Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking.

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.

Heavy meromyosin induces sliding movements between antiparallel actin filaments.

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of ATPase activity of immobilized myosin heads.

Myosin, heavy meromyosin, and myosin subfragment-1 (S-1) were immobilized on the inner surfaces of glass capillary tubes, the inside walls of which had been coated with nitrocellulose. The ATPase activities of the immobilized proteins were measured using radiolabeled ATP and electrophoretic separation of the reaction products. The activity was proportional to the amount of immobilized protein. Activation by actin of the ATPase was also observed.

In vitro motility of skeletal muscle myosin and its proteolytic fragments.

We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.

Comparison of insulinotrophic actions of nateglinide with glibenclamide dissociated from absorption in conscious dogs.

Nateglinide is more rapidly absorbed than glibenclamide. Therefore, the different absorption kinetics of both drugs were eliminated by intraportal administration in conscious fasted dogs. The plasma insulin profiles were compared under similar kinetic changes in plasma drug concentrations. After a priming dose of nateglinide (1 mg/kg. 5 min) or glibenclamide (40 microg/kg. 5 min), plasma drug concentrations reached a peak at 4 minutes (nateglinide, 80 +/- 5 micromol/L, n = 6 and glibenclamide, 263 +/- 60 nmol/L, n = 6) followed by a sustained level at approximately 30% of the peak concentration at 30 minutes. Nateglinide led to a rapid and constant reduction in arterial glucose of approximately 30% basal, while glibenclamide promoted a gradual decrease to approximately 50% basal at 120 minutes. An increase in plasma insulin level by nateglinide of 4 times basal (218 +/- 58 pmol/L v 47 +/- 3 pmol/L, P <.05, n = 6) occurred at 6 to 10 minutes followed by sustained release of 1.4 times basal (67 +/- 15 pmol/L, n = 6). The insulin surge was more than doubled (484 +/- 209 pmol/L, n = 6) under a euglycemic clamp. Insulin release by glibenclamide increased gradually reaching 10-fold basal (449 +/- 166 pmol/L, n = 6) at 60 minutes. This was not enhanced during a euglycemic clamp. Lowering the primed doses of nateglinide resulted in a diminished peak plasma insulin concentration. In contrast, glibenclamide caused only a slower increase, but eventually reaching a similar peak. By increasing the continuous infusion of nateglinide, the sustained insulin release was not altered. Glibenclamide, but not nateglinide, evoked prompt and sustained insulin release in the continuing presence of the other. These results are consistent with the concept that nateglinide produces a quick, but very short-lived, interaction with sulfonylurea (SU)-receptors on plasma membrane by free access of the drug from the cell exterior. In contrast, glibenclamide promotes a slow and longer interaction with the receptor by distribution of the drug into the cell inferior. We conclude, therefore, that not only the different kinetics of gastrointestinal (GI) absorption, but also the inherent difference in the interaction with beta cells is attributed to the different insulin release characteristics between nateglinide and glibenclamide in vivo.

Inhibitory effect of M50054, a novel inhibitor of apoptosis, on anti-Fas-antibody-induced hepatitis and chemotherapy-induced alopecia.

M50054, 2,2'-methylenebis (1,3-cyclohexanedione), was identified as a novel inhibitor of apoptosis (programmed cell death) using an in vitro cell death assay system induced in human Fas-expressing WC8 cells by soluble human Fas ligand. Furthermore, M50054 inhibited the apoptotic cell death of U937, a human monocytic leukemic cell line, induced by anticancer agents such as etoposide; it was also confirmed that M50054 inhibited apoptotic features such as DNA fragmentation and phosphatidylserine exposure in these cells. These anti-apoptotic effects were attributable to inhibition of caspase-3 activation. Additionally, M50054 significantly inhibited anti-Fas-antibody-induced elevation of plasma alanine aminotransferase and aspartate aminotransferase. Alopecia (hair loss) symptoms were also significantly improved with topical treatment with M50054. In conclusion, M50054 inhibits apoptosis induced by a variety of stimuli via inhibition of caspase-3 activation, and may thus be effective for hepatitis and chemotherapy-induced alopecia.

