Pregnancy outcomes in women with rheumatic diseases: a real-world observational study in Japan.

OBJECTIVES: We aimed to evaluate the obstetric complications and the risk factors for these events in pregnant women with rheumatic diseases (RDs). METHODS: A single-center retrospective study of women with RDs at Hokkaido University Hospital between 2007 and 2016 was conducted. Clinical features and maternal and fetal outcomes were retrospectively collected. The rate of pregnancy complications was compared with the general obstetric population (GOP) in Japan. RESULTS: Overall, 132 pregnancies in 95 women with RDs were recorded. Underlying RDs were systemic erythematosus (SLE) (n = 57), antiphospholipid syndrome (APS) (n = 35), rheumatoid arthritis (n = 9), and other RDs (n = 31). Antiphospholipid antibodies (aPL) were detected in 44 pregnancies (32%). Glucocorticoid was used in 82 pregnancies (62%), and tacrolimus in 20 pregnancies (15%). There were 24 disease flares (18%), but no RD-related death was documented. We recorded 112 live births, 6 abortions, 8 miscarriages, and 6 stillbirths. Pregnancies with RDs appeared to have frequent, emergency cesarean sections and preterm deliveries compared with GOP (30% vs 15% and 21% vs 14%, respectively). The median [interquartile range] birthweight in SLE and APS was lower than GOP (2591 [2231-2958] g and 2600 [2276-2920] g vs 2950 [2650-3250] g, respectively). In pregnancies with SLE, low complement levels presented the risk of maternal complications (odds ratio [95% CI]; 3.9 [1.0-14.9], p = 0.046) and anti-DNA antibody positivity was significantly correlated with the risk of fetal complications (3.5 [1.1-11.2], p = 0.036). In pregnancies with APS, maternal age over 35 years and duration of disease longer than 9 years (7.4 [1.3-40.8], p = 0.021, and 11.16 [1.1-118.8], p = 0.046, respectively) were significantly correlated with the risk of fetal complications. CONCLUSION: Pregnancies with RDs were at increased risk of having both maternal complications and adverse neonatal outcomes, indicating these pregnancies should be closely monitored.

Hemoglobins M: identification of Iwate, Boston, and Saskatoon variants.

Hemoglobin M variants, M Iwate, M Boston, M Saskatoon are easily and accurately identified by electron spin resonance with small amounts of patients' blood. In hemoglobin M Iwate and M Boston the electron spin resonance of both fresh blood (unprocessed) and isolated pure ferrihemoglobin M revealed similar signal shapes; whereas that of hemoglobin M Saskatoon was doublet in fresh blood and triplet in pure ferrihemoglobin M.

Weekly paclitaxel/5-fluorouracil followed by platinum retreatment for patients with recurrent ovarian cancer: a single institution experience.

PURPOSE: Since the prognosis of recurrent ovarian cancer patients is still poor, we need to establish a useful treatment strategy to achieve their long-term survival. We treated recurrent ovarian cancer patients with weekly paclitaxel (PTX)/5-fluorouracil (5-FU) followed by platinum retreatment to investigate its clinical efficacy in a preliminary manner. METHODS: Sixteen patients with recurrent ovarian cancer, pretreated with taxane and platinum, were treated with weekly paclitaxel (PTX)/5-fluorouracil (FU). PTX (80 mg/m2) on day 1, 8, and 15 was combined with a bolus injection of 5-FU (500 mg/m2) on day 2, 9, and 16. Chemotherapy was given every four weeks. Patients with stable disease or progressive disease were subsequently retreated with a platinum-containing regimen. Response was evaluated by RECIST criteria or CA125 criteria. Toxicities were evaluated according to the National Cancer Institute-common toxicity criteria (NCI-CTC) version 3. RESULTS: Among five patients with sensitive disease, one of four patients with measurable tumor and one without measurable tumor responded to weekly PTX/5-FU. Among 11 patients with resistant disease, none of five patients with measurable tumor and three of six patients without measurable tumor responded to weekly PTX/5-FU. Overall objective response rate by weekly PTX/5-FU was 31.3% (5/16). Among 16 patients, 13 patients who showed no response or progressive disease (three with sensitive disease, ten with resistant disease) received platinum retreatment after weekly PTX/5FU. All three patients with sensitive disease and three of ten patients with resistant disease revealed response to platinum retreatment. Overall objective response rate by platinum retreatment after weekly PTX/5-FU was 46.2% (6/13). CONCLUSIONS: Weekly PTX/5FU followed by platinum retreatment could be a useful treatment strategy for recurrent ovarian cancer patients. We need to establish the standard treatment strategy for recurrent ovarian cancer patients with a poor prognosis.

Diabetes mellitus is a multivariate independent prognostic factor in endometrial carcinoma: a clinicopathologic study on 313 patients.

