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Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell–extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, EXT1-silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of EXT1-silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with EXT1-silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein–transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with EXT1-silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.

The sulfate transporter gene SLC26A2 is responsible for diastrophic dysplasia, which represents skeletal dysplasia in humans. This highlights the importance of sulfate metabolism in skeletal formation. SLC26A2-related chondrodysplasia is also known to exhibit abnormalities in craniofacial and tooth development. Although the function of SLC26A2 in mammals has been investigated using genetic mouse models, the essential role of SLC26A2 during craniofacial and tooth development has not been elucidated.In this study, we demonstrate the pivotal roles of SLC26A2-mediated sulfate metabolism during tooth development. Analysis ofSlc26a2expression reveals that it is predominantly expressed in dental tissues, including odontoblasts and ameloblasts, during tooth development.Slc26a2knockout mice (Slc26a2-KO-Δexon2) exhibit a retrognathic upper jaw, small upper incisors, and hypoplasia of upper molars. Additionally, upper incisors and molars inSlc26a2-KO-Δexon2mice display flattened odontoblasts and nuclei that lack intracellular polarity. In contrast, tooth phenotype is not remarkable in lower incisors and molars.Furthermore, the expression of odontoblast differentiation markers,DsppandDmp1, is significantly decreased in the upper molars ofSlc26a2-deficient mice.Ex vivoorgan culture of tooth germs by implantation ofSlc26a2-deficient tooth germs under the kidney capsule reveals hypoplasia of the dentin matrix as well as tooth root shortening.In vitrostudies using human dental pulp stem cells (hDPSCs) show that the expression levels ofDsppandDmp1inshSlc26a2knockdown cells are significantly decreased compared to control cells.Collectively, our data demonstrate that SLC26A2-mediated sulfate metabolism is essential for tooth development. This study may provide insight into the mechanisms underlying tooth abnormalities in patients with recessively inherited chondrodysplasias caused by mutations in the SLC26A2 gene.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

The present study directly addresses the roles of the P2X(7) receptor (P2X(7)R), an ionotropic adenosine triphosphate (ATP) receptor, and cytokines in the induction of orofacial pain following chronic constriction injury (CCI) of the infraorbital nerve (IoN).Rats were anesthetized, and ligatures of 4-0 chromic gut were tied around the IoN. A438079, a P2X(7)R antagonist or SB203580, a phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) inhibitor, was infused intrathecally into CCI-treated rats. In another group of rats, 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), a P2X(7) R agonist, was infused intrathecally with A438079, SB203580 or etanercept, a tumor necrosis factor (TNF)-α receptor-binding recombinant drug.CCI of the IoN induced tactile allodynia/hyperalgesia and up-regulation of P2X(7)R, membrane-bound TNF-α (mTNF-α) and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC). Tactile allodynia/hyperalgesia or up-regulation of mTNF-α and sTNF-α in the TNC following CCI of the IoN was inhibited by A438079. SB203580 also attenuated tactile allodynia/hyperalgesia or up-regulation of mTNF-α, but not the up-regulation of sTNF-α in the TNC. Treatment of rats with BzATP induced tactile allodynia/hyperalgesia and up-regulation of sTNF-α and p-p38 in the TNC. Tactile allodynia/hyperalgesia or up-regulation of sTNF-α following BzATP treatment was inhibited by SB203580 and etanercept.Based on these findings, phosphorylation of p38 MAPK via P2X(7)R may induce tactile allodynia/hyperalgesia, which is most likely mediated by sTNF-α released by microglia.

