Visualization of ultraviolet absorption distribution beyond the diffraction limit of light by electron-beam excitation-assisted optical microscope.

We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 has absorption at ultraviolet wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film of an electron-beam excitation-assisted (EXA) optical microscope. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal. The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA optical microscope is useful for analysis of growth of a crystal, bioimaging and so on.

Improvement of (R)-3-hydroxybutyric acid secretion during Halomonas sp. KM-1 cultivation with saccharified Japanese cedar by the addition of urea.

UNLABELLED: Japanese cedar (Cryptomeria japonica) is a major species in artificial Japanese forests. The Halomonas sp. KM-1 was recently isolated and found to grow effectively on saccharified Japanese cedar wood, resulting in the intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. Under microaerobic conditions, the extracellular secretion of (R)-3-hydroxybutyric acid ((R)-3-HB) led to the degradation of intracellular PHB. In this study, the production of PHB and the secretion of (R)-3-HB using saccharified Japanese cedar were much improved in cultures that were grown in the presence of urea. The level of intracellular PHB production after 36 h under aerobic cultivation was 23·6 g l(-1) ; after shifting to microaerobic conditions for 24 h, the (R)-3-HB concentration in the medium reached 21·1 g l(-1) . Thus, KM-1 efficiently utilizes saccharified Japanese cedar to produce PHB and secretes (R)-3-HB, making it a practical candidate for use in the industrial production of (R)-3-HB. SIGNIFICANCE AND IMPACT OF THE STUDY: Japanese cedar is a major species grown in artificial Japanese forests, and its thinning is crucial for the health of artificial forests and the Japanese economy. Halomonas sp. KM-1 grew effectively on saccharified Japanese cedar wood, resulting in intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. Under microaerobic conditions, extracellular secretion of (R)-3-hydroxybutyric acid ((R)-3-HB) caused intracellular PHB degradation. (R)-3-HB is a chiral compound that is useful in the chemical, health food and pharmaceutical industries. The production of PHB and secretion of (R)-3-HB using saccharified wood was dramatically improved, which may positively affect its future industrial production.

Acute treatment with candesartan cilexetil, an angiotensin II type 1 receptor blocker, improves insulin sensitivity in high-fructose-diet-fed rats.

We aimed to determine whether acute treatment with candesartan cilexetil (CV-11974), an angiotensin II type 1 receptor blocker (ARB) can improve insulin sensitivity in high-fructose-diet (HFD)-fed rats. In vivo glucose utilization was measured by applying the euglycemic clamp technique and the expression levels of insulin-signaling molecules in skeletal muscles were examined by western blotting. A bolus injection of CV-11974 improved the glucose infusion rate (GIR) of HFD-fed rats to the level of the control rats. Furthermore, restoration of impaired tyrosine phosphorylation of insulin receptor (IR) ß, Akt phosphorylation at Ser47³ and Thr³°8, and phosphorylation of the 160-kDa Akt substrate (AS160) in the skeletal muscles of HFD-fed rats were achieved by this treatment. These results suggest that acute administration of candesartan cilexetil can increase insulin sensitivity of HFD-fed rats, which is associated with improved insulin signaling in skeletal muscles.

The influence of vulnerability on depression among Japanese university athletes.

Objective: This study examined the estimated causal relationship between vulnerability and depressive symptoms in Japanese university athletes and how the degree of vulnerability affects depressive symptoms. Materials and methods: In Study 1, 248 Japanese university athletes completed a continual survey from Time 1 to Time 3. In Study 2, 562 Japanese university athletes responded to another survey during the same period. Structural equation modeling was performed to estimate the causal relationship using the cross-lagged effects model for the three waves. Next, a binomial logistic regression analysis was performed to examine the influence of vulnerability on depression. Results: Results of the cross-lagged effects model showed that all paths from vulnerability to depressive symptoms were significant, and all paths from depressive symptoms to vulnerability were not significant. Thus, vulnerability was the causative variable and depressive symptoms were the outcome variables within the causal relationship. The logistic regression results showed that those with high vulnerability were 1.7 times more likely to have moderate or higher depressive symptoms than those with low vulnerability. Vulnerable individuals are at a higher risk for developing depressive symptoms. By verifying the causal relationship between vulnerability and depressive symptoms, we can contribute to the enhancement of mental health care in accordance with the weakest link model. Appropriate psychological support for athletes can decrease depression and improve their mental health.

Combined effects of short-term calorie restriction and exercise on insulin action in normal rats.

