Multiple vitamin deficiencies additively increase the risk of incident fractures in Japanese postmenopausal women.

The associations of multiple vitamin deficiencies on incident fractures were uncertain, the relationships between serum vitamin markers and incident bone fractures were investigated in Japanese postmenopausal women. The number of deficiencies was additively associated with incident fracture after adjustment for possible confounding factors including the treatment of osteoporosis. INTRODUCTION: To evaluate the associations of multiple vitamin deficiencies on incident fractures, the relationships between serum vitamin markers and incident bone fractures were investigated in Japanese postmenopausal women. METHODS: This analysis used a subset of the ongoing cohort maintained by a primary care institution. Inclusion criteria of the present study were postmenopausal women aged ≥ 50 years, without vitamin supplementation and secondary osteoporosis. Baseline serum concentrations of 25-hydroxyvitamin D (25(OH)D), undercarboxylated osteocalcin (ucOC), and homocysteine (Hcy) were measured to assess vitamin D, vitamin K, and vitamin B, respectively. Since 25(OH) D positively relates to vitamin D, ucOC and Hcy negatively relate to vitamin K and vitamin B nutrients, respectively, the subjects with lower (25(OH)D) or higher (ucOC or Hcy) values than each median value was defined as subjects with the corresponding vitamin deficiency. Subjects were divided into four groups according to the number of deficiency: no deficiency, single deficiency, double deficiencies, and triple deficiencies. Relationships between the vitamin deficiencies and incident fractures were evaluated by Cox regression analysis. RESULTS: A total of 889 subjects were included in this analysis; their mean and SD age was 68.3 ± 9.5 years, and the follow-up period was 6.3 ± 5.1 years. The numbers of subjects in the four groups were 139 (15.6%), 304 (34.2%), 316 (35.5%), and 130 (14.6%) for the groups with no, single, double, and triple deficiencies, respectively. Incident fractures were observed in 264 subjects (29.7%) during the observation period. The number of deficiencies was significantly associated with incident fracture (hazard ratio 1.25, 95% confidence interval 1.04-1.50, P = 0.018) after adjustment for possible confounding factors including the treatment of osteoporosis. CONCLUSION: Accumulation of vitamin deficiencies was related to incident fractures.

Increased Sylvian fissure angle as early sonographic sign of malformation of cortical development.

OBJECTIVE: To evaluate Sylvian fissure development by assessing Sylvian fissure angles in fetuses with malformation of cortical development (MCD). METHODS: This was a retrospective study of 22 fetuses with MCD. Cases with a stored three-dimensional (3D) brain volume acquired at 18 + 0 to 30 + 6 weeks of gestation at an ultrasound-based research clinic between January 2010 and December 2017 were identified through a database. Of the 22 fetuses, seven had an extracranial abnormality, such as cardiac, renal, gastrointestinal and/or digital anomalies, and five had a minor abnormality such as micrognathia, low-set ears and/or single umbilical artery. To confirm the final clinical diagnosis of brain abnormality, postmortem histological findings or prenatal or postnatal magnetic resonance images were used. For measurement of Sylvian fissure angle, an anterior coronal plane of the fetal brain on transvaginal 3D volume multiplanar imaging was visualized as a single image from the three orthogonal views. The right and left Sylvian fissure angles were measured between a horizontal reference line (0°) and a line drawn along the upper side of the respective Sylvian fissure. The Sylvian fissure angle on both sides was plotted on the graphs of the reference ranges for gestational age in weeks. RESULTS: In 21 (95.5%; 95% CI, 86.8-100.0%) of 22 fetuses with MCD, the Sylvian fissure angle on one or both sides was larger than the 90th percentile of the normal reference. There was one case with apparent focal MCD in the parietal lobe, but the Sylvian fissure angles were normal. A case with apparent unilateral cortical dysplasia and one with apparent unilateral schizencephaly had conspicuous discrepancies between the left and right Sylvian fissure angles. Abnormal genetic test results were obtained in six cases, including four cases with a mutation in a single gene. CONCLUSIONS: This study has shown that the Sylvian fissures, as defined by the Sylvian fissure angle, have delayed development in most MCD cases prior to the diagnosis of the condition. The Sylvian fissure angle may potentially be a strong indicator for the subsequent development of cortical malformation, before the time point at which the gyri and sulci become obvious on the fetal brain surface. Further research is required to validate these findings. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Stimulation of intestinal calcium absorption by orally administrated vitamin D3 compounds: a prospective open-label randomized trial in osteoporosis.