A therapeutic protocol to minimize root resorption induced by tooth replantation has not yet been universally established. In this context, noninvasive modality such as ultrasound therapy have been a focus of increased interest. This study aimed to evaluate the inhibitory effect of ultrasound therapy on root resorption of replanted rat molars. In addition, the study aimed to promote insights into the mechanism through which ultrasound mediates the metabolism of periodontal cells in vitro.An experimental model of tooth replantation in rats, involving luxation and immediate replacement of the maxillary first molars, was used to assess the inhibitory effect of an ultrasound-therapy regimen (15 min of exposure to ultrasound, each day for 21 d) on root resorption. Moreover, the effect of ultrasound on osteoclastogenesis/cementoclastogenesis was examined in vitro using a mouse osteoblastic stromal cell line (ST2) and a mouse cementoblastic cell line (OCCM-30).The area of root resorption lacunae was statistically decreased (p0.01) in the ultrasound-treated sample. In addition, immunohistochemical staining, using murine TNF-α polyclonal antibody, failed to detect tumor necrosis factor-α (TNF-α) protein in the ultrasound-treated sample compared with the control. An in vitro study showed that the lipopolysaccharide (LPS)-induced expression of Tnfalpha mRNA was significantly reduced by ultrasound therapy in both osteoblastic and cementoblastic cells. Moreover, the TNF-α-induced up-regulation of Rankl mRNA was also inhibited by ultrasound.Ultrasound may contribute to the reduction of the trauma-induced inflammatory reaction through impairment of the TNF-α signaling pathway. It is therefore suggested that ultrasound shows potential as a therapeutic tool to optimize the regenerative potential of periodontal tissues on replanted teeth.

Non-localized and localized 31P nuclear magnetic resonance (NMR) spectra of rat calf muscle during arterial occlusion and after reperfusion were concurrently observed using chemical shift imaging and ‘H magnetic resonance imaging (MRI). During ischaemia. the levels of high-energy phosphates (phosphocreatine and adenosine 5′-triphosphate) were depleted and that of inorganic phosphate was increased. In addition, the signal intensity on 1H MRI was increased. These changes were observed to be relatively homogeneous throughout the calf. In contrast, the changes during reperfusion were heterogeneous. In the central part of the calf, inorganic phosphate disappeared, phosphocreatine was restored immediately on reperfusion and the ‘H signal was decreased. However, in the regions of the tibialis anterior muscle and the superficial part of the gastrocnemius muscle, inorganic phosphate persisted for several hours, phosphocreatine was not restored and the signal intensity on T2-weighted 1H MRI was increased further. The heterogeneous changes detected by 31P NMR spectroscopy and 1H MRI showed close agreement. The susceptibility of different calf muscles to ischaemia and reperfusion seems to depend on their predominant muscle fibre type (i.e. fast-twitch or slow-twitch fibres). Reversible and irreversible ischaemic changes could be non-invasively distinguished by in vivo31P NMR spectroscopy and 1H MRI.

Amyloid β (Aβ) deposition in the brain is considered the initiating event in the progression of Alzheimer's disease (AD). Amyloid imaging is widely studied in diagnosing AD and evaluating the disease stage, with considerable advances achieved in recent years. We have developed a novel ¹⁹F-containing curcumin derivative (named FMeC1) as a potential imaging agent. This compound can exist in equilibrium between keto and enol tautomers, with the enol form able to bind Aβ aggregates while the keto form cannot. This study investigated whether FMeC1 is suitable as a ¹⁹F magnetic resonance imaging (MRI) probe to detect Aβ deposition in the Tg2576 mouse, a model of AD. In ¹⁹F nuclear magnetic resonance (NMR) spectra obtained from the whole head, a delayed decreased rate of F ¹⁹F signal was observed in Tg2576 mice that were peripherally injected with FMeC1 in comparison to wild-type mice. Furthermore, ¹⁹F MRI displayed remarkable levels of ¹⁹F signal in the brain of Tg2576 mice after the injection of FMeC1. Histological analysis of FMeC1-injected mouse brain showed penetration of the compound across the blood-brain barrier and binding to Aβ plaques in peripherally injected Tg2576 mice. Moreover, the distribution of Aβ deposits in Tg2576 mice was in accordance with the region of the brain in which the ¹⁹F signal was imaged. FMeC1 also exhibited an affinity for senile plaques in human brain sections. These findings suggest the usefulness of FMeC1 as a ¹⁹F MRI probe for the detection of amyloid deposition in the brain. Furthermore, the properties of FMeC1 could form the basis for further novel amyloid imaging probes.

This paper describes the development of a medical robot that remains in the abdominal cavity in order to monitor sites of medical interest, and discusses robot travel operations and specifications. A long and narrow piece of ferromagnetic material was placed inside the robot, after which an external magnetic field was used to set the robot in motion. We developed a prototype robot, and conducted experiments in order to verify our proposed concept and the drive principles of the robot. In vivo experiments in a rabbit model showed that solenoids produced sufficient magnetic force to enable the robot to travel through the abdominal cavity, verifying the motion principles. The appropriate shape of the robot was confirmed in the experiments, and the friction between the robot and the organs and the abdominal wall was measured. Using a modified prototype of the robot, we conducted clinical experiments in the rabbit model in which a surgeon operated the XYZ axis stages in order to adjust the position of the subject for the experiment and moved the robot to the liver. Robot travel from the insertion point to the liver was verified on X-rays. The long travel distance of the robot was enabled by its improved shape and through the use of accurate magnetic field imaging.

