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Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 --> 381, 471, 567, and 679 for GWHWE and m/z 688 --> 381, 445, 541, and 653 for GWHC*E) were detected.

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Purine contents of soybean-derived food and various other Japanese foods were quantitatively determined by high-performance liquid chromatography (HPLC). Purine contents were as follows: soybean-derived foods, 21.9-172.5 mg/100 g or 100 mL; Japanese vegetables, 2.3-171.8 mg/100 g; Japanese mushrooms, 9.5-142.3 mg/100 g. Since purine levels in these foods did not exceed 200 mg/100 g, we recommend that eating of them should be adopted and good dietary habits followed.

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The liquid chromatography-mass spectrometry (LC-MS) following on from the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.

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The smooth pursuit system moves the eyes in space accurately while compensating for visual inputs from the moving background and/or vestibular inputs during head movements. To understand the mechanisms underlying such interactions, we examined the influence of a stationary textured visual background on smooth pursuit tracking and compared the results in young and adult humans and monkeys. Six humans (three children, three adults) and six macaque monkeys (five young, one adult) were used. Human eye movements were recorded using infrared oculography and evoked by a sinusoidally moving target presented on a computer monitor. Scleral search coils were used for monkeys while they tracked a target presented on a tangent screen. The target moved in a sinusoidal or trapezoidal fashion with or without whole body rotation in the same plane. Two kinds of backgrounds, homogeneous and stationary textured, were used. Eye velocity gains (eye velocity/target velocity) were calculated in each condition to compare the influence of the textured background. Children showed asymmetric eye movements during vertical pursuit across the textured (but not the homogeneous) background; upward pursuit was severely impaired, and consisted mostly of catch-up saccades. In contrast, adults showed no asymmetry during pursuit across the different backgrounds. Monkeys behaved similarly; only slight effects were observed with the textured background in a mature monkey, whereas upward pursuit was severely impaired in young monkeys. In addition, VOR cancellation was severely impaired during upward eye and head movements, resulting in residual downward VOR in young monkeys. From these results, we conclude that the directional asymmetry observed in young primates may reflect a different neural organization of the vertical, particularly upward, pursuit system in the face of conflicting visual and vestibular inputs that can be associated with pursuit eye movements. Apparently, proper compensation matures later.

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We showed previously that smooth pursuit training combined with whole-body rotation in the orthogonal plane induces adaptive cross-axis vestibulo-ocular reflex (VOR). To gain an insight into the possible pathways and the nature of error signals for cross-axis VOR adaptation, we examined further properties of adaptive responses. In the first series, we trained monkeys for vertical pursuit during sinusoidal yaw rotation at 0.5 Hz (+/- 10 degrees) by presenting a target spot either in phase with, or with phase shifts (lead or lag) of 90 degrees to, the chair for 1 h. After training, sinusoidal or trapezoidal yaw rotation was tested in complete darkness without a target. Different training conditions resulted in different amounts of phase shift in cross-axis VOR. Trapezoidal yaw rotation (peak acceleration approximately 780 degrees/s2) revealed further differences in the direction, latency and time course of the adaptive responses depending on the conditions of the pursuit task. At least two (fast and slow) components with different latencies were induced in the cross-axis VOR by trapezoidal rotation after in-phase and phase-shift training. Adaptive responses were accurately simulated by the weighted sum of these two components. In the second series, we examined the effects of sequentially flashed (10 microseconds) targets in the horizontal plane during pitch rotation. The monkeys learned to track such targets by smooth pursuit, and cross-axis VOR was also induced after such apparent motion stimuli without retinal slip of the target image. These results indicate the importance of eye velocity for cross-axis VOR and suggest that this adaptation occurs most probably in the smooth pursuit pathways.

