Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

Stretch-induced enhancement of mechanical power output in human multijoint exercise with countermovement

Takarada, Yudai, Yuichi Hirano, Yusuke Ishige, and NaokataIshii. Stretch-induced enhancement of mechanical power output inhuman multijoint exercise with countermovement. J. Appl. Physiol. 83(5): 1749-1755, 1997.Therelation between the eccentric force developed during a countermovementand the mechanical power output was studied in squatting exercisesunder nominally isotonic load (50% of 1-repetition maximum). Thesubjects (n = 5) performed squattingexercises with a countermovement at varied deceleration rates beforelifting the load. The ground reaction force and video images wererecorded to obtain the power output of the body. Net muscle momentsacting at hip, knee, and ankle joints were calculated from videorecordings by using inverse dynamics. When an intense deceleration wastaken at the end of downward movement, large eccentric force wasdeveloped, and the mechanical power subsequently produced during thelifting movement was consistently larger than that produced without thecountermovement. Both maximal and mean power outputs during concentricactions increased initially with the eccentric force, whereas theybegan to decline when the eccentric force exceeded ~1.4 times the sumof load and body weight. Video-image analysis showed that thischaracteristic relation was predominantly determined by the torquearound the knee joint. Electromyographic analyses showed no consistentincrease in time-averaged integrated electromyograph from vastuslateralis with the power output, suggesting that the enhancement ofpower output is primarily caused by the prestretch-induced improvementof an intrinsic force-generating capability of the agonist muscle.

     

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain

Summary The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 Å. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Å from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.Abbreviations rmsd root-mean-square deviation - NOE nuclear Overhauser enhancement - 4D-SA simulated annealing in four-dimensional space - Myb-R2R3 repeats 2 and 3 of the DNA-binding domain of the c-Myb protein - DNA 16 Myb-specific binding DNA duplex with 16 base pairs - IHDD-C residues 3 to 59 of the C-chain of the engrailed homeodomain-DNA complex - DNA11 DNA duplex with base pairs 9 to 19 of the engrailed homeodomain-DNA complex     

Does Paramecium sense gravity?

In order to get an insight into the cellular mechanisms for the integration of the effects of gravity, we investigated the gravitactic behaviour in Paramecium. There are two main categories for the model of the mechanism of gravitaxis; one is derived on the basis of the mechanistic properties of the cell (physical model) and the other of the physiological properties including cellular gravireception (physiological model). In this review article, we criticized the physical models and introduced a new physiological model. Physical models postulated so far can be divided into two; one explaining the negative gravitactic orientation of the cell in terms of the static torque generated by the structural properties of the cell (gravity-buoyancy model by Verworn, 1889 and drag-gravity model by Roberts, 1970), and the other explaining it in terms of the dynamic torque generated by the helical swimming of the cell (propulsion-gravity model by Winet and Jahn, 1974 and lifting-force model by Nowakowska and Grebecki, 1977). Among those we excluded the possibility of dynamic-torque models because of their incorrect theoretical assumptions. According to the passive orientation of Ni(2+)-immobilized cells, the physical effect of the static torque should be inevitable for the gravitactic orientation. Downward orientation of the immobilized cells in the course of floating up in the hyper-density medium demonstrated the gravitactic orientation is not resulted by the nonuniform distribution of cellular mass (gravity-buoyancy model) but by the fore-aft asymmetry of the cell (drag-gravity model). A new model explaining the gravitactic behaviour is derived on the basis of the cellular gravity sensation through mechanoreceptor channels of the cell membrane. Paramecium is known to have depolarizing receptor channels in the anterior and hyperpolarizing receptors in the posterior of the cell. The uneven distribution of the receptor may lead to the bidirectional changes of the membrane potential by the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell in the gravity field; i.e. negative- and positive-going shift of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential with respect to gravity, in combination with the close coupling of the membrane potential and the ciliary locomotor activity, may allow the changes in swimming direction along with those in the helical nature of the swimming path; upward shift of axis of helix by decreasing the pitch angle due to hyperpolarization in the upward-orienting cell, and also the upward shift by increasing the pitch angle due to depolarization in the downward-orienting cell. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically an axis parallel to the gravity vector, after which the model was named as "Super-helix model". Three-dimensional recording of the trajectories of the swimming cells demonstrated that about a quarter of the cell population drew super-helical trajectory under the unbounded, thermal convection-free conditions. In addition, quantitative analysis of the orientation rate of the swimming cell indicated that gravity-dependent orientation of the swimming trajectory could not be explained solely by the physical static torque but complementarily by the physiological mechanism as proposed in the super-helix model.     

Surface ultrastructure of the tegument of Clonorchis sinensis newly excysted juveniles and adult worms.

The tegumental structures of newly excysted juveniles and adult worms of Clonorchis sinensis were studied using scanning and transmission electron microscopy. After excystation the juvenile's tegumental surface is characterized by knoblike protuberances and is armed almost entirely with numerous rows of small spines encircling the body. These spines are double- or triple-pointed on the anterior portion of the body and become single-pointed posteriorly. Four types of presumed sensory structures were observed as follow: A) ciliated knoblike papillae and B) nonciliated platelike papillae, both of which are arranged in rougly a bilaterally symmetrical pattern dorsally, ventrally, and laterally; C) rounded swellings of nonciliated papillae on the lips of the ventral and oral suckers, which are characterized in the transmission electron microscope by a rounded dense body in the apical bulb; and D) a sensory receptor with a bulbous projection having the appearance of a modified cilium, which was not found with SEM likely owing to its being enclosed by an extension of the tegument. In full-grown adult worms, the tegumental surface is knobbed or lobulated in various forms without surface spines. The tegumental structures in the adults appear to be clearly differentiated from those in the juveniles. Upraised, buttonlike papillae, each topped by a short cilium, which are similar to the Type A papillae in the juveniles, are distributed thickly around the oral and ventral suckers, and are rather randomly scattered over the remainder of the body. Some nonciliated swollen papillae were found on the lip of the ventral sucker.     

A new biologically active phenolic from Cattleya trianaei

A new phenolic, hydroxyeucomic acid, and dopamine were isolated from Cattleya trianaei and their biological activities examined.     

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

A hypotetraploid human T lymphoid cell line established by cell fusion.

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6-phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.