A statistical mechanical theory for the adsorption of protein to liposomal membranes.

The observed topology change of spherical lipid vesicles to coffee cups [Saitoh, A. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 1026] was analyzed by a statistical mechanical theory. The topology change was due to the adsorption of talin molecules to the orifices of the coffee cups. The adsorption isotherm of talin between an aqueous solution and the vesicle membrane was analyzed by taking account of the bending energy of the membrane. The equilibrium is determined by the balance of the energy gain for the adsorption of talin to the periphery of the vesicles and the change of the bending energy of the membrane due to the shape change. The observed coexistence of coffee cups and sheet-like vesicles were reproduced. Vesicles with two orifices were also analyzed and theoretically reproduced.

Dissociation between plasma and atrial content of atrial natriuretic polypeptide (ANP) following sodium load in rats.

To investigate the time course effect of sodium intake on release and synthesis of atrial natriuretic polypeptide (ANP), plasma and atrial content of ANP were measured in rats which had been fed either a high or a low salt diet for 1, 3, 7, 14 and 35 days. Plasma ANP in rats fed the high salt diet for one day was significantly higher than in those fed the low salt diet. However, there were no significant differences between the groups fed either the high or the low salt diet for 3 days or more. In contrast to the direction of change in plasma ANP, atrial content of ANP in rats fed the high salt diet for one day tended to be lower and was significantly lower in those fed for 3 and 7 days than in the low salt diet group, while there were no significant differences between both groups that were fed for 14 and 35 days. These results suggest that ANP is rapidly released into the circulation when sodium is loaded, however, the atrial storage of ANP remains depleted for about one week.

Mechanical analyses of morphological and topological transformation of liposomes.

Liposomes are micro-compartments made of lipid bilayer membranes possessing the characteristics quite similar to those of biological membranes. To form artificial cell-like structures, we made liposomes that contained subunit proteins of cytoskeletons: tubulin or actin. Spherical liposomes were transformed into bipolar or cell-like shapes by mechanical forces generated by the polymerization of encapsulated subunits of microtubules. On the other hand, disk- or dumbbell-shaped liposomes were developed by the polymerization of encapsulated actin. Dynamic processes of morphological transformations of liposomes were visualized by high intensity dark-field light microscopy. Topological changes, such as fusion and division of membrane vesicles, play an essential role in cellular activities. To investigate the mechanism of these processes, we visualized the liposomes undergoing topological transformation in real time. A variety of novel topological transformations were found, including the opening-up of liposomes and the direct expulsion of inner vesicles.

Demultiplexer for optical orthogonal frequency-division multiplexing using an optical fast-Fourier-transform circuit.

An integrated-optic device for demultiplexing optical orthogonal frequency-division-multiplexed signals is described that consists of an optical fast-Fourier-transform circuit and optical gates. The fast-Fourier-transform circuit is composed of symmetrical Mach-Zehnder interferometers that are suitable for precisely adjusting filter characteristics. This device can process four or more channels with a relatively simple configuration and reduced size. An optical demultiplexer for four subcarrier channels is realized and used to demultiplex 4 x 10 Gbit/s signals.

Tunable chromatic dispersion and dispersion slope compensator using a planar lightwave circuit lattice-form filter.

A tunable chromatic dispersion and dispersion slope compensator is proposed that has a single lattice-form filter configuration. Wavelength dependence is intentionally added to its tunable couplers, which produces dispersion slope compensation in addition to the dispersion compensation. Dispersion tunability of +/- 500 ps/nm and a slope of -4.9 ps/nm(2) over 40 GHz are successfully demonstrated, thus meeting the requirement for 40 Gbits/s differential quadrature phase shift keying transmission with an 80 km long nonzero dispersion-shifted fiber.

