Cryopreservation of in vitro Shoot Tips of Ulluco (Ullucus tuberosus Cal.) Using D Cryo-Plate Method.

　　BACKGROUND: Maintenance of in vitro collections of ulluco (Ullucus tuberosus Cal.) is cumbersome and costly in an ex-situ genebank. An alternative method for long term preservation which is safe and cost-effective is required. OBJECTIVE: To apply a novel cryopreservation procedure using the cryo-plate system to improve the long-term conservation of ulluco. MATERIALS AND METHODS: Initially V and D cryo-plate methods were tested, subsequently the D cryo-plate method was selected for ulluco cryopreservation. The D cryo-plate procedures were optimized for post-LN regrowth procedures including cold-hardening, sucrose addition in alginate gel, and duration of LS treatment. Optimized procedures were tested with 11 ulluco lines. RESULTS: Shoot tips were isolated from cold-hardened shoots for 3-4 weeks at 5 degree C were excised to 1.0-1.5 mm long and 0.5 mm wide and precultured for 16h at 25 degree C on MS with 0.3 M sucrose. The shoot tips were attached on the cryo-plates by alginate gel with 0.4M sucrose. The cryo-plates with attached shoot tips were treated with 2.0 M glycerol and 1.0 M sucrose solution for 90 min at 25 degree C and dehydrated on filter paper in a Petri dish by air current flow at 25 degree C for 45 min before direct immersion in LN. This optimized procedure was applied to shoot tips of 11 ulluco lines, resulting regrowth ranging from 73 % to 97 %, with an average of 90 % post-LN regrowth. CONCLUSION: D cryo-plate is a practical and simple procedure for cryo-storage of in vitro grown ulluco shoot tips in an ex situ genebank.

Development of body weight support gait training system using pneumatic Mckibben actuators -control of lower extremity orthosis.

Recently, robot assisted therapy devices are increasingly used for spinal cord injury (SCI) rehabilitation in assisting handicapped patients to regain their impaired movements. Assistive robotic systems may not be able to cure or fully compensate impairments, but it should be able to assist certain impaired functions and ease movements. In this study, the control system of lower extremity orthosis for the body weight support gait training system which implements pneumatic artificial muscle (PAM) is proposed. The hip and knee joint angles of the gait orthosis system are controlled based on the PAM coordinates information from the simulation. This information provides the contraction data for the mono- and bi-articular PAMs that are arranged as posterior and anterior actuators to simulate the human walking motion. The proposed control system estimates the actuators' contraction as a function of hip and knee joint angles. Based on the contraction model obtained, input pressures for each actuators are measured. The control system are performed at different gait cycles and two PMA settings for the mono- and bi-articular actuators are evaluated in this research. The results showed that the system was able to achieve the maximum muscle moment at the joints, and able to perform the heel contact movement. This explained that the antagonistic mono- and bi-articular actuators worked effectively.

Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates.

Two cryopreservation procedures using aluminium cryo-plates, termed V-Cryo-plate and D-Cryo-plate, were successfully developed for in vitro mat rush (Juncus decipiens Nakai) basal stem buds. Multiple stems induced in liquid MS medium containing 8.9 µM BA by roller culture were cut into small clumps, plated on solid MS medium and cultured for 1 week at 25 degree C. Clumps that had produced many buds were cold-hardened at 5 degree C for 1-2 months. The buds with basal stems were dissected from small clumps and precultured overnight at 25 degree C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in loading solution (2 M glycerol + 1.0 M sucrose). In the D-Cryo-plate procedure, the buds were dehydrated to 27-25% moisture content (fresh weight) by placing the cryo-plates in the air current of a laminar flow cabinet for 2 to 3 h. In the V-Cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25 degree C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in medium with 1.0 M sucrose for 20 min at room temperature. Regrowth of cryopreserved buds of line 'Kitakei 2' using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional mat rush lines. Using the V-Cryo-plate procedure resulted in regrowth ranging between 13.3 and 86.7%, with an average of 52.5%. The D-Cryo-plate led to regrowth ranging between 73.3 and 96.7%, with an average of 86.3%. The D-Cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

Stretch reflex excitability of the anti-gravity ankle extensor muscle in elderly humans.

