set of axial proteins. Thus the basis for recognition of a protein for export by the flagellum-specific pathway remains to be identified.

Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components.

The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpar strains, NMB205 and NMB201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Co-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpar phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex.

Inversion of thermosensing property of the bacterial receptor Tar by mutations in the second transmembrane region.

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.

FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium.

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus.

The Na(+)-driven flagellar motor in Vibrio alginolyticus rotates very fast. Rotation of a single flagellum on a stuck cell was measured by laser darkfield microscopy with submillisecond temporal resolution. The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s. at 300 mM NaCl. The Na+ influx through the motor should determine the rotation period (tau) and affect the speed stability. Fluctuation of the rotation period was analyzed at various rotation rates (from approximately 50 r.p.s. to approximately 1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor. At high rotation rates (over 400 r.p.s.), the observed rotation was stable, and the standard deviation of tau (sigma tau) ranged from 7% to 16% of the average rotation period (< tau >). At low rotation rates (under 100 r.p.s), the rotation period tended to fluctuate, and the distributions of tau were non-Gaussian. The value of sigma tau ranged from 10 to 30% of < tau >. However, the observed minimum value of sigma tau at various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament. These results suggest that the torque was stably generated at various Na+ influxes through the motor. We observed large fluctuations that cannot be explained by rotational diffusion. We discuss the factors that induce the large fluctuation.