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K Kinbara

Publications and source records attributed to K Kinbara.

12 recordsLinked to original sources
PubMed1999

Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system.

Human nCL-4, a digestive tract-specific calpain, was stably produced with 30K, a regulatory subunit for ubiquitous calpain as a fully active form using the baculovirus-expression system. nCL-4 showed an activity only when it was coexpressed with 30K. Expressed heterodimeric recombinant nCL-4 was purified to near homogeneity by sequential column chromatographies. Purified nCL-4 showed a calcium-dependent activity (calcium concentration at 50% maximum activity (Ka): 0.125 mM) with a sp act of 21 U/mg, which is distinct from those of ubiquitous calpains. nCL-4 exhibited calcium-dependent autolysis, but the cleavage pattern of nCL-4 was clearly different from ubiquitous calpains. Although it was inhibited by leupeptin, E-64, and calpastatin, and exhibited an optimal pH at 7.3 like other ubiquitous calpains, its optimal temperature was much lower. When overexpressed in COS-7 cells, clear asymmetric juxtanuclear, and/or nuclear staining rather than typical cytoplasmic staining was observed. Moreover, a translation product of nCL-4 was detected in rat stomach tissue by immunofluorescence analysis. In conclusion, human nCL-4 resembles ubiquitous calpain in some enzymatic properties and interacts with 30K for its activity. This is the first report on biochemical and enzymatic properties of a fully active tissue-specific calpain species expressed in the baculovirus system.

Animals↗
PubMed1998

Purification of native p94, a muscle-specific calpain, and characterization of its autolysis.

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous mu- and m-calpains, in its preference for residues at the P3 to P1' sites, indicating a distinct substrate specificity and function for the muscle enzyme.

Amino Acid Sequence↗
PubMed1998

Skeletal muscle-specific calpain, p49: structure and physiological function.

Recent studies indicate that calpain, a cytosolic Ca2+-dependent protease, constitutes a large family comprising ubiquitous, tissue-specific, and atypical calpains. p94 is a homologue of the catalytic large subunit of calpain, expressed predominantly in skeletal muscle. Recently, p94 has been found to interact with connectin/titin, a muscle elastic protein, and its gene has been identified as being responsible for limb-girdle muscular dystrophy type 2A. The loss of function of a calpain species eventually leads to the activation of proteases including other calpain species responsible for muscle degradation. p94 does not form a complex with the small subunit of calpain (30K), but exists as a homodimer. This, together with other results, led us to consider a novel mechanism for the activation of calpain, a Ca2+-induced subunit rearrangement.

Animals↗
PubMed1997

Muscle-specific calpain, p94, interacts with the extreme C-terminal region of connectin, a unique region flanked by two immunoglobulin C2 motifs.

Using the yeast two-hybrid system, we have recently reported that skeletal muscle-specific calpain, p94, binds specifically to connectin (or titin), a gigantic muscle elastic protein. Connectin has at least two binding sites for p94; one is at the N2-line region and the other is at the extreme C-terminus. In order to analyze the interaction between p94 and the C-terminus of connectin, we examined the C-terminal sequence of human skeletal muscle connectin. The sequence was essentially identical to that of heart muscle reported by Labeit and Kolmerer (1995, Science 270, 293-296), and the minimal binding site for p94 contained two IgC2 motifs and the intervening sequence called "M-is7." The exon encoding M-is7 is reported to be alternatively spliced depending on muscle tissues, resulting in the existence of both types of connectin with and without M-is7. However, the C-terminal region of connectin bound to p94 through M-is7. Our results suggest that the interaction between p94 and the C-terminus of skeletal muscle-type connectin is involved in tissue-specific myofibriogenesis.

Amino Acid Sequence↗
PubMed1995

Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence.

p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.

Amino Acid Sequence↗
PubMed1995

Calpain: novel family members, activation, and physiologic function.

The current status of calpain research is summarized on the basis of the most recent results. The main points are as follows. (i) Calpain constitutes a large family. (ii) Ca2+ ions cause the dissociation of calpain into subunits and the resulting free 80 kDa subunit is the active form of the enzyme. This dissociation corresponds to the activation of calpain. (iii) Some powerful clues have been obtained that will be helpful for analyzing the physiological function.

Animals↗
PubMed1994

Simulation study on heart failure: effects of contractility on cardiac function.