To search for a possible relationship between brain energy metabolism and mental fatiguability, 10 normal volunteers were examined by 31P-MRS (phosphorus-31 magnetic resonance spectroscopy) in the occipital cortex during photic stimulation (PS), and Uchida-Kraepelin test (UKT), a standardized serial calculation task, was used for measurement of intraindividual characteristics of mental fatigue in these subjects. More decrease of phosphocreatine measured by 31P-MRS during the PS was significantly associated with more effects of rest assessed by UKT (r = -0.86, p0.005). More decrease in pH after PS was associated with steeper decline of performance (r = 0.78, p0.01). These findings suggest that characteristics of brain energy metabolism in an individual may relate to intrinsic patterns of mental fatigability.

The authors have previously reported decreased intracellular pH (pHi) in the frontal lobes in euthymic bipolar patients treated with lithium using 31P-MRS. White matter hyperintensity (WMHI) is frequently seen in bipolar disorder. To examine a possible effect of lithium on pHi and the relationship between pHi and WMHI, seven drug-free euthymic bipolar patients were examined, and T2-weighted MRI were examined in 14 previously reported bipolar patients. Drug-free patients showed significantly lower pHi than controls. WMHI was associated with low pHi and increased phosphodiester peak. These results suggest that decrease of pHi is not an effect of lithium but is instead related to the pathophysiology of illness. Decrease of pHi and increase of the PDE peak may be the biochemical basis of WMHI in bipolar disorder.

Effects of photic stimulation (PS) on energy metabolism were examined in the occipital cortex of 25 healthy volunteers aged 23-69 years old using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). A significant effect of photic stimulation was found only for intracellular pH (p0.05 by repeated measures analysis of variance) but not for any peak area ratios. An interaction between intracellular pH and age were statistically significant (p0.005), and the interaction between phosphocreatine and age was close to significance (p=0.06). In subjects aged more than 40 years old, phosphocreatine was significantly decreased during the photic stimulation (p0.05, multiple comparison by Dunnett's method), and intracellular pH tended to be elevated just after the stimulation (p=0.07). There were no significant changes in these values in younger subjects. These results suggest that no significant effect of photic stimulation on brain energy metabolism was found in younger subjects, and that significant effects of photic stimulation on intracellular pH and phosphocreatine were found in middle-aged subjects. Metabolic response of the human brain to photic stimulation may be dependent on age.

Today's technical status as well as future developments of clinical spectroscopic imaging were overviewed in terms of hardware and software aspects. For the hardware development, discussion was focused on superconducting magnet with high static fields, new probe designs and fast acquisition of spectroscopic imaging. Some of the technical limitation was also included. In software, pulse shape design for B1 insensitive excitation, selective signal detection utilizing homo- and hetero-nuclear coherence transfer, and post processing of acquired data were briefly discussed. Especially the processed data is important to extract chemical information for the metabolite which can be utilize in the clinical application. Some of the proposed methods were summarized for this.

The main purpose of this study was to investigate whether in vivo 31P three-dimensional chemical shift imaging(3D-CSI) of the human heart could serve as a useful tool for evaluating myocardial metabolism. We performed slice-selective 3D-CSI employing nuclear magnetic resonance(NMR) imaging with slice selection in one dimension and phase encoding in two dimensions. 31P NMR images of phosphocreatine (PCr) and ATP were superimposed on a corresponding 1H image of the heart. Our study revealed that while accurate in vivo 31P 3D-CSI measurement of the human heart is difficult to achieve it is not impossible, although various additional techniques for improving signal to noise ratio are indicated.