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The smooth pursuit system interacts with the vestibular system to maintain the accuracy of eye movements in space. To understand neural mechanisms of short-term modifications of the vestibulo-ocular reflex (VOR) induced by pursuit-vestibular interactions, we used a cross-axis procedure in trained monkeys. We showed earlier that pursuit training in the plane orthogonal to the rotation plane induces adaptive cross-axis VOR in complete darkness. To further study the properties of adaptive responses, we examined here the initial eye movements during tracking of a target while being rotated with a trapezoidal waveform (peak velocity 30 or 40 degrees/s). Subjects were head-stabilized Japanese monkeys that were rewarded for accurate pursuit. Whole body rotation was applied either in the yaw or pitch plane while presenting a target moving in-phase with the chair with the same trajectory but in the orthogonal plane. Eye movements induced by equivalent chair rotation with or without the target were examined before and after training. Before training, chair rotation alone resulted only in the collinear VOR, and smooth eye movement-tracking of orthogonal target motion during rotation had a normal smooth pursuit latency (ca 100 ms). With training, the latency of orthogonal smooth tracking eye movements shortened, and the mean latency after 1 h of training was 42 ms with a mean gain, at 100 ms after stimulus onset, of 0.4. The cross-axis VOR induced by chair rotation in complete darkness had identical latencies with the orthogonal smooth tracking eye movements, but its gains were <0.2. After cross-axis pursuit training, target movement alone without chair rotation induced smooth pursuit eye movements with latencies ca 100 ms. Pursuit training alone for 1 h using the same trajectory but without chair rotation did not result in any clear change in pursuit latency (ca 100 ms) or initial eye velocity. When a new target velocity was presented during identical chair rotation after training, eye velocity was correspondingly modulated by just 80 ms after rotation onset, which was shorter than the expected latency of pursuit (ca 100 ms). These results indicate that adaptive changes were induced in the smooth pursuit system by pursuit-vestibular interaction training. We suggest that this training facilitates the response of pursuit-related neurons in the cortical smooth pursuit pathways to vestibular inputs in the orthogonal plane, thus enabling smooth eye movements to be executed with shorter latencies and larger eye velocities than in normal smooth pursuit driven only by visual feedback.

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The mechanisms underlying the diverse responses to step current stimuli of models [Edman et al. (1987) J Physiol (Lond) 384: 649-669] of lobster slowly adapting stretch receptor organs (SAO) and fast-adapting stretch receptor organs (FAO) are analyzed. In response to a step current, the models display three distinct types of firing reflecting the level of adaptation to the stimulation. Low-amplitude currents evoke transient firing containing one to several action potentials before the system stabilizes to a resting state. Conversely, high-amplitude stimulations induce a high frequency transient burst that can last several seconds before the model returns to its quiescent state. In the SAO model, the transition between the two regimes is characterized by a sustained pacemaker firing at an intermediate stimulation amplitude. The FAO model does not exhibit such a maintained firing; rather, the duration of the transient firing increases at first with the stimulus intensity, goes through a maximum and then decreases at larger intensities. Both models comprise seven variables representing the membrane potential, the sodium fast activation, fast inactivation, slow inactivation, the potassium fast activation, slow inactivation gating variables, and the intra cellular sodium concentration. To elucidate the mechanisms of the firing adaptations, the seven-variable model for the lobster stretch receptor neuron is first reduced to a three-dimensional system by regrouping variables with similar time scales. More precisely, we substituted the membrane potential V for the sodium fast activation equivalent potential Vm, the potassium fast inactivation Vn for the sodium fast inactivation Vh, and the sodium slow inactivation Vl for the potassium slow inactivation Vr. Comparison of the responses of the reduced models to those of the original models revealed that the main behaviors of the system were preserved in the reduction process. We classified the different types of responses of the reduced SAO and FAO models to constant current stimulation. We analyzed the transient and stationary responses of the reduced models by constructing bifurcation diagrams representing the qualitatively distinct dynamics of the models and the transitions between them. These revealed that (1) the transient firings prior to reaching the stationary state can be accounted for by the sodium slow inactivation evolving more slowly than the other two variables, so that the changes during the transient firings reflect the bifurcations that the two-dimensional system undergoes when the sodium slow inactivation, considered as a parameter, is varied; and (2) the stationary behaviors of the models are captured by the standard bifurcations of a two-dimensional system formed by the membrane potential and the potassium fast inactivation. We found that each type of firing and the transitions between them is due to the interplay between essentially three variables: two fast ones accounting for the action potential generation and the post-discharge refractoriness, and a third slow one representing the adaptation.