The present study examined the effect of combination of short-term calorie restriction (CR) and moderate exercise on insulin action in normal rats. Rats were divided randomly into 4 groups: ad libitum, sedentary (A-Sed); calorie restriction, sedentary (CR-Sed); ad libitum, exercise (A-Ex); and calorie restriction, exercise (CR-Ex). Rats in the exercise groups were run on a rodent treadmill. Rats in the CR groups were fed every alternate day. Oral glucose tolerance test (OGTT) showed improvements in both CR-Sed and A-Ex groups compared with the A-Sed group; no further improvement in glucose tolerance was observed in the CR-Ex group. In contrast, glucose infusion rates (GIRs) determined by the hyperinsulinemic-euglycemic clamp method indicated that the GIR of the CR and exercise combination was significantly better than that of the sole intervention of CR or exercise. There was no difference in the levels of fasting glucose, insulin, or high-molecular weight forms of adiponectin among the 4 groups. Protein expression of GLUT-4 in the skeletal muscle increased by exercise, but not by CR. Our findings indicate that the combination of exercise and CR may be effective in enhancing insulin sensitivity at the skeletal muscle in normal subjects.

Stroboscopic near-field imaging for the analysis of contraction of muscle cells.

We have developed a stroboscopic near-field optical microscope for observation of biological specimens and observed glycerinated muscles before and after muscle contraction with the developed system. In the system, the optical field distribution localized near the specimen is recorded as the surface topographic distribution of a photosensitive film surface. Our system is very useful for observing living biological specimens with high resolutions, because it is possible to get stroboscopic image by using a photosensitive film as detecting optical distributions instead of a scanning of probes. We have succeeded in observing inner structures of muscle cells with sub-wavelength resolution and achieved higher contrast than an ordinary optical microscope.

Adenosine receptor subtypes modulate two major functional pathways for hippocampal serotonin release.

To clarify the mechanisms of interaction between adenosine A(1) receptor (A1-R) and adenosine A(2) receptor (A2-R) on neurotransmitter release, this study determined the functional interactions among adenosine receptors (AD-Rs), voltage-sensitive Ca(2+) channels (VSCCs), protein kinases (PKs), and synaptic proteins [N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors] on hippocampal serotonin release using in vivo microdialysis in freely moving rat. Basal serotonin release was regulated by two functional complexes: N-type VSCC (N-VSCC)/calcium-phospholipid-dependent protein kinase (PKC)/syntaxin (major pathway) and P-type VSCC (P-VSCC)/cyclic AMP-dependent protein kinase (PKA)/synaptobrevin (minor pathway). However, K(+)-evoked serotonin release was regulated by N-VSCC/PKC/syntaxin (minor pathway) and P-VSCC/PKA/synaptobrevin (major pathway). A1-R antagonists increased basal serotonin release, which was reduced by inhibitors of N-VSCC, PKC, and syntaxin predominantly and by inhibitors of PKA and synaptobrevin weakly, but was not affected by P-VSCC inhibitor. In the presence of A1-R antagonist, A2-R agonists increased basal serotonin release, which was inhibited by inhibitors of P-VSCC, PKA, and synaptobrevin predominantly and reduced by inhibitors of N-VSCC, PKC, and syntaxin weakly. Under the condition of activation of adenylate cyclase in the absence of A1-R antagonists, A2-R agonists increased basal serotonin release. A1-R antagonist and A2-R agonist enhanced K(+)-evoked serotonin release, which was inhibited by inhibitors of P-VSCC, PKA, and synaptobrevin predominantly. These results suggest that an activation of A1-R suppresses serotonin release via inhibition of both N-VSCC/PKC/syntaxin and P-VSCC/PKA/synaptobrevin pathways, and an activation of A2-R stimulates serotonin release via enhancement of the P-VSCC/PKA/synaptobrevin pathway. Therefore, PKA activity plays an important role in the interaction between A1-R and A2-R on hippocampal serotonin release.

The guanidine-induced conformational changes of the chaperonin GroEL from Escherichia coli. Evidence for the existence of an unfolding intermediate state.

Equilibrium unfolding experiments of the E. coli chaperonin GroEL were performed in guanidine hydrochloride. A reversible unfolding intermediate was observed in very low concentrations of denaturant (< 0.5 M guanidine hydrochloride). This intermediate was characterized by a decreased light scattering intensity and an increased binding of the fluorescent probe 1-anilino-8-naphthalene sulfonate. No significant changes in circular dichroism spectra were observed for this unfolding intermediate. A second decrease in fluorescence intensity and light scattering was observed in higher concentrations of guanidine hydrochloride, with a transitional midpoint of 1.15 M. This transition was accompanied by the complete loss of secondary structure, as monitored by circular dichroism spectroscopy. This second transition agreed well with the results previously reported in this journal (Price et al. (1993) Biochim. Biophys. Acta 1161, 52-58).

High pressure conditions promote the proliferation of rat cultured mesangial cells in vitro.