The relationship between the phosphoenergetic state and gluconeogenesis in the liver after ischemic damage was investigated using living rats. The ATP level was determined with in vivo 31P nuclear magnetic resonance spectroscopy, and gluconeogenesis was evaluated with in vivo 31C NMR spectroscopy using L-[3-13C]alanine as a tracer. These two measurements were alternated repeatedly. The rats were divided into three groups: without ischemia (group A); with 10 min ischemia (group B); and with 30 min ischemia (group C). ATP was depleted to 20% of the preischemic state after 10 min ischemia and this level was maintained during 30 min ischemia. After reperfusion, the ATP level was partially restored, but the recovery was smaller in group C. Infusion of [3-13C]alanine was started immediately after the reperfusion. In vivo 13C NMR disclosed changes in the alanine C3, glutamine/glutamate C2 and C3, glucose C1-6, and glycogen C1 signals in the liver. After 60 min infusion of [3-13C]alanine, the ATP level correlated negatively with the signal intensity of alanine (r = -0.664, p = 0.008) and positively with those of glucose and glyogen (r = 0.586, p = 0.023, and r = 0.643, p = 0.011, respectively). These results suggest that the ATP level participates in gluconeogenesis and glycogenesis in the liver. Such multinuclear in vivo NMR observations might uncover new aspects of the metabolic function of the liver in the in vivo state.

We studied the subcellular mechanisms of the negative force-frequency relationship in rat myocardium by measuring 1) intracellular Ca2+ transients by indo-1 fluorometry and 2) intracellular pH (pHi) and phosphate compounds with 31P-nuclear magnetic resonance (NMR). The data were compared with those from guinea pig hearts, which show a positive force-frequency relationship. By increasing the pacing rate from 3 Hz to 5 Hz, the peak positive first derivative of left ventricular pressure (LVdP/dt) in rat heart decreased by 10 +/- 1% (n = 6). In contrast to this negative inotropic response, simultaneously measured peak Ca2+ transients increased by 6 +/- 1%. Guinea pig heart (n = 6) showed an increase in peak positive LVdP/dt (33 +/- 1%) which was associated with an increase in peak Ca2+ transients (8 +/- 1%). Under equivalent experimental conditions in an NMR spectrometer, this increase in the pacing rate did not affect intracellular levels of phosphate compounds in either rat (n = 6) or guinea pig heart (n = 6). In contrast, pHi showed a decrease of 0.031 +/- 0.006 pH units in rat heart, while no changes were observed in guinea pig heart. These results suggest that in physiological rat myocardium, pHi is susceptible to changes in the stimulus frequency and may affect the Ca(2+)-responsiveness of contractile proteins, which results in the negative force-frequency relationship.

Choline-containing compounds (Cho) were examined by proton magnetic resonance spectroscopy (1H-MRS) in the left subcortical region, including basal ganglia, in 19 euthymic patients with bipolar disorder and 19 age-matched normal controls. Ten of the patients were treated with lithium; the remaining 9 were not treated with lithium for at least 30 d. The Cho to creatine + phosphocreatine (Cr) peak ratio in the bipolar patients (0.75 +/- 0.38 [mean +/- SD]) was higher than that in the normal controls (0.52 +/- 0.26, P < 0.05). There was no significant difference in the Cho:Cr peak ratio between patients treated with lithium (0.63 +/- 0.36) and without lithium (0.89 +/- 0.35). These results do not support the hypothesis that lithium increases the brain choline-containing compounds, but rather imply that membrane breakdown may occur in the basal ganglia of patients with bipolar disorder.

The activities of cathepsin B, L, J and H in rat liver were significantly increased by starvation if compared with normal diet rats. Furthermore, the activity of cathepsin L increased with glucagon treatment, and the activities of cathepsin L and H decreased significantly with insulin treatment. The changes in cathepsin B and J activities showed the same tendencies as those of cathepsin L and H, but the differences were not statistically significant. The changes in the activities of cathepsin B and L on starvation corresponded with the changes of enzyme protein amounts judged from Western blotting analysis. The levels of the lysosomal cysteine proteinases and amino acid deaminases in the liver changed in parallel with the hormonal and dietary conditions. The increases of alanine amino transferase activity (AAT) started from a much earlier stage than those of cathepsins under the starvation condition. Although administration of prednisolone caused marked induction of the deamination enzymes such as AAT, the levels of cathepsins in the liver were not changed.