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The input-output relation of a chain of pulse-coupled oscillators is examined. The oscillators capture the essential aspect of the dynamics of pacemaker neurons. Inputs consist of pacemaker, and noisy trains impinging upon the first unit in the chain. The response of the chain is defined as the spike train emitted by the last unit. We observe two important phenomena in the response of the chain for a given input train, whether pacemaker or noisy. First, the mean output rate of the chain is equal to the mean input rate in the range of input rate in which one input pulse corresponds to one output spike without phase locking (1:1 alternation). Second, for the same input range, the output interspike intervals tend to the average of the input interpulse intervals in a long chain of oscillators. This behavior contrasts with the fact that the response of a single unit depends on both input rate and pattern. We show that the response of the chain is reproduced by the phase transition curve which represents the phase shift due to a single isolated pulse stimulus. This analysis reveals that the averaging of the output interspike intervals is due to the geometrical aspect of the phase transition curve. This geometrical aspect causes the dependence of the response of a single unit on input pattern.

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The response of a pacemaker neuron model to a train of transient inhibitory impulsive perturbations is examined. The model reproduces the heterogeneous discharge forms and abrupt switchings displayed by the crayfish slowly adapting stretch receptor organ (Segundo et al., 1994, Neuroscience 62(2), pp. 459-480). The non-monotonous aspect of the instantaneous firing rate of the model reflects the fact that in some regimes input and output rates are both increasing, despite the inhibitory effect of the former. We determine how such paradoxical acceleration takes place by analyzing the response of the model using its phase transition curve. We show that paradoxical acceleration results from the fact that the phase transition curve exhibits a large slowly increasing, almost linear section similar to that of living preparations.

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The main feature separating a living pacemaker from other oscillators is the fact that the former has not only the oscillatory property but also slow and fast changes of the membrane potential corresponding to the sub- and suprathreshold regions. We propose a simple mathematical model called mRIC and show that the model exhibited the essential feature of pacemakers generating spikes at constant time intervals. The behavior of the model driven by periodic pulse trains is analysed using the phase transition curve.

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When sera from the frogs, Rana (R.) nigromaculata and Rana (R.) brevipoda, were run on starch-gel electrophoresis (SGE), several bands were seen in an electrophoretic pattern of proteins. This pattern on SGE appeared the same at stages XIV, XV and XXI, and in the adult frog, R. niguromaculata. However, the pattern at stage X was different. A protein, designated "protein C", did not appear clearly at this stage, but afterwards. This protein was the second richest among serum proteins of mature frogs. Protein C (Mr = 180 kD, when estimated by SDS-PAGE) was obtained after SGE and then subjected to an NH2-terminal sequence analysis. Sequences of protein C from R. nigromaculata and R. brevipoda were NH2-TDPMYVIFIPQTLXE for the first 15 amino acids and NH2-TDPHYVIFKG for the first 10 amino acids, respectively. Homology search of GenBank sequences indicated no significant similarity with any known proteins. The results suggest that protein C is a new protein, and that it may play an important role(s) in the serum after stage X in these species.

