The reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3.6.1.3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, APT accelerated the reactivity of S1 and S2 much more in the myosin B system than in myosin alone. NEM-modified myosin B ATPase exhibited a shift of the KCL dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group(s) essential for Ca2+ sensitivity was discussed.
Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP‐1 and GNCP‐2 in the granules. To understand the regulation of the gene expression, the promoters for the GNCP‐1 and GNCP‐2 genes were characterized. Sequencing analysis of the genomic clones showed that the nucleotide sequences of the 5′‐upstream regions (1.7 kb) from exon 1 were homologous (90–93%) between the GNCP‐1 and GNCP‐2 genes. However, transient transfection assays using luciferase reporter gene constructs revealed that the promoter activity of GNCP‐1 was 2‐fold greater than that of GNCP‐2. Furthermore, DNase I footprint analysis demonstrated that three regions (I, II and III) were protected on the GNCP‐1 promoter, whereas only two protected regions (II and III) were identified on the GNCP‐2 promoter. Together these observations indicate that GNCP‐1 and GNCP‐2 are encoded by homologous genes, but the expression of the GNCP‐1 and GNCP‐2 genes is likely to be different at the level of transcription.
The tryptic inactivation of EDTA-ATPase activity of myosin A was studied in 0.5 M monovalent ion media at pH 7.6 and 20°. 1) Trypsin activity itself was affected by monovalent ions in the absence of divalent ions, but not by nucleotide regardless of the presence of' monovalent and divalent ions. 2) Only in the absence of divalent ions were remarkable differences in the digestibility pattern observed between K+ and Na+ ions; in the K+ medium the inactivation rate was hardly promoted by ITP but was slightly increased by ATP and ADP. In the Na+ medium, on the other hand, these nucleotides, including ITP, markedly increased the rate of inactivation. Pyrophosphate showed almost the same potent effect as ATP in both media. In the presence of divalent ions, however, nucleotides strongly accelerated the inactivation of ATPase activity and no differences between K+ and Na+ were observed in the digestibility pattern. The mode of inactivation in the absence of divalent cations was first order, while the semilogarithmic plot of the inactivation rate in the presence of Mg2+ was biphasic in the presence of ATP. 3) A plot of the degree of tryptic inactivation of ATPase [EC 3.6.1.3] activity in the presence of ATP and ITP against the crystal ionic radius of monovalent ions from 0.6 (Li+) to 1.7 Å (Cs+) was bell-shaped, with a minimum in K+ medium (1.33 Å). The accelerating effect of ITP was not observed in K+ and NH4+ media, where ATP was still effective. 4) The actions of K+ and Na+ on the tryptic digestibility were independent of each other but were competitive on the catalytic activity. 5) When a specific sulfhydryl group, S1, was blocked with N-ethylmaleimide, myosin became more susceptible to trypsin. The typical effect of nucleotides seen in intact myosin was hardly observed in K+ medium, though the nucleotides were still effective in Na+ medium. From the experimental data obtained, the ion-dependent conformations of the active site of myosin ATPase were discussed.
The structure of a tryptic peptide containing one specific sulfhydryl group (Sa), which is responsible for the activation of Mg2+-ATPase of myosin B and is present in the light meromyosin region of the myosin molecule, was studied. The amino acid sequence was deduced to be Thr (or Ser)-Asn-Ala-Ala-Cys-Ala-Leu-Asp-Lys-Lys. In addition, a space-filling model around Sa was built up by comparing Sa-peptide with the amino acid sequence around Cys 190 of α-tropomyosin, and the high reactivity of Sa with N-ethylmaleimide is considered based on this model.
Defensin has been known as a major antimicrobial component of neutrophil granules. Recently, we have purified a novel cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophilis. In this study, we have compared the biological activity and gene expression between CAP11 and defensin. When stimulated, CAP11 was extracellularly released from neutrophils, accompanied by the release of a specific granule component (lysozyme), whereas defensin was released, accompanied by the release of an azurophil granule component (β-glucoronidase) . Defensin modulated neutrophil functions such as adhesion, phagocytosis and superoxide anion generation. However, CAP11 did not affected the neutrophil functions. Both CAP11 and defensin possessed histamine-releasing activity for mast cells, but CAP11 was 10-fold less potent than defensins. CAP11 exhibited 8-fold more antibacterial activity against Escherichia coli than defensin, and the activity was retained even in the presence of physiological concentration of NaCl, although the activity of defensin was completely lost in the presence of NaCl. Sequence analysis of CAP11 cDNA showed that pre-prosequence of CAP11 was similar to the sequences of cathelicidin family polypeptides. Furthemore, in situ hybridization study revealed that CAP11 mRNA was highly expressed in metamyelocytes among bone marrow cells. In contrast, defensin mRNA was expressed in promyelocytes and myelocytes. Together these observations indicate that CAP11, a member of cathelicidin family, which is synthesized during the late stage of neutrophil maturation and stored possibly in the specific granules, is released from stimulated neutrophils and functions as an antibacterial molecule in the extracellular milieu, whereas defensin released from the azurophil granules likely participate in the modulation of neutrophil functions and mast cell histamine release.