Glomerular capillary pressure is involved in the development of chronic renal failure and has at least two effects on mesangial cells: transmembrane hydrostatic pressure and stretch. To clarify whether pure hydrostatic pressure itself affects the proliferation of cultured rat mesangial cells, we compared the cell number under atmospheric pressure condition with high pressure condition. At 24 and 48 h with 0.5% serum, cell number was significantly higher under high pressure condition than under atmospheric pressure condition. At 48 h, cell number under high pressure condition was increased in a pressure-dependent manner. Furthermore, flow cytometric assay indicated that pressure-load could promote DNA synthesis rate at S phase and enhance G1/S progression induced by low concentration of serum (0.5%). These results suggest that pure hydrostatic pressure itself can promote the proliferation of cultured rat mesangial cells by advancing cell cycle progression in vitro.

Interaction of GroEL with conformational states of horse cytochrome c.

GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytochrome c, and fragments 1-65 and 1-38, but not for intact cytochrome c or fragment 11-21. On the other hand, under conditions of high ionic strength, only apo-cytochrome c remained tightly bound. Whereas porphyrin-cytochrome c assumes a molten-globule-like compact conformation with a native-like far-UV CD, apo-cytochrome c shows a compact conformation without an ordered secondary structure. The far-UV CD spectra of the three fragments indicated that the fragments lack an ordered secondary structure. These results suggest that the fluctuating and exposed hydrophobic clusters of the substrates are responsible for the interaction, and that the interaction is modulated by electrostatic interaction. It is notable that these characteristics are similar to those of the interaction of cytochrome c derivatives with negatively charged phospholipid membranes, suggesting a common mechanism. Using fluorescence-labeled apo-cytochrome c, we also measured the kinetics of the interaction and estimated the association rate constant to be 5 x 10(7) M-1s-1. This relatively fast association rate constant indicates that the refolding rate of substrate protein is another important factor determining the interaction.

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase.

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.

Phorbol myristate acetate stimulates osteoclast formation in 1 alpha,25-dihydroxyvitamin D3-primed mouse embryonic calvarial cells by a prostaglandin-dependent mechanism.

Our previous study provided a novel assay system utilizing devitalized bone slices for study of the differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts among calvarial cells of mouse embryos. Using this assay system, we examined the effect of phorbol myristate acetate (PMA) on osteoclast formation as assessed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells and bone resorption lacunae. PMA alone was directly unable to induce the appearance of TRAP-positive cells and bone resorption lacunae of calvarial bone cells of mouse embryos. However, PMA markedly stimulated increases in the number of TRAP-positive cells and area of the resorption lacunae of the calvarial cells when the bone cells were primed by 1 alpha,25-(OH)2D3. This stimulatory effect of PMA was dose dependent. H-7, having relatively high affinity for protein kinase C, strongly inhibited in a dose-dependent fashion the stimulatory effect of PMA on the bone resorption of the hormone-primed calvarial cells. We also examined the involvement of prostaglandin in this stimulatory effect of PMA. Indomethacin, a cyclooxygenase inhibitor, markedly abolished the stimulatory effect of PMA on the bone resorption of the calvarial cells. PMA stimulated prostaglandin E2 (PGE2) production by the calvarial cells primed with 1 alpha,25-(OH)2D3 in a dose-dependent fashion. However, the PMA stimulation of the PGE2 production was significantly inhibited by H-7 and also by indomethacin. Furthermore, we observed that the addition of PGE2 to the calvarial cells primed with 1 alpha,25-(OH)2D3 for 1 or 3 days resulted in an increased number of TRAP-positive cells and increased bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro.

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10(-5) M, 10(-6) M or 10(-7) M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P < 0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10(-5) M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P < 0.05), 13.9% at 24 h (P < 0.001), and 76.5% at 48 h (P < 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P < 0.005) and 12 h (P < 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate.

Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli.

Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis.

Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation.

In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro. Without nucleotides the refolding reactions were arrested by GroEL. Addition of GroES and ADP to the reaction mixture initiated the refolding reactions and the enzyme activities were regained efficiently. The presence of GroE (GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures. These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL-facilitated protein folding.

The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES.

In order to understand the role of ATP hydrolysis of the chaperonin GroEL during protein folding, we have studied GroEL-GroES complex formation in the presence of ATP or ADP by using capillary electrophoresis and surface plasmon resonance. Capillary electrophoresis analysis showed that the GroEL 14-mer and GroES 7-mer formed a 1:1 complex in the presence of ATP. In the presence of ADP, both the association and dissociation rates of the complex were slower by about one order of magnitude than the rates in the presence of ATP at 25 degrees C. The implications of such a stable complex on the overall mechanism of chaperonin function are discussed.