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The distribution of serum albumin is of interest in the Xenopus (X.) laevis testis, since albumin is probably a major protein that binds testosterone (T) in the plasma and interstitial fluid. This study was undertaken to determine the localization and purification of serum albumin in the X. laevis testis. The interstitial tissue and spermatogenia immunoreacted strongly with a sheep antiserum raised against X. laevis albumin. A weak staining was also seen in spermatocytes and early spermatids, but there was no staining in Sertoli cells. In order to clarify whether serum albumin was really localized on the surface of testicular cells in the X. laevis testis, a membrane-rich fraction was prepared from testes and extracted with 0.6 M KCl. The KCl extract was then subjected to gel filtration, ammonium sulfate precipitation and high-performance liquid chromatography (HPLC). A protein with M(r) = 74 kD was obtained by this procedure and its NH2-terminal amino acid sequence was determined. The sequence of the first 19 amino acids was DTDADXXKXIADVYTALTE, suggesting that this protein was identical to serum albumin (M(r) = 74 kD). When the membrane fraction of blood cells in this animal was handled in the same manner, no appreciable amount of albumin was detected. These results suggest that the 74 kD serum albumin, possibly associated with bound T, may play an important role in the differentiation of germ cells during spermatogenesis of X. laevis testis.

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In accordance with physiological and electronmicroscopic evidence that, in the anterior byssal retractor muscle (ABRM) of a common mussel Mytilus edulis, Ca2+ activating the contractile system is accumulated at the inner surface of the plasma membrane and at the membrane of sarcoplasmic reticulum (Ebashi, S. and Endo, M. (1968) Prog. Biophys. Mol. Biol. 18., 123-183; Suzuki, S. and Sugi, H. (1982) in The role of calcium in biological systems, Vol. I (Anghileri, L.J. and Tuffet-Anghileri, A.M., eds.), pp. 201-207, CRC Press, Boca Raton), we have found a high-molecular-mass (450 kDa) Ca(2+)-binding protein (MCBP-450) in the membrane fractions of the ABRM by 45Ca autoradiography of proteins transferred to nitrocellulose membrane (Rüegg, J. C. (1971) Physiol. Rev. 51, 201-248). MCBP-450, purified to electrophoretic homogeneity, exhibited Ca(2+)-dependent changes in mobility, tryptophan fluorescence, UV absorption and CD spectrum, indicating its Ca(2+)-dependent conformational changes. MCBP-450 has a high content of aspartic and glutamic acid (23.8%) and a high content of basic residues (27%). It has a high capacity Ca(2+)-binding site, which binds about 38 mol of Ca2+ per mol with an adissociation constant of 10(4) M-1, and a low-capacity Ca(2+)-binding site, which binds about 7 mol of Ca2+ per mol with an association constant of 10(5) M-1. These characteristics of MCBP-450 are consistent with the view that it is actually involved in regulating the contraction-relaxation cycle in the ABRM.

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Recently, we purified a Ca(2+)-binding protein from rat spermatogenic cells [Biochem. Biophys. Res. Commun. 176, 135-1364, 1991]. In the present study, this protein was identified as calreticulin, which is a resident protein of the endoplasmic reticulum (ER). Immunohistochemical studies revealed that calreticulin was present in the acrosome of both round spermatids and mature sperm. However, under immunoelectron microscopy, gold-particles were seen over other subcellular structures of spermatocytes, spermatids, and Sertoli cells. When the labeling density in subcellular structures of spermatids was analyzed, the acrosome was found to be most heavily labeled and the Golgi apparatus was second. The complete amino acid sequence of calreticulin, deduced from the cDNA sequence, shares a high degree of identity with that of the analogous mouse protein. The cDNA encoded a protein of 416 amino acids, including a 17-residue NH2-terminal signal sequence. The mature protein contains a KDEL sequence as an ER signal at the COOH terminus. Sperm calreticulin contained no glycosyl moiety. Northern blot analysis of RNAs from purified populations of rat spermatogenic cells indicated that the calreticulin mRNA was present in both pre- and postmeiotic cells. Immunoblot analysis of calreticulin during developmental stages showed that calreticulin was detected in the testis between the ages of 5 and 50 days. Furthermore, purified rat calreticulin contained two Ca(2+)-binding sites, a low affinity/high capacity site and a high affinity/low capacity site. These results suggest that calreticulin, which is not specific to testis, is closely associated with spermatogenesis of rats. This ER protein may be incorporated into the acrosomal vesicle via the Golgi apparatus, without glycosylation, during spermiogenesis, and may play an important role in the regulation of cell functions such as sperm motility and acrosome reaction.