Journal Article The Amino Acid Sequence at the Active Site of Myosin A Adenosine Triphosphatase Activated by Ca++ Get access TATSUHISA YAMASHITA, TATSUHISA YAMASHITA From the Department of Biochemistry, School of Medicine, Juntendo UniversityTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar YAEKO SOMA, YAEKO SOMA From the Department of Biochemistry, School of Medicine, Juntendo UniversityTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar SHIZUKO KOBAYASHI, SHIZUKO KOBAYASHI From the Department of Biochemistry, School of Medicine, Juntendo UniversityTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar TAKAMITSU SEKINE TAKAMITSU SEKINE From the Department of Biochemistry, School of Medicine, Juntendo UniversityTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 57, Issue 3, March 1965, Pages 460–461, https://doi.org/10.1093/oxfordjournals.jbchem.a128103 Published: 01 March 1965
Adult patients with type 2 diabetes were classified using the Temperament and Character Inventory (TCI). This classification was used to propose effective therapeutic approaches based on subtypes of psychological characteristics.The TCI and various psychological tests were administered to 89 patients (54 men and 35 women). Cluster analysis was performed using three temperament factors of the TCI as variables for subclassification: novelty seeking (NS), harm avoidance (HA) and reward dependence (RD).The patients were divided into two clusters based on the TCI results. Cluster 1 had a low NS/high HA/low RD pattern, which indicated resistance to change and lack of cooperativeness, and results of other tests indicated that patients in cluster 1 were obsessional and had weak intention and personality disorder symptoms. In contrast, cluster 2 had a high NS/low HA/high RD pattern, indicating a socially active person who easily becomes dependent on others, and results of other tests indicated that these patients were histrionic and less anxious. In cluster 1 there was a significant negative correlation between hemoglobin A1c (HbA1c) and SD-1 (TCI self-directedness) 'responsibility' and a significant positive correlation between HbA1c and State anxiety of State-Trait Anxiety Inventory. In cluster 2 there was a significant positive correlation between HbA1c and HA-2 'fear of uncertainty'.Patients with type 2 diabetes were classified using the TCI into two subgroups. These two groups differed in psychological characteristics and had a different pattern in correlation with glycemic control.
Cognitive behavioral therapy (CBT) is one of the recommended therapeutic approaches for anorexia nervosa (AN). CBT for AN often needs to be designed for individual cases,1 and previously we have developed a behavioral therapy program combined with liquid nutrition for AN: Kyoto Prefectural University of Medicine Behavior Therapy (KPT).2 Here, we describe a newly designed video-assisted CBT for a patient with AN refractory to KPT. The patient was a 16-year-old girl who had been diagnosed with restricting-type AN at age 14, based on DSM-IV criteria, and was refractory to KPT. To acquire credits for graduation, she attended school during the day and returned to hospital at night. The patient repeatedly stated that she could not communicate with her classmates easily because she was too fat. Psychological interventions were started to address this belief, in addition to KPT. However, she continued to maintain her belief. We focused on her maladaptive eating behavior related to AN, rather than on her distorted cognition. At meals, she took too much time to eat and tended to mash her food into paste using chopsticks and a spoon. The therapist suggested to her that this abnormal eating behavior might be strange to those around her, and that it tended to complicate her communication with others. The patient resisted the verbal advice regarding her maladaptive eating behavior. We then tried a video-assisted visual tool to bring awareness of her abnormal eating behavior. We proposed following a structured CBT and the patient accepted this treatment. Once a week, we filmed her eating dinner at hospital, and on another day the patient and her therapist watched the video together and talked about her approach to eating. In the first three sessions, the patient responded, ‘My way of eating is normal.’ However, at the fourth session, she laughed at a scene in which she spilled mashed food onto herself from her mouth, and said to the therapist that she always crushed sandwiches into small parts. She then asked, ‘Do my friends find me strange in any way?’ and the therapist replied, ‘I think so’. In that session, the patient agreed to the challenge to eat food in normally sized pieces and to spend less time at meals. By the seventh session, she commented, ‘My way of eating might have caused a curious impression and alienated my classmates from me; since I have now changed my eating habits, I can start to talk to my classmates in a more friendly way.’ The patient gradually developed a more normal eating manner and her bodyweight increased. After 10 sessions over 6 months she had completed her inpatient treatment. In the present case we kept records of abnormal eating behavior using a video-assisted visual tool. Being asked to watch a scene of herself eating a meal contributed to objective reorganization of the abnormality of the patient’s eating behavior and acceptance of her illness. Because patients with AN often show cognitive impairment and disturbed self-awareness in association with low bodyweight, a video-assisted approach might be more effective than the use of verbal psychotherapy only.