Intestinal fractional calcium absorption (FCA) was assessed before and after vitamin D3 treatment. Serum 1,25(OH)2D concentration was significantly increased by plain vitamin D3 and reduced by eldecalcitol. The 1α hydroxyl calcidiol and eldecalcitol treatments increased FCA, which may be induced through direct stimulation of vitamin D receptors in the intestine. INTRODUCTION: To assess the effects of vitamin D3 compounds on intestinal FCA and calcium-regulating hormones in post-menopausal osteoporosis, a randomized open-label prospective study was conducted. METHODS: Forty eligible patients were allocated randomly into four groups: eldecalcitol (ELD; 0.75 µg/day), 1α hydroxyl calcidiol (ALF; 1 µg/day), plain vitamin D3 (800 IU/day), and control. Before and after the 4-week treatment, intestinal FCA was estimated by using a double isotope method, and serum concentrations of calcium-regulating hormones and a bone turnover marker were measured. RESULTS: The baseline FCA value of the participants was 21.5 ± 7.9% (mean ± SD) and was significantly correlated with serum 1,25(OH)2D (calcitriol) concentration. After the treatment, the FCA significantly increased by 59.5% (95% CI, 41.6 to 77.4%) in the ELD group and by 45.9% (27.9 to 63.8%) in the ALF group, whereas no significant change in the plain vitamin D3 group was found. Unlike the baseline FCA, post-treatment FCA exhibited no significant correlation with serum calcitriol concentration. Parathyroid hormone levels were suppressed by ALF and plain vitamin D3 but were sustained in the ELD and control groups. Serum calcitriol tended to be suppressed by ELD, whereas plain vitamin D3 treatment increased both serum 25(OH)D and calcitriol concentrations. CONCLUSION: These findings suggest that oral administration of vitamin D3 analogues (ALF and ELD) stimulates FCA but plain vitamin D3 does not. Those effects of vitamin D3 compounds on FCA were independent of serum calcitriol concentration, suggesting that ALF and ELD may directly stimulate intestinal vitamin D receptors.

Correction to: Stimulation of intestinal calcium absorption by orally administrated vitamin D3 compounds: a prospective open-label randomized trial in osteoporosis.

There were two errors in this article. 1. In the section "Ethical considerations", the registration number of the study was incorrectly given as UMIN000024492. The correct number is UMIN0000 20267. 2. The Acknowledgments paragraph was incomplete.

Relationships between bone mass and dietary/lifestyle habits in Japanese women at 3-4 months postpartum.

OBJECTIVES: The relationships between calcaneal bone mass and dietary/lifestyle habits in women at 3-4 months postpartum were examined in the context of osteoporosis prevention. STUDY DESIGN: Cross-sectional survey. METHODS: We measured bone mass using calcaneal ultrasound in mothers who brought their 3- to 4-month-old babies to healthcare centers in Japan for health examination and administered a self-report questionnaire on physical characteristics and dietary/lifestyle habits to those who agreed to participate in the survey. Valid data were available for 1220 women (valid response rate, 97.5%). RESULTS: Based on their stiffness score, a measure of bone mass, 70.9% (n = 865) of the participants were classified as 'no apparent abnormality (stiffness score ≥78.8)' (low-risk group), 18.2% (n = 222) as 'guidance required (≥70.1-<78.8)' (intermediate-risk group), and 10.9% (n = 133) as 'complete examination required (<70.1)' (high-risk group), according to the criteria for osteoporosis screening test results. The percentage of individuals with a history of fracture was higher in the guidance required/complete examination required than in the no apparent abnormality group (P = 0.016). The analysis of relationships between the consumption frequency of certain foods, such as calcium-rich foodstuffs, and bone mass found that women who reported lower frequencies of milk and dark-colored (beta-carotene rich) vegetables for breakfast consumption had a significantly lower bone mass than those who consumed these foods more often. Furthermore, the guidance required/complete examination required group had a significantly lower calcium intake than the no apparent abnormality group (P = 0.022). CONCLUSIONS: These results indicate the need to provide postpartum women with dietary education programs to promote healthy eating habits, such as increased consumption of calcium-rich foods, and prevent osteoporosis.