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Thanks to the advancement of medical technology, there has been a significant decline in stroke mortality in Japan. This means that many stroke victims could gain additional years of survival with several disabilities. It is necessary that nurses assist these disabled patients upon discharge for shifting smoothly from the hospital to the home. The purpose of this study was to clarify the factors of continuity of care for the disabled patients who has had a stroke, between hospital and community. The 504 patients who were discharged from the Kanagawa Prefectural Rehabilitation hospital and were in need of visiting nursing services upon their discharge were selected in this study. The survey was conducted through discharge summaries and visiting nursing reports. The data were compared at discharge with the first nursing visit with respect to the level of independence in activities of daily living (ADL) and their care needs. The results were summarized as follows: 1. As for the functional level of ADL, most patients remained unchanged. Feeding was improved; however, grooming (including bathing), using the toilet, and dressing were worse. Hence, hospital nurses should strongly educate the patients and repeat ADL so that dressing is a rehabilitation exercise itself. Also, nurses, prior to discharge, should consider dealing with the housing problems, especially in the bathrooms and washrooms, due to the patients' physical disabilities. Nurses should pay particular attention to the aged group upon the continuity of care since their level of independence in walking changed variously among individuals. 2. Hospital nurses identified nursing problems mostly in the physical aspects, whereas public health nurses did it in terms of family-caregivers and social resources rather than physical needs. The reasons why nursing problems did not correspond between hospital nurses and public health nurses were probably owing to the differences in their practice settings, job content, and working conditions between the hospital and community. Thus, both nurses should reciprocally provide one's information and introduce other's information in order to understand and employ the other's standpoints.

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The purpose of this study was to purify a calcium-binding protein (CalBP) from human spermatozoa of 226 men and to determine the localization of this protein in spermatozoa. The sperm cells were extracted with 0.6 M KCl, and the KCl extract was then subjected to gel filtration and high performance liquid chromatography. Two CalBPs of Mr = 38 kDa and 52 kDa were found by 45Ca(2+)-binding on blotted proteins. Only a 52 kDa CalBP (CalBP-52) was purified to a final yield of 0.007%. The isoelectric point of this protein was 5.1. The human CalBP-52 reacted with rabbit antiserum directed against rat 52 kDa CalBP. An immunocytochemical study showed that this protein was localized on the sperm head. It is postulated that this protein may have important functions related to Ca(2+)-transport into sperm cells.

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A Ca(2+)-binding protein of Mr = 52000, estimated by SDS-PAGE, was purified to a final yield of 0.04% from rat spermatogenic cells. Purification steps included gel filtration, ammonium sulfate precipitation and HPLC. Amino acid analysis showed the content of 34% acidic residues and 15% basic residues. The isoelectric point of this protein was 4.7. Dot-blot analysis indicated that the Ca(2+)-binding protein bound 2 mol of calcium per mol of protein. This protein had two binding sites with dissociation constants of 4.8 microM and 0.2 microM. No appreciable amount of hexose was observed (less than 1 microgram of hexose/70 micrograms of protein). This protein may play an important role such as the Ca(2+)-transport in the plasma membrane of spermatogenic cells.

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Calmodulin was purified from the anterior byssal retractor muscle (ABRM) of a mollusc Mytilus edulis. Ca2+-induced conformational changes in the ABRM calmodulin could be demonstrated by polyacrylamide gel electrophoresis, by u.v. absorption spectrum and by circular dichroic spectrum. The amino acid composition of the ABRM calmodulin closely resembled that of other invertebrate calmodulins. The ABRM calmodulin was less effective in activating rat brain phosphodiesterase than vertebrate calmodulins.

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