AIM: To examine whether the stretch reflex excitability of the soleus muscle changes with age, stretch reflexes at rest (REST) and during weak voluntary contractions (ACT) were elicited in 18 older and 14 younger subjects. METHOD: The amplitude of the stretch reflex responses and gain, defined as the gradient of the regression line for the relation between stretch reflex responses against the angular velocity of the applied perturbation, were evaluated in each short-latency (M1) and two long-latency components (M2 and M3). RESULTS: It was found that in the older group, both the amplitude and gain of the M1 component did not change from the REST to the ACT conditions, whereas in the younger group both variables significantly increased from the REST to ACT conditions. The latency of the M1 component was significantly shorter under the REST condition (older vs. younger: 51.8 +/- 7.37 vs. 55.1 +/- 8.69 ms), while no group differences were found in those variables under the ACT condition, suggesting that the muscle-tendon complexes of SOL muscles of the older subjects were less elastic and had less slack, probably due to age-related histochemical alterations. Further, the Hoffman reflex (H-reflex), elicited during the REST condition in 10 older and 11 younger subjects showed no significant differences, suggesting that the soleus motoneuron response to the Ia input was comparable between the two subject groups. CONCLUSION: The histochemical alterations occurring with the ageing process might augment the short-latency stretch reflex in the SOL muscle without enhancement of motoneuronal excitability, and this effect might be masked when the muscle is voluntarily activated.

Neural control: novel evaluation of stretch reflex sensitivity.

We evaluated the stretch reflex activities of the elbow flexor and extensor muscles considering the relationship between the reflex electromyographic (EMG) responses and their corresponding standardized muscle stretch velocities. Specifically, muscular stretch velocity was estimated by using ultrasonograms. Stretch reflex EMG responses were elicited in the biceps brachii, brachioradialis and triceps brachii with a ramp-and-hold rotation at the elbow joint, which consisted of various angular velocities for the extension- or flexion-direction. The whole muscle stretch velocity induced by each ramp-and-hold rotation was calculated on the basis of fibre length changes associated with the elbow joint angle. A linear regression equation was fitted to the relation between the whole muscle stretch velocity and the reflex EMG responses, and the variables from the equation were used to quantify sensitivity of each reflex EMG component. The reflex EMG responses were increased as the ramp-and-hold rotational velocity increased. There were no significant differences in the recorded magnitudes of reflex EMG responses with equivalent joint rotational velocity between the brachioradialis and the triceps brachii medial head. These muscles showed the highest reflex responses in the flexor and extensor muscles, respectively. To the contrary, the reflex EMG response elicited by the standardized muscle stretches was significantly greater in the extensor muscles, indicating a higher reflex sensitivity. This was because of the lower muscle stretch velocity of the triceps brachii with an equivalent elbow joint rotation. The stretch reflex sensitivity in both the elbow flexor and extensor muscles might be regulated so as to make the reflex responses the same when the equivalent joint rotational velocity is applied to these muscles.

Phase-dependent electromyographic activity of the lower-limb muscles of a patient with clinically complete spinal cord injury during orthotic gait.

We examined the lower-limb electromyographic (EMG) activity from a patient with clinically complete spinal cord injury during orthotic gait. A newly developed gait orthosis was used to obtain bipedal locomotion. The surface EMG data during the gait together with the biomechanical variables were collected by way of a radio EMG system. A cyclic EMG activation pattern corresponding to the gait cycles were observed in each of the paralyzed lower-limb muscles during the orthotic gait. Although the EMG activation did not seem to contribute toward generating the gait, it showed some similarities to that of the infant stepping or immature gait. These results might be regarded as one of the indirect pieces of evidence that suggest the existence of a spinally originating motor mechanism underlying human locomotion.

Short- and long-latency reflex responses during different motor tasks in elbow flexor muscles.