PURPOSE: To investigate the early changes in diffusion-weighted MR images in the sustained limbic seizures. METHOD: Intraperitoneal injection of kainic acid was used to induce sustained limbic seizures in seven rats. The animals were investigated with serial 2.0-T MR imaging beginning immediately after kainic acid-induced seizures, and at 24 hours, 3 days, and 7 days after the kainic acid injection. Diffusion-weighted spin-echo and T2-weighted images and apparent diffusion coefficients were sequentially assessed and compared with histologic changes. The results were compared with eight control animals given buffered saline intraperitoneally. RESULTS: Diffusion-weighted MR images revealed an increase in signal intensity bilaterally in the amygdala and the piriform cortices immediately after the sustained seizures, whereas T2-weighted images did not show changes in signal intensity at this time. Both diffusion-weighted and T2-weighted images showed marked increase in signal intensities in these same areas 24 hours after kainic acid injection. The apparent diffusion coefficient values were significantly lower in the area of the amygdala and the piriform cortex immediately after and lower again 24 hours after the sustained seizures. The area of hyperintensity in diffusion-weighted images was concordant with the histologic distribution of neuronal pyknosis and neuropile vacuolation. CONCLUSION: Diffusion-weighted MR revealed focal abnormalities in the limbic system after 1 hour of sustained seizures induced with kainic acid, before changes on T2-weighted imaging. Diffusion-weighted MR is a potential method for studying the mechanisms of brain damage caused by sustained seizures.

Lithium concentrations in the brain were measured in 14 manic patients with bipolar disorder (12 with bipolar disorder, manic, and 2 with bipolar disorder, not otherwise specified, by DSM-III-R) by the use of lithium-7 magnetic resonance spectroscopy ([7Li]MRS). The reduction of Petterson Mania Rating Scale 4 weeks after the initiation of lithium treatment was not significantly correlated with concentrations in serum (r = 0.33), but was correlated with concentrations in the brain (r = 0.64; p0.05). These results suggest that the treatment response to lithium in manic patients with bipolar disorder is more closely related to the lithium concentration in the brain as measured by [7Li]MRS than to the concentration in serum.

We obtained 202.5-MHz 31P-nuclear magnetic resonance (NMR) spectra of isolated perfused rat lungs, degassed and inflated, and of lung extract. The spectra included those of ATP, ADP, phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters, phosphodiesters, and a broad component due to the membrane phospholipids. The line width at one-half peak height for beta-ATP was 1.0 ppm for the degassed lung and 1.2 ppm for the inflated lung. This suggests that the air-water interfaces in inflated lung, which produce proton NMR line broadening, do not act prominently in 31P-NMR spectroscopy. In a degassed lung, when perfusion was stopped for up to 30 min, PCr and ATP peaks decreased progressively with time while Pi and phosphomonoester peaks increased. On return of flow, these changes reversed. The intracellular pH values calculated from the difference in magnetic field between PCr and Pi peaks of inflated and degassed lungs were 7.16 +/- 0.10 (SD; n = 4) and 6.99 +/- 0.10 (n = 4), respectively. The change of intracellular pH caused by 30 min of ischemia was -0.2 pH units. Our findings indicate that air-water interfaces should not broaden lung 31P peaks in vivo.

Effects of calcium antagonists, Nicardipine and Nilvadipine, on neurologic deficits and size of infarct were studied in the rat middle cerebral artery (MCA) occlusion model. Each drug was administered immediately after induction of ischemia, and neurologic grade was evaluated 1 to 24 hours after MCA occlusion. At 24 hours post-occlusion, size of the infarct was compared with that of the control group. In addition, evolution of cerebral infarction was studied at 6 hours and 12 hours post-occlusion by magnetic resonance imaging (MRI). In the Nilvadipine-treated group, neurologic deficits improved more rapidly and the size of infarct was significantly smaller than in the Nicardipine-treated group. MRI showed a progressive extension of cortical infarct in the untreated rat, whereas the infarct size remained unchanged in the Nilvadipine-treated rat. These results suggest the potential therapeutic usefulness of calcium antagonist for acute cerebral ischemia.

A hospital-based case-control study was conducted at 8 hospitals in Osaka in order to clarify risk factors among non-smoking women. A total of 129 lung cancer cases and 519 controls were investigated. An elevated risk was observed for women who had used wood or straw as cooking fuels at 30 years of age. Maternal smoking in childhood also seemed to raise the risk. No elevated risks were observed for husband smoking and use of heating apparatus so far.

