Short review of human prolonged horizontal bed rest studies in Japan

As many as 700,000-1,000,000 Japanese are spending a substantial part of their life in bed not being able to resume standing position due to disease and/or accident. Our objectives were urgently derived from that fact. The research project group at Faculty of Medicine, University of Tokyo, in Japan have studied effects of prolonged horizontal bed rest on humans since 1990. Financial supports from the Japanese Ministry of Education (Grant-in aide for scientific research), National Aero Space Development Agency of Japan and Japan Federation for Health Sciences have enabled us to perform five prolonged bed rest experiments for 5 years from 1990 to 1994. Many scientists in Europe, America and Russia have already made a heap of studies on this subject since the latter half of 1940s (eg. 39). However, our studies mainly focused upon the effects of inactivity on human health to determine how to bring a human from inactivity back to an unrestricted active life. This short review is going to summarize our results obtained from 1990 to 1993.     

Activated renin-angiotensin-aldosterone system and its effects on cardiovascular system during 20-days horizontal bed rest

Change in circulating blood volume by bed rest has been suggested to effect on many cardiovascular responses after bed rest including orthostatic intolerance and exercise performance. However, there is a lack of consensus on effect of renin-angiotensin-aldosterone system (RAAS) on baseline heart rate and blood pressure during bed rest, although RAAS is the most potent fluid regulating system. The purpose of this study was to clarify the effect of RAAS on changes in baseline cardiovascular system and urine excretion.     

A method with three triangular flaps as a secondary operative procedure after reconstruction of the lower lip by Estlander's method.

We have devised a new method for secondary commissuroplasty after reconstruction of the lower lip using Estlander's method with both aesthetically and functionally satisfactory results. This method consists of forming two equilaterally triangular mucosal flaps on the vermilion and a small triangular skin flap in the new position of the commissure and transposing these three flaps to reconstruct the commissure. In the present paper, we reported the procedure and the results. This method produces extremely good results, obtaining favorable commissure form and reconstruction of the mucosa of both upper and lower lips without leaving an unnatural-looking color change in the mucosa or a step deformity in the vermilion. It can be expected that our method will improve the results of Estlander's operations after tumor resection in the lower lip.     

Robot control with biological cells

At present there exists a large gap in size, performance, adaptability and robustness between natural and artificial information processors for performing coherent perception-action tasks under real-time constraints. Even the simplest organisms have an enviable capability of coping with an unknown dynamic environment. Robots, in contrast, are still clumsy if confronted with such complexity. This paper presents a bio-hybrid architecture developed for exploring an alternate approach to the control of autonomous robots. Circuits prepared from amoeboid plasmodia of the slime mold Physarum polycephalum are interfaced with an omnidirectional hexapod robot. Sensory signals from the macro-physical environment of the robot are transduced to cellular scale and processed using the unique micro-physical features of intracellular information processing. Conversely, the response form the cellular computation is amplified to yield a macroscopic output action in the environment mediated through the robot's actuators.     

Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution

Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.     

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.     

Improvement of L-lysine production by Methylophilus methylotrophus from methanol via the Entner-Doudoroff pathway, originating in Escherichia coli

To improve the amino acid production by metabolic engineering, eliminating the pathway bottleneck is known to be very effective. The metabolic response of Methylophilus methylotrophus upon the addition of glucose and of pyruvate was investigated in batch cultivation. We found that the supply of pyruvate is a bottleneck in L-lysine production in M. methylotrophus from methanol as carbon source. M. methylotrophus has a ribulose monophosphate (RuMP) pathway for methanol assimilation, and consequently synthesized fructose-6-phosphate is metabolized to pyruvate via the Entner-Doudoroff (ED) pathway, and the ED pathway is thought to be the main pathway for pyruvate supply. An L-lysine producer of M. methylotrophus with an enhanced ED pathway was constructed by the introduction of the E. coli edd-eda operon encoding the enzyme involving the ED pathway. In this strain, the overall enzymatic activity of ED pathway, which is estimated by measuring the activities of 6-phosphogluconate dehydrogenase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, was about 20 times higher than in the parent. This strain produced 1.2 times more L-lysine than the parent producer. Perhaps, then, the supply of pyruvate was a bottleneck in L-lysine production in the L-lysine producer of M. methylotrophus.     

Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration.

The Tek/Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. To study the signal transduction pathways that are mediated by this receptor, we have used the yeast two-hybrid system to identify signaling molecules that associate with the phosphorylated Tek receptor. Using this approach, we demonstrate that five molecules, Grb2, Grb7, Grb14, Shp2, and the p85 subunit of phosphatidylinositol 3-kinase can interact with Tek in a phosphotyrosine-dependent manner through their SH2 domains. Mapping of the binding sites of these molecules on Tek reveals the presence of a multisubstrate docking site in the carboxyl tail of Tek (Tyr(1100)). Mutation of this site abrogates binding of Grb2 and Grb7 to Tek in vivo, and this site is required for tyrosine phosphorylation of Grb7 and p85 in vivo. Furthermore, stimulation of Tek-expressing cells with Angiopoietin-1 results in phosphorylation of both Tek and p85 and in activation of endothelial cell migration and survival pathways that are dependent in part on phosphatidylinositol 3-kinase. Taken together, these results demonstrate that Angiopoietin-1-induced signaling from the Tek receptor is mediated by a multifunctional docking site that is responsible for activation of both cell migration and cell survival pathways.     

Changes in force-velocity and power output of upper and lower extremity musculature in young subjects following 20 days bed rest

Ten healthy sedentary students were exposed to 20 days bed rest (BR) to study the effect of simulated weightlessness on force(F)-velocity(V) characteristics and power(P) output of upper and lower limb movements. F, V and P were measured using a special dynamometer applicable to single joint movements [elbow flexion(EF) and extension(EE), knee flexion(KF) and extension(KE), and hip flexion] or multi-joint movements (squatting). Physiological cross-sectional areas(PCSA) of KF and KE muscles were measured by a magnetic resonance imaging technique. After BR, attenuation in P were observed in lower limb movements (decreased by 19.8-43.6% for squatting, KF and KE), in upper limb movements on the other hand, the decreases in P were not significant (approximately -5% for EF and EE). Decrease in P in lower limb were more pronounced in heavier loaded conditions which were characterized by decreases in both F and V. For KF and KE, decreases in maximal static F (-18.9 to approximately -26.8%) were more pronounced than the decreases observed in the PCSA (approximately -7%), resulting in decreases in specific tension (-12.3 to approximately -22.1%). Neural excitation potentials to generate maximal muscle tension or P might also be influenced by weightlessness.     

Sociality of an agent during morphogenetic canalization: Asynchronous updating with potential resonance

Canalization is a typical self-organization process leading to complementarity between parts and the whole. In the field of developmental biology, concerns about morphogenesis canalization are often framed as the French flag problem, questioning how each cell knows its own position in the whole system. Although chemical gradients have been suggested to provide positional information, there is no direct evidence that gradients are used to gain positional information. The chemical gradient hypothesis is based on the assumption that agents (e.g., cells) in a domain that locally interact with each other can generate a chemical gradient thanks to a global reference point. Instead of a chemical gradient, we here propose a model based on agents that are equipped with sociality that is based not on a global reference but rather on the ability to sense other neighboring agents, or potential resonance. The interaction among the agents with sociality, developed from undifferentiated types or tokens, is implemented using asynchronous updating automata equipped with potential resonance. We show that these automata can generate a French flag pattern that is very robust against perturbations without positional information by comparing automata with synchronous updating and asynchronous automata without potential resonance.