Understanding a molecular motor walking along a microtubule: an asymmetric Brownian motor driven by bubble formation with a focus on binding affinity

In recent years, many studies on a molecular motor have been conducted in the fields of biorheology and nanoengineering. The molecular motor is a molecule that converts the chemical energy obtained by ATP hydrolysis into mechanical energy. Explaining this mechanism is important for nanoengineering. A kinesin, which is a type of molecular motor, has the characteristics to move on a microtubule with hand-over-hand steps. The kinesin walking behaviour is explained by the ‘asymmetric Brownian ratchet model’. Previously, we had suggested that the walking mechanism was achieved by the bubble formation in a nanosized channel surrounded by hydrophobic atoms with the transition between the two states – bubble state and liquid state. However, the walking behaviour of the model motor was different from that of a single molecule measurement of a kinesin. In this study, we constructed a new motor system focused on the asymmetric binding affinity of a motor protein and performed a model simulation using the dissipative particle dynamics method. As a result, it was observed that hand-over-hand walking depends on the transition position ratio and the transition frequency coefficient. Moreover, the efficiency of the new motor system is higher than that of the previous motor systems. The new motor model can provide a simulation guide for the design of biomimetic nanomachines.     

Sse9I restriction-modification system: Organization of genes and structural comparison of proteins

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C.Sse9I), restriction endonuclease (R.Sse9I), and DNA methyltransferase (M.Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C.Sse9I and R.Sse9I were compared with those of related proteins. In the case of R.Sse9I, the highest homology was observed with the R.MunI (5′-CAATTG-3′) and R.EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R.Sse 9I was isolated.     

Patterns of autotomy and regeneration in Hemigrapsus nudus

Leg autotomy and regeneration can have severe impacts on survival and reproduction, and these impacts may be even more pronounced in animals with multifarious legs, such as decapods. Thus, determining the patterns and frequency of autotomy and regeneration could reveal the effects of these processes on the individual and population level. We investigated whether some legs are lost more often than others and if all legs are equally likely to be regenerated. We sampled nearly 500 purple shore crabs (Hemigrapsus nudus) and showed that (1) most animals are found with at least one injured leg, (2) the patterns of autotomy differ between males and females, and (3) successful claw regeneration is unlikely in both males and females. Future work with H. nudus and other grapsid crabs will elucidate how patterns seen here relate to other developmental and ecological factors.     

Locomotory activity and exploration behaviour of juvenile European lobsters (Homarus gammarus) in the laboratory

The behaviour of juvenile European lobsters (Homarus gammarus) in the wild is little understood. A laboratory system was designed with a maze-like environment as an alternative to the large technological expenditure of a field study. It provided an apparently endless runway with uniform thigmotactical cues. Juvenile lobsters having a total length of 68 to 115 mm were studied. The lobsters showed an extensive nocturnal locomotory activity. They established home shelters in which they spent the day and covered distances of 1200 to 1600 m during the night. On average, the lobsters performed 136 excursions from their shelters, of which 10% led only to the immediate surroundings of the shelters. Of all the excursions 90% were shorter than 16 m. In some exceptional cases distances of several hundred meters were covered in the maze between shelter visits. Excursions of less than 16 m lasted on average less than 5 min. The frequencies of shelter visits during the dark phase were highest in small lobsters (300 visits) and lowest in larger lobsters (50 visits). The time spent within shelters decreased from 10% to less than 2% with lobster size. A distinct change in behaviour was obvious at a body length of 75 to 80 mm. Smaller lobsters behaved defensively and relied on shelter protection. Larger lobsters were less dependent on shelter protection and thus were able to explore and utilize their environment more intensively.     

The effect of uphill and downhill walking on gait parameters: A self-paced treadmill study

It has been shown that gait parameters vary systematically with the slope of the surface when walking uphill (UH) or downhill (DH) (Andriacchi et al., 1977; Crowe et al., 1996; Kawamura et al., 1991; Kirtley et al., 1985; McIntosh et al., 2006; Sun et al., 1996). However, gait trials performed on inclined surfaces have been subject to certain technical limitations including using fixed speed treadmills (TMs) or, alternatively, sampling only a few gait cycles on inclined ramps. Further, prior work has not analyzed upper body kinematics. This study aims to investigate effects of slope on gait parameters using a self-paced TM (SPTM) which facilitates more natural walking, including measuring upper body kinematics and gait coordination parameters.Gait of 11 young healthy participants was sampled during walking in steady state speed. Measurements were made at slopes of +10°, 0° and −10°. Force plates and a motion capture system were used to reconstruct twenty spatiotemporal gait parameters. For validation, previously described parameters were compared with the literature, and novel parameters measuring upper body kinematics and bilateral gait coordination were also analyzed.Results showed that most lower and upper body gait parameters were affected by walking slope angle. Specifically, UH walking had a higher impact on gait kinematics than DH walking. However, gait coordination parameters were not affected by walking slope, suggesting that gait asymmetry, left-right coordination and gait variability are robust characteristics of walking. The findings of the study are discussed in reference to a potential combined effect of slope and gait speed. Follow-up studies are needed to explore the relative effects of each of these factors.     

Measuring plasma fatty acid oxidation with intravenous bolus injection of 3H- and 14C-fatty acid

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-¹⁴C]palmitate versus [9,10-³H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (¹⁴C- or ³H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [³H]acetate recovery as ³H₂O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-¹⁴C]acetate recovery as expired ¹⁴CO₂ was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-¹⁴C]palmitate and [9,10-³H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [³H]palmitate. In conclusion, intravenous boluses of [9,10-³H]palmitate versus [1-¹⁴C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.     

Evaluation of PCR-based methods for isolating flanking regions of genes

Several polymerase chain reaction (PCR)-based methods are available for isolation of unknown genomic fragments. In the present study, a comparative evaluation of a few methods of ligation-mediated PCR methods and a ligation-independent one were made by isolating promoter fragment for N-methyltransferase gene involved in the caffeine biosynthetic pathway of Coffea canephora. The benefits of tertiary PCR and the effects of a 4-base cutting restriction endonuclease on the size of the PCR products obtained were demonstrated in one of the ligation-mediated PCR methods. The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions.     

Correlates of ecological dominance within Pheidole ants (Hymenoptera: Formicidae)

1. In any group of organisms, one can almost invariably find some species that are ecologically dominant (i.e. disproportionately more abundant and widespread), whereas others are comparatively less prevalent. Understanding of the causes of variation in ecological dominance has been elusive, particularly given that dominant and subordinate species often lack obvious features that could predict their abundance in nature. 2. In this study, physiological, behavioural, morphological, and phylogenetic information is integrated in an effort to understand the mechanisms underlying ecological dominance in ants using the hyperdiverse ant genus Pheidole (Formicidae: Myrmicinae) as a model system. Field estimates of the relative abundance of 10 Pheidole species were compared with potential correlates, which included behavioural (walking velocity), physiological (tolerance to high and low temperatures and desiccation), and morphological traits (body size and degree of dimorphism in the worker caste). A molecular phylogeny of the tested species was also generated to account for potential confounding effects of phylogenetic non‐independence. 3. Dominant Pheidole species were characterised by higher environmental tolerance with respect to temperature and humidity, as well as faster walking speeds. On the other hand, no morphological correlates of ecological dominance were detected. Interestingly, subordinate species showed no evidence of trade‐off in performance, being both more fragile to environmental challenges and slower in their walking speeds. 4. These results provide important insights into the mechanisms involved in local species coexistence in Pheidole.     

Sequence characterization,in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2–4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.