Mitochondrial macrohaplogroup associated with muscle power in healthy adults.

The present study was undertaken to examine the effect of mitochondrial haplogroups on aerobic and anaerobic performance phenotypes such as maximum oxygen consumption, muscle power, and muscle mass. We recruited 474 healthy Japanese individuals and measured their physical performance phenotypes such as peak oxygen uptake, muscle power, and muscle mass. The genotypes for 186 polymorphisms in the mitochondrial DNA were determined, and the haplotypes were classified into 2 macrohaplogroups (i. e., N and M) and 12 haplogroups (i. e., F, B, A, N9a, N9b, M7a, M7b, G1, G2, D4a, D4b, and D5). When we compared the 2 major Japanese macrohaplogroups, leg extension power (P=0.0395), leg extension power based on body weight (P=0.0343), and vertical jump performance (P=0.0485) were significantly higher in subjects with mitochondrial macrohaplogroup N than in those with macrohaplogroup M. However, peak oxygen uptake was similar between the 2 groups. When we analyzed the 12 haplogroups, all of the measured parameters were similar among them. In conclusion, mitochondrial macrohaplogroup N may be one of the determinant factors of anaerobic physical performance phenotype such as muscle power.

A cross-sectional study of sarcopenia in Japanese men and women: reference values and association with cardiovascular risk factors.

In this study of Japanese men and women, we determine reference values for sarcopenia and test the hypothesis that sarcopenia is associated with risk factors for cardiovascular disease, independent of waist circumference. A total of 1,488 Japanese men and women aged 18-85 years participated in this study. Appendicular muscle mass (AMM) was measured by dual-energy X-ray absorptiometry. Reference values for classes 1 and 2 sarcopenia (skeletal muscle index: AMM/height2, kg m-2) in each sex were defined as values one and two standard deviations below the sex-specific means of reference values obtained in this study from young adults aged 18-40 years. The reference values for class 1 and class 2 sarcopenia were 7.77 and 6.87 kg m-2 in men and 6.12 and 5.46 kg m-2 in women. In subjects both with class 1 and class 2 sarcopenia, body mass index and % body fat were significantly lower than in normal subjects. Despite whole-blood glycohaemoglobin A1c in men with class 1 sarcopenia was significantly higher than in normal subjects, and brachial-ankle pulse wave velocity in women both with class 1 and class 2 sarcopenia were significantly higher than in normal subjects, using one-way ANCOVA with adjustment for the covariate of waist circumference. Although sarcopenia is associated with thin body mass, it is associated with more glycation of serum proteins in men and with greater arterial stiffness in women, independent of waist circumference.

Acclimation responses to high light by Guazuma ulmifolia Lam. (Malvaceae) leaves at different stages of development.

The re-composition of deforested environments requires the prior acclimation of seedlings to full sun in nurseries. Seedlings can overcome excess light either through the acclimation of pre-existing fully expanded leaves or through the development of new leaves that are acclimated to the new light environment. Here, we compared the acclimation capacity of mature (MatL, fully expanded at the time of transfer) and newly expanded (NewL, expanded after the light shift) leaves of Guazuma ulmifolia Lam. (Malvaceae) seedlings to high light. The seedlings were initially grown under shade and then transferred to full sunlight. MatL and NewL were used for chlorophyll fluorescence and gas exchange analyses, pigment extraction and morpho-anatomical measurements. After the transfer of seedlings to full sun, the MatL persisted and acclimated to some extent to the new light condition, since they underwent alterations in some morpho-physiological traits and maintained a functional electron transport chain and positive net photosynthesis rate. However, long-term exposure to high light led to chronic photoinhibition in MatL, which could be related to the limited plasticity of leaf morpho-anatomical attributes. However, the NewL showed a high capacity to use the absorbed energy in photochemistry and dissipate excess energy harmlessly, attributes that were favoured by the high structural plasticity exhibited by these leaves. Both the maintenance of mature, photosynthetically active leaves and the production of new leaves with a high capacity to cope with excess energy were important for acclimation of G. ulmifolia seedlings.

Effects of habitual aerobic exercise on the relationship between intramyocellular or extramyocellular lipid content and arterial stiffness.

