Programming an in vitro DNA oscillator using a molecular networking strategy

Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well‐controlled environment, the corresponding chemical network is easily amenable to quantitative mathematical analysis. These synthetic systems may thus accelerate our understanding of the underlying principles of biological dynamic modules.     

Changes in center-of-pressure dynamics during upright standing related to decreased balance control in young adults: fractional Brownian motion analysis

We investigated the relationships between the ability to maintain balance in an upright stance and center-of-pressure (COP) dynamic properties in young adults. Included in this study were 10 healthy male subjects in each of two groups with respect to balance ability. Balance ability was evaluated according to the length of time a subject stood on one leg with his eyes closed. The means and ranges of this one-leg balancing time were 17.9 s (3-43 s) and 118.3 s (103-120 s) for the off-balance and balance groups, respectively. The time-varying displacements of the COP under a subject's feet during quiet two-leg (normal) standing were measured by an instrumented force platform. Each subject was tested in both the eyes-open and eyes-closed conditions. The COP trajectories were analyzed as fractional Brownian motions according to the procedure of 'stabilogram-diffusion analysis', proposed by Collins and De Luca (1993). The extracted parameters were the effective diffusion coefficients (D) for the short-term (less than about 1.0 s) and long-term intervals, respectively, as well as the Hurst exponents (H) for the short-term and long-term intervals, and some critical-point coordinates (i.e., critical mean square displacements and critical time intervals). The off-balance group showed significantly higher values for short-term D, short-term H, and critical mean square displacements than the balance group. No significant differences between the groups were found in the long-term D and H or in the critical time intervals. That is, for the off-balance subjects, an increase in the stochastic activity and positively correlated (persistent) behavior of the postural sway during shorter timescales may cause postural instability. These results suggest that the difference in balance ability for young adults is related to the open-loop (i.e., short-term) control mechanisms but not to the corrective feedback (i.e., long-term) mechanisms used to maintain balance in an upright stance.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes. Received: 26 June 2000 / Accepted: 20 September 2000     

Effects of Intracellular Vanadate on Electrogenesis, Excitability and Cytoplasmic Streaming in Nitellopsis obtusa

Vanadate, which is known to inhibit the plasma membrane H^$-ATPaseof Neurospora, was applied intracellularly to internodal cellsof Nitellopsis by use of the intracellular perfusion technique.It inhibited electrogenesis and H^$-extrusion, evidence thatthe electrogenic pump of the Characeae plasmalemma is the H^$-extrudingone. The concentration of vanadate for the half-maximal inhibitionof the activity of the pump was 5 µM. The membrane potentialand H^$-extrusion occasionally recovered from vanadate inhibitionsfor reasons that are unknown. Membrane excitability, which isdependent on Mg?ATP, was not inhibited by vanadate, which suggeststhat the ATPase involved with membrane excitability differsfrom that of the H^$ pump. Cytoplasmic streaming took place evenwhen the cell was perfused with the medium containing 1 mM vanadate,which indicates that the vanadate-insensitive actomyosin systemis concerned with the motive force generation of the streaming. (Received August 27, 1981; Accepted April 13, 1982)