Association between Dairy Product intake and Risk of Osteoporotic Fractures in Postmenopausal Japanese Women: Secondary Analysis of 15-Year Follow-Up data from the Japanese Population-Based Osteoporosis (JPOS) Cohort Study.

OBJECTIVES: Few prospective cohort studies have evaluated the relationship between dairy product intake frequency and risk of osteoporotic fractures in Asians. This study aimed to investigate the association between habitual dairy product intake and risk of osteoporotic fractures. DESIGN: Secondary analysis of prospective cohort study. SETTING: Five municipalities of Japan. PARTICIPANTS: This study included 1,429 postmenopausal Japanese women (age ≥45 years at baseline). MEASUREMENTS: Baseline milk-intake frequency was obtained using nurse-administered questionnaires. Intakes of yogurt and cheese, and estimated calcium intake, were assessed using a validated food frequency questionnaire. Osteoporotic fracture was defined as a clinical fracture diagnosed using radiography. Hazard ratios (HRs) with 95% confidence intervals (CIs) were estimated using Cox proportional hazards models. RESULTS: Over a median follow-up period of 15.1 years (interquartile range [IQR], 10.1-15.4 years; total, 18,118 person-years), 172 women sustained at least one osteoporotic fracture. The proportions of participants with milk intakes <1, 1, and ≥2 cups/d were 34.4%, 48.0%, and 17.6%, respectively. After adjustment for age, frequency of yogurt intake, frequency of cheese intake, body mass index, history of osteoporotic fractures, and frequency of natto intake, the HRs compared with that for milk intake <1 cup/d were 0.71 (95% CI: 0.51-0.98) and 0.57 (95% CI: 0.35-0.92) for 1 cup/d and ≥2 cups/d, respectively. After adjustment for bone mineral density, HR significance for milk intakes ≥2 cups/d remained significant. Yogurt and cheese intakes were not related to the risk of osteoporotic fractures. CONCLUSION: High habitual milk intake, but not a habitual yogurt or cheese intake is associated with a decreased risk of osteoporotic fractures, independent of bone mineral density, in postmenopausal Japanese women.

Misreporting of dietary energy, protein, potassium and sodium in relation to body mass index in young Japanese women.

OBJECTIVE: Although under-reporting of dietary intake is more common in persons with a high body mass index (BMI), it is not well known whether or not misreporting is selective for different foods (and hence energy and nutrients), particularly in non-Western populations. We examined misreporting of dietary intake against biomarkers and its relation with BMI in young Japanese women. DESIGN: Cross-sectional study. SUBJECTS: A total of 353 female Japanese dietetic students aged 18-22 years (mean BMI: 21.4 kg/m(2), mean fat intake: 29.8% of energy). METHODS: Misreporting of dietary energy, protein, potassium and sodium (assessed by a self-administered diet history questionnaire) was examined against respective biomarkers (estimated energy expenditure and 24-h urinary excretion). Reporting accuracy was calculated as the ratio of reported intake to that estimated from corresponding biomarkers (complete accuracy: 1.00). RESULTS: Mean reporting accuracy of absolute intake (amount per day) varied considerably (0.86-1.14). Reporting accuracy of absolute intake decreased with increasing BMI (P for trend <0.001). However, no association was observed between reporting accuracy of energy-adjusted values and BMI (P for trend >0.15), indicating that BMI-dependent misreporting was canceled by energy adjustment. This was owing to positive correlation between the reporting accuracy of energy intake and that of absolute intake of the three nutrients (Pearson correlation coefficient: 0.49-0.67, P<0.0001). CONCLUSIONS: Although differential misreporting of absolute intake was associated with BMI, differential misreporting of energy-adjusted value was not. These findings support the use of energy-adjusted values in the investigation of diet-disease relationships among lean populations with a low-fat intake.

Nutritional status in Japanese renal transplant recipients with long-term graft survival.

