Novel site on sodium channel alpha-subunit responsible for the differential sensitivity of grayanotoxin in skeletal and cardiac muscle.

We searched for sites on the alpha-subunit of the fast Na(+) channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na(+) current. cDNA clones, encoding the skeletal or cardiac isoforms of the alpha-subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na(+) currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (mu 1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of alpha-subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type mu 1). Successive replacement of Ser-251 (D1S4-S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.

Novel wasp toxin discriminates between neuronal and cardiac sodium channels.

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta-PMTX modifies rat brain type II Na+ channel alpha-subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha-subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta-PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta-PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

Three-dimensional measurement of rolling in tetraplegic patients.

OBJECTIVE: To investigate the differences between traumatic tetraplegic patients who can roll and those who cannot. DESIGN: Motion analysis using 3-dimensional measurement. SETTING: Rehabilitation centers in southwestern Japan. PARTICIPANTS: Nineteen male participants, all of whom had traumatic C6 complete injury. METHODS: We used an electromagnetic device to examine the degree of spinal movement in axial rotation during rolling (shifting from supine to side lying). This system (3-Space Win) measures the position and orientation of sensors in space. Two sensors were mounted on a subject over the spinous process of T1 and L5. RESULTS: The spinal rotation of patients who could not roll was significantly lower than that of patients who could roll. (The average rotation of non-rollers was 31.5+/-17.5 degrees, while the average rotation of rollers was 66.3+/-17.3degrees). In this study, there were no statistically significant differences in the members of the two groups in terms of age, height, weight or time after injury. CONCLUSION: Rolling requires greater and adequate flexibility in the back of tetraplegic patients.

The effect of low calcium diet on bone in ovariectomized mice.

This study investigates the effects of Ca on bone in the ovariectomized mice. Twenty-six female ICR mice aged 5 weeks were used. They were ovariectomized (OVX) or sham-operated (SHAM) and fed standard mouse diet (SF) or special low calcium diet (L.Ca), respectively. All animals were sacrificed at day 100 after operation. Mechanical strength of the left femur and tibia was measured by the three-point bending strength test. The bones were dried, weighed and burned to ash. Correlation between mechanical strength and ash content was found. A specimen of the right tibia was prepared. Mechanical strength, ash content and ratio of dry bone weight to body weight of the femur and tibia in OVX and L.Ca mice were significantly less than in SHAM and SF mice. SHAM/SF mice and OVX/L.Ca group showed highest and lowest values in all cases. The values for the femur and tibia in OVX/SF mice were lower than in SHAM/SF group and in OVX/L.Ca group were less than in OVX/SF mice. Correlation coefficients for mechanical strength and ash content were 0.704 and 0.776 for the femur and tibia. Ca is thus related to inhibition of bone loss and maintenance of bone mass and effective prevention of osteoporosis.

Modification of postural response caused by footwear conditions.

The purpose of this study was to clarify the effect of changing footwear conditions on postural response against postural perturbation. Twenty-three healthy subjects participated in this study. Postural response was induced by moving a platform forward, hereafter referred to as forward-perturbation of a platform. The center of pressure (COP) from the force plate and the electromyograms (EMG) of the tibialis anterior (TA) and quadriceps femoris (QUAD), which are both agonists of the response, were measured. The effect of plantar material and shape of footwear on postural response was examined as footwear condition. Changing plantar materials had an effect on integrated EMG of the agonists (IEMG) but not on the response pattern. On the other hand, the shape of footwear had an effect on the response pattern but not on IEMG. It was supposed from this result that changes in somatosensory input, caused by coupling of plantar material and shape of footwear, modifies postural response variously.

Oxygen releasing from cellular hemoglobin.

The oxygen-releasing behavior of hemoglobin vesicles (HbV) was measured in order to study the difference in oxygen dynamics inside and outside the cellular Hb using a conventional stopped flow method and a newly developed stopped flow flash photolysis method. The partial pressure of oxygen in the solution outside the HbV was monitored with the lifetime of the triplet state of meso-tetraphenylporphinatozinc(II) bound to human serum albumin excited by the laser flash. The change in the partial pressure of oxygen outside the HbV showed a biphasic profile and was slower than that inside the HbV. The first phase shows the oxygen-releasing process from Hb near the phospholipid bilayer membrane, and the second phase is considered the process in which oxygen diffuses to the bulk aqueous region and reaches the equilibrium value.

Possibility of independence in ADL (Activities of Daily Living) for patients with cervical spinal cord injuries--an evaluation based on the Zancolli Classification of Residual Arm Functions.

For patients with cervical spinal cord injuries to become independent in their ADL (Activities of Daily Living), residual arm function is very important. Also, age, sex, physical strengths, obesity, spasticity, pain, contracture and motivation are related. We investigated the possibility of independence in ADL for patients with cervical spinal cord injuries, carrying out our evaluation based on the Zancolli Classification of Residual Arm Functions. Zancolli classification C6BII is taken as the boundary level for ADL independence. Rehabilitation is not only controlled by the patients with cervical spinal cord injuries themselves but also by the ability of the rehabilitation staff. This implies that taking responsibility in rehabilitation important.

Properties of and oxygen binding by albumin-tetraphenylporphyrinatoiron(II) derivative complexes.

A hydrophobic tetraphenylporphyrinatoiron(II) derivative bearing a covalently bound axial imidazole [Fe(II)P] was efficiently and noncovalently bound into human serum albumin (HSA) up to an average of eight Fe(II)P molecules per HSA molecule. The aqueous solutions of the HSA-Fe(II)P complex provided a reversible and relatively stable oxygen adduct under physiological conditions (pH 7.4 and 37 degrees C). The half-life of the oxygen adduct (tau 1/2) was 1 h at 37 degrees C in an air atmosphere. With Fe(II)-TpivPP (the so-called "picket-fence heme") having no axial base, an oxygenated HSA-Fe(II)TpivPP complex was obtained using a 20-fold molar excess of 1,2-dimethylimidazole, but the tau 1/2 was very short (ca. 10 min at 37 degrees C). The oxygen affinity [P 1/2(O2)] and oxygen transporting efficiency (OTE) of HSA-Fe(II)P at 37 degrees C were 30 Torr and 22%, respectively. Furthermore, the oxygen-binding and dissociation rate constants (kon and koff) are extremely high in comparison with those of hemoglobin. The HSA molecule binding eight Fe(II)P molecules can transport about 3.4 mL/dL of oxygen under physiological conditions, corresponding to about 60% of the oxygen transporting amount of human blood.

Organ culture of psoriatic skin: effect of TGF-alpha and TGF-beta on epidermal structure in vitro.

Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3-4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O2 plus 5% CO2, and rotated at 60 rpm at 37 degrees C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF-alpha caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF-alpha as well as with the duration of the culture, but without disappearance of their granular layers. TGF-beta caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serum-containing medium or with their disappearance in the serum-free medium. TGF-beta also antagonized the acanthotic and degenerative effect of TGF-alpha. The results suggest that TGF-alpha and TGF-beta may partially be related to the induction of psoriatic epidermal lesions.