Water-suppressed proton nuclear magnetic resonance spectra were generated (by using 360 and 500 MHz systems) from human plasma and serum samples taken from 35 apparently healthy individuals, 52 patients with overt malignancies, and 37 patients with hypertriglyceridemia (triglycerides greater than 200 mg/dL or 2.26 mmol/L). The line widths from the lipoprotein-lipid methylene and methyl resonances at approximately 1.3 and 0.9 ppm were averaged by the method of Fossel et al. (N Engl J Med 1986;315:1369-76), but, contrary to their findings, we were unable to distinguish normal individuals from those with malignant tumors (e.g., mean +/- SD line width at 360 MHz: normal group = 32.9 +/- 3.6 Hz, malignant group = 28.3 +/- 4.9 Hz). The average line-width measurements (y), however, varied with the triglyceride content (x, mg/dL) of the plasma or serum as follows (logarithmic transformation of the data determined at 360 MHz and regression analysis): y = 110 (x-0.27). Data from both nonmalignant and malignant specimens fit this equation, the coefficient of correlation being -0.91. These findings suggest that considerable caution should be used in interpreting water-suppressed proton NMR spectra for cancer detection.

5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.

Transition metal ions, Mn2+ and Co2+, are incorporated into nerve fibers when they are applied externally. For nerve fibers in the resting state, however, extracellular and intracellular water may be distinguished by applying transition metal ions externally. NMR spectra of water protons from nerve fibers in high potassium media, which contain transition metal ions, consist of three or more components, reflecting a complex distribution of these ions around the nerve membranes. In the case of Co2+, three components may be identified.

1H and 31P NMR spectra of a variety of phosphorylated myo-inositols have been analyzed using a Bruker WH-360 spectrometer. Proton and phosphorus chemical shifts and coupling constants are reported for myo-inositol 1-phosphate, myo-inositol 2-phosphate, myo-inositol 5-phosphate, myo-inositol 1,2-cyclic phosphate, myo-inositol 1,4-bisphosphate, myo-inositol 1,4,5-trisphosphate, and myo-inositol 1,3,4,5-tetrakisphosphate. These data provide the basis for the chemical identification and characterization of biologically relevant inositol phosphates.

220 MHz proton Fourier transform (FT) NMR with quadrature phase detection (QPD) technique is applied to observe largely hyperfine-shifted signals of various hemoproteins and hemoenzymes in ferric high-spin state. The binding of F-, OCN-, SCN-, and CH3OH to the ferric heme iron in high-spin state in various hemoproteins has been studied by the use of FT/QPD technique at 220 MHz. The binding of formate ion to metmyoglobin (metMb) has also been studied. The spectrum of the formate complex was compared with that of hemoglobin M Milwaukee where carboxylate groups are bound to the hemes of the beta subunits. The acid-base transition of ferric myoglobin (Mb) was confirmed by monitoring the pH-dependent shift of the heme side methyl signals with the reflection point at pH 9.1. This finding is analyzed on the basis of rapid exchange between alkaline (low spin) and acidic (high spin) forms accompanied by the dissociation and association of one proton in the ferric Mb. The structure of the heme environment of ferric horseradish peroxidase (HRP) was studied. The pH-dependent features of NMR spectra of the ferric enzyme and its complexes with cyanide and azide were discussed in terms of heme environmental structures, comparing with the case of metMb. The results were interpreted as follows: There exists an ionizable amino group near the heme responsible for the ligand binding reactions of the enzyme, which modulates the entry of external azide to the heme iron through protolytic equilibrium of this group. The pK value of this group was determined to be 5.9 by monitoring the pH-dependent shift of the heme peripheral methyl signals of the native enzyme, indicating that the group is probably a histidyl residue. Acid-alkaline transition of metMb was confirmed to associate with the proton dissociation of an iron-bound water molecule, whereas in HRP, pH-dependent spin state change characterized by pK 11 is attributed not to the simple protolytic reaction of the iron-bound water but to the direct coordination of an amino acid residue of the polypeptide chain to the ferric heme iron. Histidyl imidazole is a possible candidate for the new sixth iron ligand in alkaline peroxidase above pH 11. Interaction of HRP with electron donor(indolepropionic acid, IPA) was also studied. The hyperfine-shifted proton signals of the heme peripheral groups of the enzyme showed a small but significant shift with stepwise additions of IPA, indicating that the donor binds at a specific site of HRP. There results are interpreted in terms of the interaction between the enzyme and the donor at the heme edge site.

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.

5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.