Using the model proposed by Beyar and Sideman, the effect of maximum isometric active stress at optimal sarcomere length (sigma 0) on left ventricular (LV) function was examined. Comparing the results of calculated LV function with those of reported experiments, sigma 0 was shown to be a potential indicator of myocardial contractility, and the model of Beyar and Sideman successfully predicted LV function with various myocardial contractilities. The LVP compensation curve, which describes the relationship between sigma 0 and maximum LV pressure, was then hypothesized. The combination of the Beyar-Sideman model and the LVP compensation curve enabled the prediction and approximation of the actual process of deterioration in heart failure. These models represent a step towards a fundamentally new concept in the current clinical situation of compensated heart failure and also in evaluating the process of heart failure.

Computer Simulation↗
PubMed1994

Simulation study on the effect of external vibration on left ventricular function: potential indicator of LV tolerance to the sudden reduction of myocardial contractility.

In a previous study the authors reported that external mechanical vibration applied to the left ventricular (LV) epicardium induces contractility-dependent depression in LV pressure, stroke volume and stroke work. It was suggested that this depression may be caused by the direct effect of external vibration on contractile protein. In another paper in this issue, it is proved that LV function with various myocardial contractilities and the actual process of deterioration in heart failure are well simulated in the model proposed by Beyar and Sideman, after some modifications have been made. In the study reported here it is assumed that an external mechanical vibration induces sudden reduction in myocardial active stress in the model of Beyar and Sideman; in this way the contractility-dependent effect of external vibration on LV function has been simulated. The results of this simulation support the suggestion that external mechanical vibration directly affects contractile protein and reduces LV function, and it is further suggested that the reduction of LV function induced by external vibration reflects the reserve or tolerance capacity of LV to a sudden reduction of myocardial contractility.

Animals↗
PubMed1994

Processing and secretion of Alzheimer's disease amyloid precursor protein.

We studied in vivo expression and in vitro secretion of the Alzheimer's disease amyloid precursor protein (APP). The results indicate that secretion of APP is mediated by PKC and the initial step of the processing may occur in the acidic secretory granules of the glial cells. Our results suggest that a metabolic switch of APP in neural cells is critical in amyloid deposition.

Alzheimer Disease↗
PubMed1992

Effects of NIK-247 on CO-induced impairment of passive avoidance in mice.

The effect of NIK-247 on carbon monoxide (CO)-induced amnesia were investigated. A step-down type passive avoidance task with mice was used to compare the effects of NIK-247 with those of tacrine. Two types of CO-induced amnesia model, acute and delayed models, were used. The acute amnesia model was developed using mice exposed to CO before memory consolidation, just after training, and a retention test carried out 24 h after training. The delayed amnesia model was prepared 7 days after CO exposure even when the animals were exposed to CO 4 h after training, after memory had consolidated. NIK-247 administered post-training at 0.03-0.3 and 3 mg/kg or pre-retention test (24 h after training) at 0.3 and 10 mg/kg attenuated the acute amnesia. In addition, NIK-247 (0.03, 0.1, 1 and 10 mg/kg) and tacrine (0.03, 0.1 and 1 mg/kg) administered before the retention test (7 days after CO exposure) improved retrieval in the delayed amnesia model. Tacrine (0.01-0.3 and 3 mg/kg), administered post-training, attenuated the acute amnesia but pre-retention test administration did not. The dose-response curves for NIK-247 and tacrine were biphasic bell-shaped. These results indicated that NIK-247 has an improving effect on hypoxia-induced acute and delayed cognitive dysfunction, and suggest that NIK-247 has promise as a nootropic drug for therapy of memory deficits in patients with cerebrovascular-type dementing disorders.

Aminoquinolines↗
PubMed1980

Epidemic of hand, foot and mouth disease in Gifu Prefecture in 1978.

During the period from May to August, 1978, an epidemic of hand, foot and mouth disease (HFMD) occurred in Gifu prefecture. Epidemiological, virological and serological investigations were performed. Cases involved ranged from 0 to 31 years of age, and 80.2% of them were under 5 years of age. The incidence of HFMD with neurological complication (3.7%) was lower than that in 1973. Enterovirus 71 (EV71) was isolated from 83 of 108 cases (75.9%) and a significant rise in the neutralization antibody titer against the isolate was found in 14 of 25 cases (56%). Thus, it was confirmed that the causative agent of the epidemic of HFMD in Gifu prefecture in 1978 was enterovirus 71.

Adolescent↗