Stretch reflex responses in three elbow flexor muscles - the brachioradialis and the short and long heads of the biceps brachii - were studied during different motor tasks. The motor tasks were iso-velocity (8 deg/s) elbow flexion movements in which the muscles performed shortening or lengthening contractions, or were isometric contractions. Care was taken to maintain constant background electromyographic (EMG) activity in the brachoradialis muscle at a 50-deg elbow angle across the tasks by changing the magnitude of the initial load. During each task, mechanical perturbations (duration 170 ms) were applied at pseudorandom intervals when the elbow angle was 50 deg. The magnitude of the perturbation was varied across tasks in order to induce an elbow extension velocity of 80 deg/s over the first 50 ms after the onset of perturbation. The stretch reflex EMG responses in all muscles varied across the three tasks, despite a constant EMG level and similar perturbation-induced angular velocity in the direction of elbow extension. In particular, both the short- and long-latency reflex EMG components were reduced during the lengthening contractions. Further, the task-dependent variations in the early (M2) and the late (M3) components of the long-latency reflex were different, i.e., the magnitude of M3 was considerably enhanced during the shortening task as compared with that of M2. These findings suggest that central modification was responsible for the task-dependent modulation of late EMG responses.

A new concept of dynamic orthosis for paraplegia: the weight bearing control (WBC) orthosis.

A new concept of device, termed the weight-bearing control (WBC) orthosis, has been designed with three major needs in mind; a rigid frame that supports the user's body weight, a special hip joint device that reciprocally propels each leg forward, a gas powered foot device that varies the sole thickness of the device for foot/floor clearance, and control system of the orthosis. A paraplegic (T7 level complete paraplegia, sensory evoked potential silent, response to electro-stimulation on the cortical area of the brain also silent) who has tested this WBC orthosis has been able to walk without fatigue at a high speed for a greater distance than before. In walking tests of this WBC orthosis, he achieved a maximum walking speed of 34.1 m/min for a distance of 10 metres. The walking distance reached 521 m with an average walking speed of 21.2 m/min without rest on one small tank of CO2 liquid gas, measuring 10 cm in length and 3 cm in diameter.

Na+-gradient-dependent transport of L-proline and analysis of its carrier system in brush-border membrane vesicles of the guinea-pig ileum.

Transport of L-proline was studied with membrane vesicles prepared from the brush borders of the guinea-pig ileum. The presence of an Na+ gradient from outside to inside of the vesicles stimulated L-proline uptake. Accumulation of amino acid in the vesicles reached a maximum 30 s after incubation, then decreased due to efflux and finally equilibrated at a level nearly identical to that shown in the absence of an Na+ gradient in 30 min. The peak level of the uptake was 3.5-times greater than the final equilibrium level. The equilibrium level of L-proline uptake decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, that is, under the condition of zero intravesicular space, showed no uptake, indicating transport of L-proline into membrane vesicles. The initial rate of uptake for 15 s was enhanced with increasing concentrations of Na+ in the external medium. A small part of the L-proline transport occurred by simple diffusion in addition to Na+-gradient-dependent transport. When L-proline concentrations were varied and transport due to diffusion was subtracted, the initial rate of uptake dependent on Na+ gradient (out greater than in) obeyed Michaelis-Menten kinetics with Km and V values of 0.67 mM and 2.73 nmol/15 s per mg protein, respectively. Evidence was obtained which indicates that L-cysteine is a substract specific for transport through system ASC (alanine-, serine-, and cysteine-preferring) and that transport in the presence of an Li+ gradient (out > in) also takes palce by the ASC system. The uptake of L-proline in the presence of an Na+ gradient (out > in) was inhibited 90% by a large excess of alpha-(methylamino)-isobutyrate, the model substrate specific for the A system (alanine-preferring). This indicates than 90% of Na+-gradient-dependent L-proline uptake is supported by the A system. The remaining 10% of L-proline uptake was found to be catalyzed by the ASC system, since L-proline uptake equivalent to this alpha-(methylamino)-isobutyrate-uninhibited part was demonstrated in the presence of Li+ gradient.

Studies on cholera carriers.

Cholera carrier studies in the Philippines in 1964-66 showed a prevalence rate of 21.7% among household contacts of cholera patients, and 8.4% in occupants of houses next door to one where a cholera patient lived, as opposed to 0.34% in the general population. The duration of the carrier state among 19 household carriers isolated for examination varied from 5 to 19 days. The vibrio concentration in the stool of contact carriers was 10(2)-15(5) per gram, as compared with 10(6)-19(9) per ml of rice-water stool in cholera cases.The agglutinin titre increased with time for carriers, as it does for patients. It declined to a very low level 8-12 weeks after recovery, with the exception of one proved long-term carrier.The strains isolated from carriers were identical in all respects, including virulence in infant rabbits, with strains isolated from patients-except that 3 carrier strains were rough.