Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.     

Mechanomyogram and electromyogram responses of upper limb during sustained isometric fatigue with varying shoulder and elbow postures

To investigate the behavior of mechanomyogram (MMG) and electromyogram (EMG) signals in the time and frequency domains during sustained isometric contraction, MMG and surface EMG were obtained simultaneously from four muscles: upper trapezius (TP), anterior deltoid (DL), biceps brachii (BB), and brachioradialis (BR) of 10 healthy male subjects. Experimental conditions consisted of 27 combinations of 9 postures [3 shoulder angles (SA): 0 degree, 30 degrees, 60 degrees and 3 elbow angles (EA): 120 degrees, 90 degrees, 60 degrees] and 3 contraction levels: 20%, 40%, and 60% of maximum voluntary contraction (MVC). Subjective evaluations of fatigue were also assessed using the Borg scale at intervals of 60, 30, and 10 sec at 20%, 40%, and 60% MVC tests, respectively. The mean power frequency (MPF) and root mean square (RMS) of both signals were calculated. The current study found clear and significant relationships among physiological and psychological parameters on the one hand and SA and EA on the other. EA's effect on MVC was found to be significant. SA had a highly significant effect on both endurance time and Borg scale. In all experimental conditions, significant correlations were found between the changes in MPF and RMS of EMG in BB with SA and EA (or muscle length). In all four muscles, MMG frequency content was two or three times lower than EMG frequency content. During sustained isometric contraction, the EMG signal showed the well-known shift to lower frequencies (a continuous decrease from onset to completion of the contraction). In contrast, the MMG spectra did not show any shift, although its form changed (generally remaining about constant). Throughout the contraction, increased RMS of EMG was found for all tests, whereas in the MMG signal, a significant progressive increase in RMS was observed only at 20% MVC in all four muscles. This supports the hypothesis that the RMS amplitude of the MMG signal produced during contraction is highly correlated with force production. Possible explanations for this behavioral difference between the MMG and EMG signals are discussed.     

Effects of static stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

In-vitro tissue culture experiments were performed to study the effects of static stress on the mechanical properties of collagen fascicles obtained from the rabbit patellar tendon. After collagen fascicles having the diameter of approximately 300 microm were cultured for 1 and 2 wk under static stress between 0 and 3 MPa, their mechanical properties and crimp morphology were determined using a micro-tensile tester and a light microscope, respectively. The tensile strength and tangent modulus of the fascicles were significantly decreased by culture under no load compared to control fascicles. A statistically significant correlation, which was described by a quadratic curve, was observed between applied stress and tensile strength. The maximum tensile strength (16.7 MPa) was obtained at the applied stress of 1.2 MPa; the strength was within a range of control values. There was a similar correlation between applied stress and tangent modulus, and the modulus was maintained at control level under 1.3 MPa stress. The stress of 1.2 to 1.3 MPa is equivalent to approximately 50 percent of the peak stress developed in the intact rabbit patellar tendon by running. Strain at failure of cultured collagen fascicles was negatively correlated with applied stress, and that at 1.2 to 1.3 MPa stress was almost the same as the control value. Crimp morphology in the fascicles cultured under about 1.2 MPa stress was similar to that in control fascicles. These results indicate that cultured collagen fascicles change the mechanical properties and structure in response to static tensile stress. In addition, their mechanical properties and structure are maintained at control level if the static stress of 50 percent of in-vivo peak stress is applied.     

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.     