The accumulation of intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) is associated with arterial stiffness in middle-aged and older adults. Habitual aerobic exercise induces the improvement of arterial stiffness with reduction in fat accumulation. However, the relationship between aerobic exercise-induced changes in muscular lipids and arterial stiffness remains unclear. The purpose of this study was to investigate whether habitual aerobic exercise-induced changes in IMCL and EMCL content would lead to an improvement of arterial stiffness. First, in a cross-sectional study, we investigated whether cardiorespiratory fitness level affects the association between IMCL or EMCL content and arterial stiffness in 60 middle-aged and older subjects (61.0±1.3 years). Second, in an intervention study, we examined whether aerobic exercise training-induced changes in IMCL and EMCL content are associated with a reduction in arterial stiffness in 18 middle-aged and older subjects (67.0±1.7 years). In the cross-sectional study, IMCL content was negatively correlated with brachial-ankle pulse wave velocity (baPWV) (r=-0.47, P<0.05), whereas EMCL content was positively correlated with baPWV (r=0.48, P<0.05) in the low-fitness group, but was not correlated in the high-fitness group. Furthermore, 8-week aerobic exercise training in older adults increased IMCL content and reduced EMCL content. The training-induced change in baPWV was negatively correlated with training-induced changes in IMCL but was positively correlated with training-induced changes in EMCL. These findings suggest that aerobic exercise training-induced changes in IMCL and EMCL content may be related to a reduction in arterial stiffness in middle-aged and older adults.

Elevated pentraxin 3 level at the early stage of exercise training is associated with reduction of arterial stiffness in middle-aged and older adults.

Regular exercise improves aging-induced deterioration of arterial stiffness, and is associated with elevated production of pentraxin 3 (PTX3) and anti-inflammatory as well as anti-atherosclerotic effects. However, the time-dependent effect of exercise training on arterial stiffness and PTX3 production remains unclear. The purpose of this study was to investigate the time course of the association between the effects of training on the circulating PTX3 level and arterial stiffness in middle-aged and older adults. Thirty-two healthy Japanese subjects (66.2±1.3 year) were randomly divided into two groups: training (exercise intervention) and sedentary controls. Subjects in the training group completed 8 weeks of aerobic exercise training (60-70% peak oxygen uptake (VO2peak) for 45 min, 3 days per week); during the training period, we evaluated plasma PTX3 concentration and carotid-femoral pulse wave velocity (cfPWV) every 2 wk. cfPWV gradually declined over the 8-week training period, and was significantly reduced after 6 and 8 week of exercise intervention (P<0.05). Plasma PTX3 level was significantly increased after 4 weeks of the intervention (P<0.05). In addition, the exercise training-induced reduction in cfPWV was negatively correlated with the percent change in plasma PTX3 level after 6 week (r=-0.54, P<0.05) and 8 weeks (r=-0.51, P<0.05) of the intervention, but not correlated at 4 weeks. Plasma PTX3 level was elevated at the early stage of the exercise training intervention, and was subsequently associated with training-induced alteration of arterial stiffness in middle-aged and older adults.

Role of circadian activation of mitogen-activated protein kinase in chick pineal clock oscillation.

A circadian pacemaker generates a rhythm with a period of approximately 24 hr even in the absence of environmental time cues. Several photosensitive neuronal tissues such as the retina and pineal gland contain the autonomous circadian pacemaker together with the photic-input pathway responsible for entrainment of the pacemaker to the daily light/dark cycle. We show here that, in constant darkness, chick pineal mitogen-activated protein kinase (MAPK) exhibited an in vivo circadian rhythm in tyrosine phosphorylation and in enzymatic activity with a peak during subjective night. Phosphorylated and hence activated MAPK was rapidly dephosphorylated after light illumination during the nighttime when light induces a phase-shift of the pacemaker. The circadian rhythmicity in MAPK phosphorylation was also observed in the cultured pineal gland, and importantly, MAPK kinase inhibitor treatment during subjective night not only shifted the time-of-peak of MAPK phosphorylation but also induced a remarkable phase-delay of the circadian pacemaker. These results indicate an important role of MAPK for time keeping in circadian clock systems.

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S.

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.

Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland.

Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.

Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation.

In rod photoreceptor cells, Ca2+-bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+-induced membrane association of recoverin has not been fully evaluated. We found that Ca2+-bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+-recoverin-rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva.

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.

Further fractionation of basic proline-rich peptides from human parotid saliva and complete amino acid sequence of basic proline-rich peptide P-H.

Three basic proline-rich peptides were newly isolated from human parotid saliva, and designated as P-G, P-H, and P-I. The amino acid sequence of P-H was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Glu-Gly-Asn-Asn- Pro-Gln-Gly-Pro-Pro-Pro-Pro-Ala-Gly-Gly-Asn-Pro-Gln-Gln-Pro-Gln-Ala-Pro-Pro- Ala-Gly-Gln-Pro-Gln-Gly-Pro-Pro-Arg-Pro-Pro-Gln-Gly-Gly-Arg-Pro-Ser-Arg-Pro- Pro-Gln by conventional methods. The amino terminal ten residues of P-H were the same as those of proline-rich peptides P-D, P-E, and P-F reported previously. Comparison of the amino acid sequences between P-H and P-D revealed that there are two deletion parts and several amino acid substitutions in the sequence of P-H. Homology between P-H and P-D was as high as 70%.