BACKGROUND: Nutritional status affects clinical outcomes in patients with chronic renal failure. Glucose intolerance, dyslipidemia, obesity, hypertension, and a calcium-phosphorus-vitamin D imbalance are the major nutritional and metabolic problems that occur in posttransplant patients. In this study, we assessed the daily intake in long-term renal transplant recipients to determine whether they have sufficient nutrients based on the Japanese nutrition recommendations (recommended dietary allowances [RDA] in Japan 2010). SUBJECTS AND METHODS: Thirty-one renal allograft recipients followed for >10 years (median, 16.3) were recruited. The median serum creatinine level was 1.2 g/dL (95% CI, 0.6-3.4). We estimated the intake of nutrients, including protein and salt, using a simple food frequency questionnaire. RESULTS: The median body mass index was 20.1 kg/m(2). The median total energy intake was 1566 kcal/d (95% CI, 892-2556). The daily intake of protein and salt was 65.1 and 9.1 g/d, respectively. The calcium, iron, vitamin D, and vitamin K intakes were 423 mg, 7.0 mg/d, 9.7 µg/d, and 197 µg/d, respectively. Patients with dyslipidemia displayed greater amounts of lipid and calcium than those with normal lipid levels. DISCUSSION: Our findings suggest that long-term renal transplant recipients in Japan seem to restrict caloric intake, while maintaining appropriate intake of protein, lipids, carbohydrates, and vitamins A, D, and K. However, daily calcium and iron intake were insufficient; salt intake was greater than the recommended dietary allowances in all subjects. In patients with dyslipidemia, calcium intake was lower than those in patients without dyslipidemia, although their intake of lipids was also lower than those without dyslipidemia. CONCLUSION: Nutritional guidance beginning during the early posttransplant phase helps to foster a healthy body mass index and nutritional balances for long-term renal transplant recipients. However, greater salt restriction was needed, and additional nutritional guidance aiming to prevent osteoporosis seems to be considered.

[Intestinal calcium absorption].

Calcium which is required by our body must be absorbed from the diet through the intestine. Food and body conditions influence on calcium absorption from the intestine. Even when the same diet is eaten, the calcium absorption rate is different in each subject. It is assumed that calcium absorption is affected by many factors including acidity in the stomach, transit time of the intestine, intestinal flora, vitamin D, PTH and the other hormones.

Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women.

UNLABELLED: Effect of milk basic protein on bone metabolism in healthy young women. INTRODUCTION: Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone mineral density and bone metabolism of healthy young women. METHODS: Thirty-five healthy young women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indexes of bone metabolism were measured at 0, 3 and 6 months. All subjects completed the study in accordance with the protocol. RESULTS: The mean rate of gain of lumbar BMD in the MBP group (1.57%) was significantly higher than in the placebo group (0.13%, P=0.042). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased, and serum osteocalcin was significantly increased in the MBP group at 6 months. CONCLUSION: These results suggested that MBP supplementation was effective in increasing BMD in young women and that this increase in BMD may be primarily mediated through the promotion of bone formation and inhibition of bone resorption by MBP supplementation.

A comparison of body perceptions in relation to measured body composition in young Japanese males and females.

The body composition of 139 Japanese females and 84 Japanese males (aged 18-30 years) was measured using anthropometry to assess gender differences in body perceptions in relation to their measured values. Participants were asked to rate perceptions of their own "heaviness" and "fatness" and these were compared to their BMI and percent body fat (%BF). Japanese females showed a significantly greater desire to lose body weight (-4.20+/-0.6 kg) compared to males (0.27+/-1.4 kg). Females also showed poor understanding of their "heaviness" and "fatness" in relation to actual body composition compared to males. The results confirmed distinct gender differences in body perception in relation to actual body composition and attitudes to weight management. Further promotion of "healthy" body image is recommended for the Japanese population.

Effect of excess phenylalanine diet during pregnancy on fetal brain growth in rats.

The effects of 10 and 20% casein diets containing 7% phenylalanine (Phe) during pregnancy on fetal brain growth were examined in rats. Control pregnant rats were fed the casein diets ad libitum or in restricted amounts. Total food intakes during 21-day period in the Phe excess groups decreased to about 50% of those of the liberally fed control groups. In control rats given 10 and 20% casein diets, fetal brain weights (Y, mg) were significantly and hyperbolically correlated to total food intakes (g/21 days), conforming to the following equations: Y = -10283/X + 130.5 (n: 13, r = 0.89, p < 0.001) and Y = -4396/X + 130.4 (n: 15, r = 0.68, p < 0.005), respectively. Similar plots for rats fed 10 and 20% casein diets with Phe fell below these lines, meaning that fetal brain growth was impaired by both the decreased food intake due to excess Phe (nonspecific effect) and the toxicity of excess Phe per se (specific effect). Total amounts of RNA and protein and protein/DNA ratio decreased in the fetal brain from excess Phe dams, suggesting that protein synthesis of brain cells was disturbed. This may be partly due to the disruption of normal patterns of free amino acids observed in the fetal brains. Reduction of total DNA was also seen in fetal brain from excess Phe groups, meaning impaired proliferation. From above findings we concluded that proliferation and hypertrophy were impaired in fetal brain from excess Phe dams.