Muscular performance modeling of the upper limb in static postures

The purpose of the present paper is to describe and evaluate the polynomial models for predicting the muscular work capacity of the upper limb during sustained holding tasks. This research was concerned with the relationship between indicators of performance, i.e., specific posture or specific level of maximum voluntary contraction (MVC), and then modeling the functional data based on experimental results to estimate factors that may have an effect on task performance. To this end, we designed an experiment using 10 subjects in which each subject performed sustained isometric shoulder and elbow flexion endurance exercise under 27 conditions [3 shoulder angles (SA)x3 elbow angles (EA)x3 levels of %MVC]. Throughout all experiments, subjective perception of effort was assessed using the Borg scale, every 60, 30, and 10 s during the 20%, 40%, and 60% MVC tests, respectively. Proposal models were represented by three approaches: model A: estimation of endurance time (ET), with input variables such as SA, EA, and %MVC; model B: estimation of recommendation time (RT, the time during which the operator was able to maintain a position under the desired condition), with input variables such as SA, EA, %MVC, and required rate on the Borg scale; and model C: estimation of limit strength or %MVC, with input variables such as SA, EA, request limit time for work (LT), and required rate on the Borg scale. Statistical analysis indicated that the three proposal estimation models based on polynomial regression functions showed high significance (p<0.0001). The proposal models suggested and recommended the possibility of finding the best positions entailing the reduction and minimization of total muscular strain from manual material handling tasks in different work situations, with the consequent increase in work efficiency.     

No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of DiI

In regions of focal adhesion, cells adhere to a substrate through the interaction of extracellular matrix proteins and transmembrane integrins which are coupled to the cell skeleton. It is generally assumed that the plasma membrane is brought to close proximity to the substrate there. We used the novel method of fluorescence interference contrast (FLIC) microscopy to measure the distance of the plasma membrane of GD25 fibroblasts on silica coated with fibronectin. We correlated the distance map with the distribution of vinculin tagged with green fluorescent protein. We found that the major part of the membrane was separated by 50 nm from the substrate. With respect to this plateau, we found spots of upward deformation and of close adhesion as well as a general ruffling of the membrane. There was no correlation between the areas of close adhesion and the distribution of vinculin. We conclude that focal adhesion does not imply a close attachment of membrane and substrate.     

Ultramicroanalysis of reducing carbohydrates by capillary electrophoresis with laser-induced fluorescence detection as 7-nitro-2,1,3-benzoxadiazole-tagged N-methylglycamine derivatives

A method for ultramicroanalysis of carbohydrates using capillary electrophoresis with laser-induced fluorescence detection was developed, based on precapillary conversion to 7-nitro-2,1, 3-benzoxadiazole (NBD)-tagged N-methylglycamines. Although the derivatization involves two-step reactions, i.e., reductive N-methylamination followed by condensation with NBD-F, they can be carried out in a one-pot fashion and proceed quantitatively within ca. 50 min in total. Since the reaction conditions are mild, it does not cause desialylation. The derivatives can be well separated by capillary electrophoresis and sensitively detected by argon laser-induced fluorescence. It allowed detection of monosaccharides of down to nanomolar concentrations for analytical sample solution, which correspond to the attomole injected amounts, and good linearity was observed over a wide range. It was also successfully applied to analysis of N-glycans in a microgram quantity of a glycoprotein. Studies on the cleanup of derivatized product are also described in relation to N-glycan analysis.     

Characterization of p-chloroamphetamine-induced penile erection and ejaculation in anesthetized rats

Methodological shortcomings present in elicitation of male sexual reflexes in anesthetized animals. The present study has demonstrated, however, that intraperitoneal (i.p.) injection of p-chloroamphetamine (PCA), an indirect serotonin (5-HT) agonist, elicited simultaneously both penile erection and ejaculation in anesthetized rats. PCA (2.5-10.0 mg/kg, i.p.) caused an intermittent cluster of genital responses consisting of penile erection, glans erections, and penile cups, which closely resembles the response observed during the ex copula tests in unanesthetized rats. Measurements of intracavernous penile pressure showed that rhythmic changes in penile pressure were produced by PCA, together with glans erections and penile cups. PCA also caused a frequent ejaculations and the weighing of ejaculate accumulated over 0.5 hr was increased in a bell-shaped pattern, and the maximum effect was observed at 5.0 mg/kg. Pretreatment with p-chlorophenylalanine, a serotonin (5-HT)-synthesis inhibitor, significantly inhibited the expression of PCA-induced penile erection and ejaculation, while acute spinal transection at thoracic level did not affect the sexual responses. These results indicate that PCA-induced penile erection and ejaculation in anesthetized rats are mainly produced by the release of 5-HT, which is limited to the lower spinal cord and/or the peripheral sites. Furthermore, the sexual responses can be easily and reliably elicited by administration of PCA, which may be useful for the study of the mechanisms underlying male sexual functions.