Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives.

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

[Risk factors for falls and survival after falling in elderly people in a community].

OBJECTIVES: The purpose of this study was to assess the risk factors associated with falls and to examine the effects of falls on survival of elderly people in a community. METHODS: A questionnaire survey was conducted in 16,462 urban elderly dwellers aged 65 years or more in City A in September 2001. A follow-up survey was carried out in September 2004. We analyzed the data of 8,285 subjects who answered both questionnaires and had not relocated by August 2007. Baseline assessments of health and functioning were carried out in 2001. Falls experienced during the 1-year period before September 2004 were recorded, and the deaths were recorded until August 2007. Statistical analysis was performed using a logistic regression model and Cox's proportional hazards analysis. RESULTS: A total of 6,420 subjects (3,127 men and 3,293 women) who had provided complete answers about their falls were included in the analyses. Of these, 27.8% of women and 16.4% of men had experienced falls, while 6.2% of women and 2.1% of men had experienced falls that caused fractures. We found that the likelihood of fall, with or without fracture development, was greater in women than in men (P < 0.001). The rate of falls tended to increase with age in both women and men. Risk factors associated with falls, in addition to age and gender, were pain (odds ratio [OR], 1.75), lack of instrumental activities of daily living (IADL; OR, 1.45), poor self-rated health status (OR, 1.42), and presence of disease (OR, 1.35). Risk factors associated with falls that caused fracture were pain (OR, 1.85) and lack of IADL (OR, 1.61). Cox's proportional hazards analysis showed a significant increase in mortality in both men and women who had experienced falls than in those who had not (hazard ratio [HR], 1.94, 1.43). CONCLUSION: Aging, pain and disease, lack of IADL, and poor self-rated health status were all significant risk factors for falls in elderly people, and a fall was related to subsequent mortality.

[Chronological evaluation of physical, psychological and social health of urban elderly dwellers over 6 years and assessment of causal inter-relationships].

OBJECTIVES: The purpose of this study is to make a chronological evaluation over 6 years of physical, psychological and social health of urban elderly dwellers. METHODS: A questionnaire survey was conducted with all urban elderly dwellers of 65 years old or more in A City in September, 2001. Answers were obtained from 13,195 people (response rate of 80.2%) in the first survey. Then 3 year and 6 year follow-up surveys of 2,375 members were performed in September 2004 and 2007. Causal relationships were analyzed using a Structural Equation Model based on the Cross-Lagged Effects Variation Model. RESULTS: According to this research, a chronological six year trend in ADL (Activities of Daily Living) was found for "physical factor" (" " means latent variable) as an observed variable, with a shifted from 91.0% to 82.9%. A trend for self-rated health with healthy as an observed variable of "psychological factor" was similarly apparent, shifting from 85.4% to 77.0%. "Social factor" conducted on the follow-up survey in 2007 was significantly affected by the "psychological factor" investigated in 2001 and "physical factor" in the follow-up survey in 2004, indirectly based on the Cross-Lagged Effects Variation Model. "Social factor" totals of 25% for men and 19% for women were explained by this model with high validity levels (NFI = 0.935, IFI = 0.950, RMSEA = 0.036). CONCLUSION: It was suggested that social health was affected by psychological health directly and physical health indirectly during six years follow-up of urban elderly dwellers. Future research is needed to encompass other generations and also to improve the external validity of the results.

Role of the N- and C-terminal domains in binding of apolipoprotein E isoforms to heparan sulfate and dermatan sulfate: a surface plasmon resonance study.

The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.

Angiotensin converting enzyme inhibitors attenuated the expression of G-protein coupled receptor kinases in heart failure patients.

BACKGROUND: There are few biological markers, which strictly show the severity of congestive heart failure (CHF). METHODS AND RESULTS: Lymphocyte G-protein coupled receptor kinase (GRK) mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction in 15 CHF patients: 5 patients classified as New York Heart Association class-II treated with angiotensin converting enzyme inhibitor (ACEI) (IIA), 5 patients in class-II without ACEI (IIC), and 5 patients in class-III treated with ACEI (IIIA). GRK mRNA level in IIIA was significantly higher than those in IIA (p<0.05). GRK mRNA level in IIA were significantly lower than those in IIC (p<0.05). CONCLUSIONS: The expression level of lymphocyte GRK might show the severity of CHF, and ACEI treatment could reduce the level of GRK in CHF patients.

Chronic beta-adrenergic receptor stimulation enhances the expression of G-Protein coupled receptor kinases, GRK2 and GRK5, in both the heart and peripheral lymphocytes.

BACKGROUND: Enhanced expression of G protein-coupled receptor kinase (GRK) has been reported in failing hearts and in the present study the stability of enhanced GRK mRNA expression, and the correlation between the expression level of GRK mRNA in peripheral lymphocytes and in the heart were both evaluated. METHODS AND RESULTS: Isoproterenol was injected into rats for 2 weeks, and then GRK5 mRNA was assessed by quantitative reverse transcriptase-palymerase chain reaction. An enhanced expression of cardiac GRK5 mRNA was observed even after 4 weeks of recovery. The isoproterenol-induced increased expression of GRK2 and GRK5 mRNA was equally observed in the heart and lymphocytes, and there was a close correlation between the heart and lymphocytes in the level of expression of each GRK mRNA. CONCLUSIONS: The GRK mRNA level is maintained at a high level for a long period without continuous beta-adrenergic receptor stimulation. The level in circulating lymphocytes could be used as a surrogate marker to estimate the level of cardiac GRK expression and, presumably, the beta-adrenergic receptor function of cardiomyocytes.

Alpha-melanocyte-stimulating hormone stimulates prolactin secretion through melanocortin-3 receptors expressed in mammotropes in the mouse pituitary.

The intermediate lobe of rodent pituitaries is involved in the regulation of prolactin (PRL) secretion from the anterior lobe. In a previous study, we demonstrated the stimulatory effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on PRL release and the expression of melanocortin-3 receptors (MC3-Rs) in cultured mouse pituitary cells. The aim of the present study was to clarify whether alpha-MSH directly stimulates PRL release through the MC3-Rs by determining the cell type of MC3-R-expressing cells in the mouse pituitary anterior lobe. Northern blot analysis revealed a 2.7-kb transcript for MC3-R mRNA in the anterior and neurointermediate lobes of pituitary glands of adult male and female mice. Dual cellular localization of MC3-R mRNA and PRL or growth hormone (GH) in the mouse pituitary glands was performed by in situ hybridization analysis of MC3-R mRNA followed by immunocytochemical detection of PRL or GH. MC3-R mRNA was detected in most mammotropes and some somatotropes. alpha-MSH increased PRL release and stimulated DNA replication in mammotropes, and these effects were blocked by SHU9119, an antagonist of MC3-R and MC4-R. These results indicate that alpha-MSH stimulates PRL release and proliferation of mammotropes through MC3-Rs, and suggest that alpha-MSH from intermediate lobes can regulate mammotrope functions in the mouse pituitary.