Effect of dietary proteins on zinc bioavailability in pregnant rats.

In order to clarify the effects of dietary proteins on zinc bioavailability during pregnancy, two experiments were carried out. In Experiment 1, changes in zinc retention due to pregnancy (difference in retention between pregnant and nonpregnant animals) during early-mid and late pregnancy were examined in rats fed 10 and 20% egg white diets. Total amounts of retained zinc due to pregnancy were about 1000 micrograms or slightly more, equal to the zinc content in the products of conception at term. However, extra zinc retention during late pregnancy ranged between only 20 to 40% of overall retention, suggesting that almost all zinc retained during early-mid pregnancy moved from the mothers to the fetuses near term. Zinc retention in early-mid and late periods of pregnancy was higher in pregnant than nonpregnant rats, due mainly to increases in intake and bioavailability. In Experiment 2, to examine the effects of quality and quantity of dietary proteins, pregnant rats were fed either 10 or 20% egg white (EW), whole egg (WE), casein (C) and soy protein isolate with or without methionine (SM and S, respectively) diets. Total zinc retention during pregnancy was affected by both zinc and nitrogen intakes, though the former effect was greater than the latter. Because rats fed the EW diets retained dietary zinc efficiently, a relationship between zinc retention (Y, microgram/100 g BW/21 d.) and zinc intake (X, microgram/100 g BW/21 d.) was also examined in the non-EW protein groups, resulting in the following regression equation: Y = 0.471X-1790 (n: 51, r = 0.81, p < 0.001). Dietary protein quality affected the food intake resulting in different zinc intake and retention during pregnancy. Zinc from EW diets was more available than from the other four protein diets, because similar plots for rats fed the 10 and 20% EW diets fell above this line. Reasons for efficient bioavailability of zinc in EW were discussed in connection with the forms of zinc in diets and the pancreatic or intestinal responses to ingested EW.

Nutritional improvements of soy protein isolate by different levels of methionine supplementation in pregnant rats.

The effects of various levels (0, 0.3, 0.4, 0.5, 0.7, 1.0 and 1.5%) of methionine supplementation to a 10% soy protein isolate (SPI) diet were investigated in pregnant and nonpregnant rats in terms of body weight gain, food efficiency, net protein utilization (NPU), growth of the conceptuses and free methionine concentration in the dam's plasma. Supplementation of 0.3 to 0.7% methionine resulted in increases in the weights of the body, fetus and placenta and improvements of food efficiency and NPU in both groups of rats. The extents of increases or improvements of these parameters were more in pregnant rats than in nonpregnant rats, though they were similar in each group independent of the dietary methionine level. No beneficial effects on body weight gain, food efficiency or reproductive performance were observed in rats fed diets with 1.0 or 1.5% methionine. The concentrations of free methionine in the plasma were increased by methionine supplementation to the diet and were consistently higher in nonpregnant rats than in pregnant rats at each level of methionine supplementation. From these results, we proposed that a level of 0.5% methionine (intermediate between 0.3 to 0.7%) is optimal for supplementing 10% SPI diet for rats. The appropriateness of this level is discussed in relation with the methionine equipment of growing rats.

Regulation of nucleoside cyclic 3':5'-monophosphate phosphodiesterase activity from rat brain by a modulator and Ca2+.