Integrated photonic decoder with complementary code processing and balanced detection for two-dimensional optical code division multiple access.

We propose a novel integrated photonic decoder for two-dimensional (time spreading, wavelength hopping) optical code division multiple access. The decoder is composed of multiplexers-demultiplexers, variable delay lines, and a coupler, which processes complementary codes and utilizes balanced detection to reduce unwanted cross-correlation interference. We successfully carried out a 10 Gbit/s transmission that demonstrated its effectiveness.

Arrayed-waveguide grating with uniform loss properties over the entire range of wavelength channels.

We propose an arrayed-waveguide grating that shows uniform loss properties over the entire range of wavelength channels by combining two light waves with adjacent diffraction orders at its output. We applied this configuration to an arrayed-waveguide grating with 32 channels and a 50 GHz spacing and were able to reduce the deviations in loss to less than 1.0 and 1.8 dB over one and three free spectral ranges, respectively.

Integrated-optic variable delay line and its application to a low-coherence reflectometer.

We propose a large-scale variable delay line based on planar light-wave circuit technology and its application as a reference arm in an optical low-coherence reflectometer. This variable delay line is composed of 16 asymmetrical delay arm pairs sandwiched between 2 optical switches, which select the path for a needed delay. This configuration enables us to eliminate the need for a moving part in the reflectometer. We can scan the reference arm over a length of 262.1 mm with a step of less than 1.0 microm in air and achieve reflectometer sensitivity of about -47 dB.

[Hygienic effect of periodical draining from auto-watering piping for laboratory animal breeding].

To prevent the bacterial contamination of the drinking water, we developed a periodical draining machine system which were composed of the electromagnetic valve and the time switch. The machine system is able to replace standing water with fresh water on optional volumes and intervals. The total bacterial numbers of standing water were counted as an indicator of the bacterial contamination. The number of total bacteria were reduced to less than 5 per ml by working the machine system on 6-hour-interval with replacing twice as much as standing water, although more than 10(3) per ml of the bacteria were found when the system was not operated. It was demonstrated that the periodical draining of the machine prevented the drinking water in auto-watering piping from bacterial contamination.

Influenza C virus infection in rats.

Four-week-old rats (WKA/Hkm strain) were infected intranasally with the Ann Arbor/1/50 strain of influenza C virus and examined for clinical symptoms, virus replication, and serum antibody response. Although the animals showed no definite signs of illness, the virus replicated in the nose, and the hemagglutination-inhibiting (HI) and neutralizing antibodies were produced in their sera. When the inoculum sizes of 10(6.2) and 10(3.2) PFU were used, virus was recovered from nasal homogenates between days 1 and 10, and serum HI antibody became detectable by 10 days after infection. The rats infected with 10(1.2) PFU of the virus continued to shed virus until as late as day 20 without producing serum HI antibody. The amount of virus recovered from the nose was not affected significantly by either sex, age, or strain of the rat except that a slower virus growth was seen in the LE strain. It was also observed that the rats, previously inoculated with 10(3.2) PFU of the virus, showed no virus shedding when reinfected 7 weeks later but produced virus though in low titers when reinfected 50 to 55 weeks later. Virus was also recovered from rats once inoculated with 10(1.2) PFU of the virus when challenged 7 weeks later. Thus repeated infections characteristic of human influenza C can be produced in rats under the restricted conditions.

[Gland formation and pepsinogen expression in the gizzard endoderm as affected by the proventricular mesenchyme in the chick embryo]. / Formation de glandes et expression de pepsinogène dans l'endoderme du gésier sous l'influence du mésenchyme proventriculaire chez l'embryon de Poulet.

Studies of epithelio-mesenchymal recombination between the proventriculus and gizzard of 6-day chick embryos revealed that the proventricular mesenchyme can induce the morphogenesis of complex glands and cause the expression of embryonic pepsinogen in the gizzard endoderm, while the gizzard mesenchyme inhibits both of them.