Experimental study of seita-fitting

The purpose of this study was to verify the difference between carrying a load on the sacrum (LOS) and on the lumbar vertebrae (LOL) in oxygen uptake, muscle activities, heart rate, cadence, and subjective response. Nine males (26.7 +/- 3.1 years old), each carrying a 7.5 kg carrier frame and a 40 kg load, walked on a treadmill at a speed of 50 m/min. EMGs were recorded from the trapezius, rectus abdominis, erector spinae, vastus lateralis, rectus femoris, vastus medialis, biceps femoris long head, tibial anterior, soleus, medial head of gastrocnemius, and the lateral head of gastrocnemius. For each subject the integrated EMG (IEMG) was normalized by dividing the IEMG in the LOL and LOS by the IEMG in a no-load condition (NL) for each investigated muscle. The following was significantly higher in LOL than in LOS: oxygen uptake; IEMG of the tibial anterior, soleus, and medial head of gastrocnemius; cadence; and rated perceived exertion. However, IEMG of the erector spinae was significantly lower in LOL than in LOS. These results suggest that seita-fitting in LOS causes a decrease of leg muscle activities, which causes oxygen uptake to decrease beyond the increase of the erector spinae activity.     

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

Ac as a tool for the functional genomics of rice

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.     

Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the Rho family of small G proteins.

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane-associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease-mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and -independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA-induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.     

Carbachol-induced Suppression of Contraction Rhythm in Spontaneously Beating Cultured Cardiac Myocytes From Neonatal Rats

Nitric oxide (NO) and reactive oxygen species (ROS) are known to play various functional and pathophysiological roles as an intracellular messenger in the heart. In this study, we investigated whether the increased production of NO and/or ROS was involved in the cholinergic regulation of rhythmic contraction in spontaneously beating cultured cardiac myocytes from neonatal rats. Exposure of cultures to carbachol, an agonist of muscarinic acetylcholine receptors (mAchR), produced a dose-dependent decrease in the beat rate of cultured cardiac myocytes, and such a effect was significantly attenuated by pre-treatment with an NOS inhibitor, as well as an NO scavenger. In addition, exposure to an NO donor (SNAP) also decreased the beat rate dose-dependently. Carbachol- or SNAP-induced suppression of the contraction rhythm was significantly attenuated by co-treatment with 5-hydroxydecanoate (5-HD). In contrast, treatment with diazoxide decreased the beat rate dose-dependently. Carbachol treatment increased the intensity of 2',7'-dichlorodihydrofluorescein fluorescence, suggesting that the production of ROS was enhanced by the treatment. In addition, the carbachol- or diazoxide-induced suppression of contraction rhythm was attenuated by co-treatment with 2-mercaptopropionyl glycine, a scavenger of ROS. The present study has suggested that the mAchR-NO-mitoK ATP -ROS pathway is a factor responsible for carbachol-induced suppression of contraction rhythm in cultured cardiac myocytes.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.     

Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997