OBJECTIVE: The aim of this study was to analyse the influence of diabetes mellitus as a prognostic factor for overall survival in endometrial cancer. MATERIALS AND METHODS: Charts were reviewed from patients with endometrial carcinoma from 1985 to 2003. Data on clinicopathologic variables, adjuvant treatment, site of recurrence and survival were collected. The chi-square test was used to examine associations between variables. The Kaplan-Meier method was used for survival analysis and Cox's proportional hazards model for multiple regression analysis. RESULTS: Multivariate analysis revealed that diabetes mellitus, FIGO stage and depth of myometrial invasion were significantly associated with overall survival.

Measurements of in vivo 31P nuclear magnetic resonance spectra in neuroectodermal tumors for the evaluation of the effects of chemotherapy.

The effects of chemotherapy on living tumor tissue in hamsters and rats were investigated by measuring the 31P nuclear magnetic resonance spectra using topical magnetic resonance. Human neuroblastoma, human glioblastoma, and rat glioma tumor cells were inoculated s.c. in the lumbar region of the animals. After the diameter of the tumors increased to 1.5 cm, in vivo 31P nuclear magnetic resonance spectra were measured selectively in the tumors with a TMR-32 spectrometer. Adenosine triphosphate, inorganic phosphate (Pi), phosphodiester, and phosphomonoester peaks were observed. The phosphocreatine peak was hardly detectable, adenosine triphosphate and phosphomonoester peaks were high, and tissue pH, calculated from the chemical shift of Pi, declined. Regardless of the tumor origin or the histological type, the spectral pattern of each neuroectodermal tumor was found to be essentially the same. After i.v. injection of a large dose of a chemotherapeutic agent, adenosine triphosphate peaks decreased and Pi increased gradually, resulting in a dominant Pi peak pattern after 6 to 12 hours. However, during the same period, there were no observable changes in the spectra of normal organs. These findings indicated that the drugs have a selective and direct action on the energy metabolism of tumor cells. With lower drug doses, no remarkable changes were seen in the spectrum. Measurement of in vivo 31P nuclear magnetic resonance spectra is valuable not only to investigate the energy metabolism in tumor tissue but also to evaluate the effects of chemotherapy.

Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering.

23Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter 23Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial 23Na were observed in the perfused gland at 25 degrees C. The resonance due to intracellular 23Na consisted of two Lorentzian signals stemming from the [1/2 mean value of -1/2[ coherence (sharp resonance) and the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences (broad resonance). The transverse relaxation rate constant corresponding to the [1/2 mean value of -1/2[ coherence was 95 +/- 4 s-1 and that corresponding to the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences was 1360 +/- 75 s-1 (mean +/- S.E., n = 5). The resonance due to the interstitial 32Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N",N",N"'-hexaacetic acid.

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR.

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.

Application of the transition state theory to water transport across cell membranes.

We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.

Phosphorus nuclear magnetic resonance of perfused salivary gland.

Phosphorus nuclear magnetic resonance (31P-NMR) was used for the sequential measurement of phosphorus energy metabolites in perfused canine submandibular gland. Under resting conditions, ATP and creatine phosphate levels were 0.42 +/- 0.11 mM and 0.62 +/- 0.16 mM (mean +/- S.D., in nine glands). When perfusion of the gland was stopped, the tissue contents of ATP and creatine phosphate decreased, that of ADP increased and tissue pH decreased. Restarting perfusion led to recoveries of the tissue content of the phosphorus compounds and tissue pH to normal. Acetylcholine administration induced secretion of saliva, decreased the level of ATP, creatine phosphate and tissue pH, and increased the ADP level.

Comparative 1H-NMR studies on the physical state of water in soft contact lens and mouse lens.

The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin-lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%.

In vivo studies of energy metabolism in experimental cerebral ischemia using topical magnetic resonance. Changes in 31P-nuclear magnetic resonance spectra compared with electroencephalograms and regional cerebral blood flow.

The energy state of the brain during and after transient cerebral ischemia was examined in rats by in vivo measurement of 31P-nuclear magnetic resonance (NMR) spectra using a topical magnetic resonance spectrometer. EEGs and regional CBF (rCBF) were monitored on the same ischemic models. Immediately after the induction of ischemia, the height of the ATP and phosphocreatine peaks in the spectrum began to decrease with a concurrent increase of the inorganic phosphate (Pi) peak. The calculated pH from the chemical shift of Pi decreased during ischemia. The EEG pattern became flat immediately after ischemic induction. The rCBF decreased below the sensitivity level of the measuring instrument. With 30-min ischemia, the 31P-NMR spectrum returned to a normal pattern rapidly after recirculation. However, recovery of the EEG was delayed. The rCBF after recirculation showed postischemic hyperemia followed by hypoperfusion. In cases of 120-min ischemia, none of the spectra showed recovery. Thus, we could investigate the dynamic process of pathophysiological changes occurring in the ischemic brain in vivo.