Gel filtration of the 40,000 rpm supernatant fraction of a homogenate of rat cerebral cortex on a Sepharose 6B column yielded two fractions: fraction II with the "Ca(2+) plus Mg(2+)-dependent" phosphodiesterase activity and fraction III containing its modulator. The activity of fraction II was stimulated by micromolar concentrations of Ca(2+) and the modulator when present together; the modulator stimulated the activity of fraction II only when the Ca(2+) concentration was above a threshold value (about 2 muM with 0.4-1 muM substrate), and the stimulatory effect of Ca(2+) was dependent upon the presence of the modulator. A possibility is discussed that the modulator may reversibly bind to the enzyme, which by itself is inactive, to form an active enzyme-modulator complex and that Ca(2+) stimulates the activity of phosphodiesterase by shifting the equilibrium between these three species towards the formation of the active enzyme-modulator complex. Although fraction II hydrolyzed both cyclic AMP and cyclic GMP, hydrolysis of the latter was more significantly influenced by Ca(2+) and the modulator than that of the former, and the "Ca(2+) plus Mg(2+)-dependent" phosphodiesterase is likely to be a cyclic GMP enzyme. This conclusion is based on the following evidence: (a) Ca(2+) stimulated hydrolysis of cyclic GMP by fraction II more than that of cyclic AMP. (b) In the presence of Ca(2+) and the modulator, fraction II hydrolyzed cyclic GMP about 8 times faster than cyclic AMP when incubated with 0.4 muM substrate. (c) Half-maximal stimulation of hydrolysis of cyclic GMP was attained at a lower concentration of Ca(2+) (4 muM) than that of cAMP (8 muM). (d) Increase in the concentration of Ca(2+) from 0.06 muM to 12 muM in the presence of the modulator caused a decrease in the K(m) value of cyclic GMP hydrolysis by fraction II from 20 muM to 2 muM accompanied by 4-fold increase in the V(max) value. Under similar conditions, there was only a slight decrease in the K(m) value of cylic AMP hydrolysis (90 muM --> 50 muM), although the V(max) value increased 7-fold. The anomalous shape of the kinetic plot of cyclic GMP hydrolysis became linear when the Ca(2+) concentration was increased in the presence of the modulator. The modulator seems to be a protein, but it is heat stable. It is probably identical to the protein activator of phosphodiesterase first described by Cheung.

Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator.

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.

Ca-2+/Mg-2+-dependent cyclic nucleotide phosphodiesterase and its activator protein.

(1) Ca-2+/Mg-2+-dependent cyclic nucleotide phosphodiesterase was found in the supernatant fluids of a variety of tissues, including cerebral cortex, cerebellum, kidney, liver, and heart. (2) this enzyme required Ca-2+, Mg-2+, and an activator protein (PAF) for the activity. In the presence of these ingredients the enzyme hydrolyzed cyclic GMP preferentially when incubated with a low concentration (0.4 muM) of substrate. (3) The enzyme devoid of PAF was eluted in fraction II with a molecular weight of approximately 150,000 after Sephadex G-200 gel filtration of the supernatant fluids using medium containing EGTA. PAF thus separated from the enzyme was eluted in a fraction corresponding to a molecular weight of approximately 28,000 by gel filtration. Stimulation of the activity of fraction II by Ca-2+ was completely dependent on the addition of PAF. (4) Formation of an active enzyme-PAF complex with an estimated molecular weight of 200,000 was demonstrated by gel filtration of a mixture of the enzyme and PAF in medium containing Ca-2+. It is likely that the activity of the Ca-2+/Mg-2+ in a concentration range of approximately 1 to 10 muM, as shown in the following equation: [Enzyme] inactive + PAF + Ca-2+ in equilibrium [enzyme - PAF - Ca-2+] active. More than one PAF protein may bind to one molecule of enzyme to form an active complex. Equilibrium of the above equation is probably determined mainly by the intracellular concentration of Ca-2+ in vivo. (5) The enzyme-PAF complex was more labile after heat treatment than the free form of enzyme. (6) PAF was isolated from all tissues listed in (1). The levels of PAF and of fraction II did not correspond exactly.

Identification of an activator protein for myosin light chain kinase as the Ca2+-dependent modulator protein.

Myosin light chain kinase which phosphorylates g2 light chain of skeletal muscle myosin requires an activator for the activity (Yazawa, M., and Yagi, K (1977) J. Biochem. (Tokyo) 82, 287-289). This activator has now been identified as the modulator protein known to be a Ca2+-dependent regulator for phosphodiesterase, adenylate cyclase, and ATPases. The identification is based on the quantitative cross-reactivity of muscle activator protein and brain modulator protein in activating myosin light chain kinase and brain phosphodiesterase and identical properties of both proteins in regard to sensitivities to Ca2+, UV absorption spectra, UV absorption difference spectra with or without Ca2+, and mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of modulator protein, the activity of myosin light chain kinase was reversibly controlled by the physiological concentration of Ca2+. We suggest that two Ca2+-receptive proteins, i.e. modulator protein and troponin-C, may play roles in the contraction-relaxation cycle of skeletal muscle.