Fractionation and characterization of basic proline-rich peptides of human parotid saliva and the amino acid sequence of proline-rich peptide P-E.

Basic proline-rich peptides of human parotid saliva were fractionated and characterized. The amino acid sequence of one of the purified peptides, P-E, was determined to be (formula: see text). The results demonstrate the repetitiveness of the partial sequence within the molecule and the occurrence of structures common to those of other salivary polypeptides such as P-C and Protein C.

The amino acid sequence of a salivary proline-rich peptide, P-C, and its relation to a salivary proline-rich phosphoprotein, protein C.

A basic proline-rich peptide, P-C, was isolated from human whole saliva and its amino acid sequence was determined to be Gly-Arg-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Gly-Gly-His-Gln-Gln-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Gly-Arg-Pro-Gln-Gly-Pro-Pro-Gln-Gly-Gln-Ser-Pro-Gln. P-C was also found to be present in stimulated parotid saliva. The sequence determined appears to correspond to the C-terminal 44 residues of a proline-rich phosphoprotein, Protein C, the partial sequence of which has been elucidated, and suggests that P-C might be derived from Protein C.

Isolation and amino acid sequences of proline-rich peptides of human whole saliva.

Three basic peptides with extremely high proline contents were isolated from human whole saliva. The amino acid sequences of two of these proline-rich peptides comprising 57 and 38 residues were determined by conventional methods. The sequence suggested that the smaller peptide was derived from the larger one and also revealed the occurrence of characteristic repeating units within the molecules. The present study is the first to describe this structural feature of proline-rich proteins or peptides.

Amino acid sequences of glycopeptides obtained from basic proline-rich glycoprotein of human parotid saliva.

A proline-rich glycoprotein, in which proline, glutamic acid, and glycine represent about 80 per cent of the total residues, was obtained from human parotid saliva. The amino acid sequences of glycopeptides obtained from digests of the glycoprotein with clostripain were determined to be Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro-Gln-Gly-Gly-Asn(CHO)-Gln-Ser-Gln-Gly-Pro- Pro-Pro-Arg and Pro-Pro-Gln-Gly-Gly-Asn(CHO)-Gln-Ser-Gln-Gly-Pro-Pro-Pro-His-Pro-Gly-Lys-Pro -Glu-Arg. The structural relationship between the glycopeptides and the known salivary peptide is discussed.

Complete amino acid sequence of a basic proline-rich peptide, P-F, from human parotid saliva.

The complete amino acid sequence of a basic proline-rich peptide, P-F, isolated from human parotid saliva was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Gly-Asn-Gln-Pro-Gln-Gly-Pro-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Asn-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Ser-Lys-Ser-Arg-Ser-Ala by conventional methods. P-F contains a number of repeating sequences and oligo-proline structures identical with those in other proline-rich peptides such as P-C, P-D, P-E, and Protein C. P-F has the highest degree of homology with P-E among these proline-rich peptides.

Primary structure of cat osteocalcin.

A bone Gla-containing protein (osteocalcin) of cat has been isolated and the complete primary structure has been determined to be YLAPGLGAOAPYPDPLXPKRXICXLNPDCDELADHIGFQDAYRRFYGTV. The protein consists of 49 amino acid residues (Mr. 5,641) containing three Gla residues and a single disulfide bond. Although the C-terminal 30 residues (20th to 49th) of the sequences of cow, monkey, and human osteocalcins are identical, three amino acid substitutions occur at positions 22, 40, and 48 in the case of cat. These substitutions can be explained by single point mutations.

Isolation and amino acid sequence of SAP-1, an acidic protein of human whole saliva, and sequence homology with human gamma-trace.

A low-molecular-weight acidic protein was isolated from human whole saliva by DE32 column chromatography and designated as SAP-1. The amino acid sequence was determined by conventional methods to be (sequence in text). The protein consisted of 113 residues and the calculated molecular weight was 12,552. Computer analysis revealed the presence of 54% sequence homology between SAP-1 and gamma-trace, a basic microprotein present in cerebrospinal fluid and in urine of patients with renal failure.

Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.

Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.

Cystatin S: a cysteine proteinase inhibitor of human saliva.

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.