Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error‐prone PCR to introduce single point mutations into the sodium‐powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium‐channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C‐terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion‐channel blockers.     

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.     

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.     

A possible balance of magnesium accumulations among bone, cartilage, artery, and vein in single human individuals

It is known that a large quantity of magnesium contains bones, and the magnesium contents in spongy bones decrease gradually with advancing age. To elucidate the relationships between a decrease of mineral contents in human bones and an accumulation of minerals in the other human tissues, the content of magnesium was analyzed by inductively coupled plasma-atomic emission spectrometry among human bones, arteries, veins, and cartilages in 27 subjects (17 men and 10 women). These were resected from the subjects who died in the age range 40–98 yr. Calcanei were chosen for analysis of magnesium contents in contrast with femoral, popliteal, and common carotid arteries, internal jugular and femoral veins, superior and inferior venae cavae, and pubic symphyses. The magnesium contents in the calcanei decreased gradually with aging, whereas they increased progressively in the arteries, veins, and pubic symphyses with aging. It was found that as the magnesium contents decreased in the calcanei, they increased in the arteries, such as the femoral, popliteal, and common carotid arteries, whereas they decreased inversely in the veins, such as the internal jugular and femoral veins and superior and inferior venae cavae. Furthermore, as the magnesium contents decreased in the calcanei, they hardly changed in the pubic symphyses. These suggest that magnesium released from bones is accompanied by accumulation of magnesium in the arteries.     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.     

Endothelin-1 activates p38 mitogen-activated protein kinase via endothelin-A receptor in rat myocardial cells

In myocardial cells (MCs), endothelin-1 (ET-1) exerts various effects such as hypertrophy, and causes cellular injury. Long-term treatment with an endothelin-A (ET_A) receptor antagonist improves the survival of rats with heart failure, suggesting that myocardial endothelin system contributes to the progression of heart failure. p38 mitogen-activated kinase (MAPK) is a member of the MAPK family and activated by several forms of environmental stresses. We show here the effect of ET-1 on p38 MAPK activation and the role of ET-1-activated p38 MAPK on morphological changes in MCs. ET-1-stimulated p38 MAPK phosphorylation was detectable within 2 min and maximal at 5 min and was concentration dependent. The maximum effect was obtained at 10 nM. An ET_A receptor antagonist, BQ-123, but not an endothelin-B receptor antagonist, BQ-788, inhibited these reactions. A p38 MAPK inhibitor, SB203580, failed to inhibit the morphological changes associated with ET-1-induced myocardial cell hypertrophy. These results indicate that p38 MAPK is activated by ET-1 but does not contribute to the development of ET-1-induced myocardial cell hypertrophy.     

Endogenous Dopamine Maintains Synchronous Oscillation of Intracellular Calcium in Primary Cultured-Mouse Midbrain Neurons

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.     

Analysis of mechanical impedance in human arm movements using a virtual tennis system

The dynamic characteristics of human upper extremities are usually expressed by mechanical impedance. Although many studies have discussed human impedance characteristics, there are no reports on control abilities of task-related impedance in skilled human hand movements. This paper proposes a virtual sports system using a virtual reality technique to examine human movements. The differences in movements between skilled and unskilled subjects are investigated through a series of experiments. Then, the human impedance of a skilled player is estimated and analyzed in the preliminary phase of motion.     

Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes

Staphylococcal gamma-hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg.The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, psiPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, psiSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.