Determination of naringin and naringenin in human plasma by high-performance liquid chromatography

An HPLC method for determining a flavonoid, naringin, and its metabolite, naringenin, in human plasma is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using genistin (for naringin) or daidzein (for naringenin) as an internal standard and solid-phase extraction using a Sep-Pak t C₁₈ cartridge. For the determination, HPLC was carried out using an Inertsil ODS-2 column (250x4.6 m I.D., 5 μm particle size). The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v; pH 7.1) for naringin and acetonitrile-0.1 M ammonium acetate solution-acetic acid (30:69:1, v/v; pH 4.9) for naringenin. The flow-rate was 1 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 280 nm for naringin and at 292 nm for naringenin. The detection limits on-column were about 0.2 ng for the two flavonoids.     

Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro

To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.     

Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos

The expression of heparan sulfate glycosaminoglycan (HS-GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS-GAG in the ³⁵S-labeled embryos. By western blot analysis using a specific anti-HS monoclonal antibody, HS-GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS-GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS-GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS-GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat-inactivated heparitinase nor chondroitinase showed such abnormality. The HS-GAG-eliminated embryos showed decreased expression of both muscular and neural-specific markers. These results suggest that HS-GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.     

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.     

Variation in selectivity of univalent cations in slime moldPhysarum polycephalum caused by reception of polyvalent cations

Summary Specificity of reception on 11 electrolytes in the slime moldPhysarum polycephalum was investigated in the presence of polyvalent cations in media. Membrane potential and motive force of tactic movement were examined with the aid of the double chamber method, and the zeta potential at the membrane surface of the slime mold was measured by electrophoretic mobility. The results obtained are summarized as follows: (1) The presence of polyvalent cations (e.g., Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, La³⁺, Th⁴⁺) in medium led to an increase in threshold concentration,C _th , determined from the potential measurements for Na- or Li-salts, and to a decrease inC _th for K-, Rb-, or NH₄-salts,C _th for 11 electrolytes changed discontinuously when the concentration of polyvalent cations in medium exceeded their respective thresholds. (2) TheC _th determined from chemotaxis agreed with that from the potential response both in the presence and absence of polyvalent cations. (3) Sequence of selectivity of univalent cations varied extensively in the presence of polyvalent cations. (4) Changes in the zeta potential induced by NaCl reception agreed with those in the membrane potential even in the presence of Ca²⁺ in medium. (5) TheC _th for reception of NaCl changed sharply at about 12 °C in the presence of polyvalent cations, while that for KCl was independent of the temperature.Conformational changes in surface membrane of the slime mold in response to reception of polyvalent cations were then discussed in relation to the discrimination of univalent cations.