Spontaneous Detachment of the Leading Head Contributes to Myosin VI Backward Steps

Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.     

Excitability Changes in Intracortical Neural Circuits Induced by Differentially Controlled Walking Patterns

Our previous single-pulse transcranial magnetic stimulation (TMS) study revealed that excitability in the motor cortex can be altered by conscious control of walking relative to less conscious normal walking. However, substantial elements and underlying mechanisms for inducing walking-related cortical plasticity are still unknown. Hence, in this study we aimed to examine the characteristics of electromyographic (EMG) recordings obtained during different walking conditions, namely, symmetrical walking (SW), asymmetrical walking 1 (AW1), and asymmetrical walking 2 (AW2), with left to right stance duration ratios of 1:1, 1:2, and 2:1, respectively. Furthermore, we investigated the influence of three types of walking control on subsequent changes in the intracortical neural circuits. Prior to each type of 7-min walking task, EMG analyses of the left tibialis anterior (TA) and soleus (SOL) muscles during walking were performed following approximately 3 min of preparative walking. Paired-pulse TMS was used to measure short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) in the left TA and SOL at baseline, immediately after the 7-min walking task, and 30 min post-task. EMG activity in the TA was significantly increased during AW1 and AW2 compared to during SW, whereas a significant difference in EMG activity of the SOL was observed only between AW1 and AW2. As for intracortical excitability, there was a significant alteration in SICI in the TA between SW and AW1, but not between SW and AW2. For the same amount of walking exercise, we found that the different methods used to control walking patterns induced different excitability changes in SICI. Our research shows that activation patterns associated with controlled leg muscles can alter post-exercise excitability in intracortical circuits. Therefore, how leg muscles are activated in a clinical setting could influence the outcome of walking in patients with stroke.     

The Vibrio parahaemolyticus pvuA1 gene (formerly termed psuA) encodes a second ferric vibrioferrin receptor that requires tonB2

We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.     

Esterification of xanthophylls by human intestinal Caco-2 cells

We recently found that peridinin, which is uniquely present in dinoflagellates, reduced cell viability by inducing apoptosis in human colon cancer cells. Peridinin is also found in edible clams and oysters because the major food sources of those shellfish are phytoplanktons such as dinoflagellates. Little is known, however, about the fate of dietary peridinin and its biological activities in mammals. The aim of the present study was to investigate the enzymatic esterification of xanthophylls, especially peridinin which is uniquely present in dinoflagellates, using differentiated cultures of Caco-2 human intestinal cells. We found that peridinin is converted to peridininol and its fatty acid esters in differentiated Caco-2 cells treated with 5 μmol/L peridinin solubilized with mixed micelles. The cell homogenate was also able to deacetylate peridinin and to esterify peridininol. Other xanthophylls, such as fucoxanthin, astaxanthin and zeaxanthin, were also esterified, but at relatively lower rates than peridinin. In this study, we found the enzymatic esterification of xanthophylls in mammalian intestinal cells for the first time. Our results suggest that the esterification of xanthophylls in intestinal cells is dependent on their polarity.     

Identification and characterization of two contiguous operons required for aerobactin transport and biosynthesis in Vibrio mimicus

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron-regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin-mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA ) is contiguous to the operon (matCDB ) encoding components of an ATP-binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.     

p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus

p57Kip2 is the only cyclin-dependent kinase (Cdk) inhibitor shown to be essential for mouse embryogenesis. The fact suggests that p57 has a specific role that cannot be compensated by other Cdk inhibitors. LIM-kinase 1 (LIMK-1) is a downstream effector of the Rho family of GTPases that phosphorylates and inactivates an actin depolymerization factor, cofilin, to induce the formation of actin fiber. Here we demonstrate that p57 regulates actin dynamics by binding and translocating LIMK-1 from the cytoplasm into the nucleus, which in turn results in a reorganization of actin fiber. The central region of p57, a unique feature among the Cdk inhibitors, and the N-terminal region of LIMK-1, which contains the LIM domains were essential for the interaction. Expression of p57, but not p27Kip1 or a p57 mutant, with a deletion in the central region was shown to induce marked reorganization of actin filament and a translocation of LIMK-1. Our findings indicate p57 may act as a key regulator in embryogenesis by bearing two distinct functions, the regulation of cell cycle through binding to Cdks and the regulation of actin dynamics through binding to LIMK-1, both of which should be important in developmental procedure.     

Amino acid sequence of galactosamine-containing glycopeptides in the hinge region of a human immunoglobulin D

Amino acid sequence and the location of seven galactosamine oligosaccharide moieties of the hinge region of the δ chain of human IgD NIG-65 have been determined. These oligosaccharide moieties are distributed in two distinct fashions: 1) three clusters each consisting of five amino acid residues with two consecutive attachment sites either Ala-X-Ala-Ser-Ser or Ala-X-Ala-Thr-Thr, where X can be any amino acid including proline, 2) one triplet sequence Val-Pro-Thr with one attachment site. We propose two rules with regard to the acceptor sequence for galactosamine oligosaccharides, the quintet sequence rule and triplet sequence rule.     

Tubulin seeds alpha-synuclein fibril formation.

Increasing evidence suggests that alpha-synuclein is a common pathogenic molecule in several neurodegenerative diseases, particularly in Parkinson's disease. To understand alpha-synuclein pathology, we investigated molecules that interact with alpha-synuclein in human and rat brains and identified tubulin as an alpha-synuclein binding/associated protein. Tubulin co-localized with alpha-synuclein in Lewy bodies and other alpha-synuclein-positive pathological structures. Tubulin initiated and promoted alpha-synuclein fibril formation under physiological conditions in vitro. These findings suggest that an interaction between tubulin and alpha-synuclein might accelerate alpha-synuclein aggregation in diseased brains, leading to the formation of Lewy bodies.