High resolution 23Na-nuclear magnetic resonance study of stroke-prone spontaneously hypertensive rat erythrocytes.

The intracellular Na+ content of washed erythrocytes from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) was measured by a high resolution 23Na-nuclear magnetic resonance (NMR) technique using a non-permeant aqueous shift reagent, dysprosium triethylenetetramine hexaacetic acid, Dy(TTHA)3-. The initial intracellular Na+ of freshly isolated and washed erythrocytes was very low (approximately 5 mmol/l) and increased progressively with prolonged incubation in isotonic salt solution at 37 degrees C. There was no significant difference in the erythrocyte Na+ concentration between SHRSP and WKY over the entire period of measurement, nor was any difference detected in their osmotic fragility or total cellular volume, although the osmotic fragility decreased with incubation time. The high energy phosphate metabolites were also studied in the same erythrocytes by 31P-NMR. The level of intracellular ATP decreased with incubation at 37 degrees C but showed no difference between the SHRSP and WKY samples. Inclusion of 1 mmol/l ouabain in the incubation medium substantially retarded the breakdown of intracellular ATP and resulted in a concomitant increase in intracellular Na+. However, neither the ouabain-sensitive nor the ouabain-insensitive component of Na+ influx altered in SHRSP erythrocytes compared with WKY erythrocytes in paired experiments. Our results do not support the hypothesis that altered Na+ transport, resulting in an increase in erythrocyte Na+ concentration, is associated with spontaneous hypertension.

NMR relaxation characteristics of rubidium-87 in perfused rat salivary glands.

Rubidium is a good substitute for potassium in many biological systems, and it has been suggested that rubidium-87 nuclear magnetic resonance (87Rb-NMR) spectroscopy could be used to measure K+ fluxes across membranes in intact tissues. To evaluate this possibility, isolated rat mandibular salivary glands were perfused with solutions containing Rb+ in place of K+. The 87Rb signals arising from the intra- and extracellular compartments were first separated by spectral subtraction and then subjected to line-shape analysis. The narrow extracellular signal was a single Lorentzian (line-width 156 Hz), whereas the broader intracellular signal consisted of two Lorentzian components (ca. 530 and 3080 Hz). Double-quantum filtering of the 87Rb signal from the glands revealed two components of transverse relaxation in antiphase (rate constants 1.8 and 13.3 ms-1), showing the probable involvement of quadrupolar interactions in the relaxation of intracellular Rb+. We conclude, therefore, that both line-shape analysis and double-quantum filtering could provide a basis for the measurement of unidirectional K+ fluxes in intact tissues.

Dome formation induced by v-H-ras oncogene in a human choriocarcinoma cell line.

To investigate the role of ras genes in trophoblastic cell lineage, we transfected the viral H- or K-ras oncogene into a human choriocarcinoma cell line, CCI, and analysed the biological properties of CCI cells expressing an activated ras oncogene. All v-H-ras-expressing clones distinctively formed the hemispherical domes, which represents an in vitro morphological expression of vectorial transport function and are characteristic of the polarized epithelial cells, but none of v-K-ras-expressing clones and control clones did. Microscopic observation demonstrated that those domes were cavities filled with fluid which accumulated between the cell layer and the surface of culture dish. Scanning electron microscopy revealed that the domes were aggregates of round cells with long numerous microvilli and were morphologically similar to a blastocyst. Furthermore, Na(+)-K(+)-ATPase activity, which is associated with the vectorial fluid transport in transporting epithelial cells, was significantly higher in the v-H-ras-expressing clones than that in the v-K-ras-expressing clones and the parental cells. Those domes flattened within 24 h after treatment with a specific inhibitor of Na(+)-K(+)-ATPase, ouabain, and the number of domes decreased in dose-dependent manner, indicating that Na(+)-K(+)-ATPase activity was required for maintainance of domes. These results suggest that up-regulated activity of H-ras but not of K-ras facilitates the vectorial fluid transport through a chorionic cell layer and leads to the dome formation. The function of II-ras in trophoblasts, may therefore, be essential for embryogenesis, especially for supplying the nutrients.

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein.

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.

Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex.

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.

31P nuclear magnetic resonance study on perfused brain slices of guinea pig.

31P-NMR measurements of brain slices of guinea pig were successfully carried out for the first time with a new perfusion and spinning technique. The 31P-NMR spectrum showed resonance lines of phosphocreatine, fructose-6-phosphate, ATP, and inorganic phosphate. In the time course of the 31P-NMR spectra, the concentrations of phosphocreatine and fructose-6-phosphate, which were estimated from the initial spectrum, were 4.3 and 1.1 mumol/g of fresh tissue, respectively, and the estimates from the following ones showed that the concentration of phosphocreatine decreased to 2.7 mumol/g of fresh tissue while that of fructose-6-phosphate did not change significantly throughout the measurements.

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins.

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.

Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system.

To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.