STAT6 and IL-10 are required for the anti-arthritic effects of Schistosoma mansoni via different mechanisms.

To investigate possible roles of T helper type 2 (Th2) cytokines in the anti-arthritic effects of a blood fluke, Schistosoma mansoni (Sm), for mouse collagen-induced arthritis (CIA), wild-type (WT), signal transducer and activator of transcription 6 (STAT6) knock-out (KO) and interleukin (IL)-10 KO mice were infected with Sm. Three weeks after infection, the mice were immunized with bovine type II collagen (IIC). Arthritis severity was monitored by scoring, measurement of paw thickness and the presence of ankylosis. Serum anti-IIC IgG levels, splenic cytokine production and cytokine gene expression in the popliteal lymph nodes (PLNs) were measured and compared among WT and gene-KO mice. Consistent with our previous findings, Sm infection reduced the arthritis severity in WT mice. Splenic production of IL-17A and tumor necrosis factor (TNF)-α was reduced by the infection. In contrast, Sm infection markedly exacerbated CIA in STAT6 KO mice. In the KO mice, IL-17A production was increased by the infection. Conversely, Sm infection did not affect the exacerbated arthritis in IL-10 KO mice, although IL-17A production was reduced by the helminth. Our results suggest that signaling via STAT6 (presumably IL-4 and/or IL-13) and IL-10 is required for the suppression of CIA by Sm infection, but through different mechanisms. STAT6 was essential for helminth-induced reduction of IL-17A, whereas regulation of the basal arthritis severity by IL-10 was needed in order for it to be sufficiently suppressed by the helminth.

Variations of the cephalic vein anterior to the clavicle in humans.

BACKGROUND: Clinicians should understand that jugulocephalic vein (JCV) variants may be occasionally found. This study aims to classify JCV variants and obtain their frequency. MATERIALS AND METHODS: We investigated anatomical variants of the cephalic vein in 55 human cadavers during a gross anatomy course at our medical school. RESULTS: The percentage of JCVs that pass through the anterior part of the clavicle and anastomose to the jugular vein as per previous studies and our study was 2-5%. Five cases with anastomosis between the cephalic and external jugular veins that pass through the anterior part of the clavicle were found. The courses were classified into 1A, 1B, 2A, and 2B. Type 1 extends beyond the clavicle and anastomoses with the external jugular vein. Type 2 follows the same course as type 1, but anastomoses with the subclavian vein. Subtype A does not have a branch that anastomoses with the axillary vein, whereas subtype B does. We encountered two cases of type 1A and three of type 1B. CONCLUSIONS: Four anatomical variants of the cephalic vein around the clavicle were identified. Clinicians' knowledge of these variants is expected to decrease possible complications if venous access via the cephalic vein is needed.

A splicing-regulatory polymorphism in DRD2 disrupts ZRANB2 binding, impairs cognitive functioning and increases risk for schizophrenia in six Han Chinese samples.

The rs1076560 polymorphism of DRD2 (encoding dopamine receptor D2) is associated with alternative splicing and cognitive functioning; however, a mechanistic relationship to schizophrenia has not been shown. Here, we demonstrate that rs1076560(T) imparts a small but reliable risk for schizophrenia in a sample of 616 affected families and five independent replication samples totaling 4017 affected and 4704 unaffected individuals (odds ratio=1.1; P=0.004). rs1076560(T) was associated with impaired verbal fluency and comprehension in schizophrenia but improved performance among healthy comparison subjects. rs1076560(T) also associated with lower D2 short isoform expression in postmortem brain. rs1076560(T) disrupted a binding site for the splicing factor ZRANB2, diminished binding affinity between DRD2 pre-mRNA and ZRANB2 and abolished the ability of ZRANB2 to modulate short:long isoform-expression ratios of DRD2 minigenes in cell culture. Collectively, this work implicates rs1076560(T) as one possible risk factor for schizophrenia in the Han Chinese population, and suggests molecular mechanisms by which it may exert such influence.

Common and separate origins of the left and right inferior phrenic artery with a review of the literature.

In a 94-year-old male cadaver, upon which routine dissection was being conducted, a rare variation was found in the gastrophrenic trunk (GPT), the common trunk of the left gastric artery (LGA), right inferior phrenic artery (RIPA), and left inferior phrenic artery (LIPA); the GPT arises from the abdominal aorta. A hepatosplenic trunk accompanied the variation. In this variation, the RIPA first branched from the GPT and then to the LIPA and LGA. Variations in the common trunk of the LIPA and RIPA in the GPT are common, but to our knowledge, a variation (separate inferior phrenic artery in the GPT) similar to our findings has not been previously reported. We discuss the incidence and developmental and clinical significance of this variation with a detailed review of the literature. Knowledge of such a case has important clinical significance for invasive and non-invasive arterial procedures. Therefore, different variations concerning the LGA and inferior phrenic artery should be considered during surgical and non-surgical evaluations.

Use of smartphone attached mobile thermography assessing subclinical inflammation: a pilot study.

OBJECTIVE: To verify the reliability and validity of FLIR ONE, a device connected to a smartphone, for the assessment of inflammation based on relative temperature increase compared with the thermography routinely used in pressure ulcer (PU) and diabetic foot assessment. METHOD: Participants in this pilot cross-sectional observational study were recruited from the patients in the PU team rounds and the diabetic foot outpatient clinic at the university hospital in January 2015. Cohen's kappa coefficient with its 95% confidence intervals was used to evaluate the criterion-related validity and inter- and intra-rater reliability for the thermal imaging assessment. For assessing criterion-related validity, a hand-held high-end infrared thermography device was used to provide reference data. Comparison of thermal images between the smartphone-connected device and the hand-held device was performed with both a 'predetermined range' and an 'automatically-set range.' For assessing inter-rater reliability, two assessors evaluated the thermal images taken by the mobile thermography. For assessing intra-rater reliability, one assessor evaluated the thermal images twice. The thermal images were shown to the assessors at random. RESULTS: Among 16 thermal images obtained from eight patients, kappa coefficients for each value were as follows: for the predetermined range and automatically-set range, respectively, the criterion-related validity was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00); the inter-rater reliability was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00); and the intra-rater reliability was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00). CONCLUSION: This pilot study suggests that FLIR ONE can work as an alternative device for assessing subclinical inflammation in PUs and the diabetic foot in clinical settings. Our results may facilitate clinicians in accepting the routine use of thermal imaging assessment at the patients' bedside.

Species differences in the metabolism of ritobegron in vitro and assessment of potential interactions with transporters and cytochrome P450 enzymes.

Ritobegron, a selective ß3-adrenoceptor agonist, is the prodrug of the active compound, KUC-7322. We investigated species differences in its metabolism in vitro and the potential for drug-drug interactions with ritobegron. In rat, dog, monkey, and human liver microsomes, ritobegron was not metabolized by cytochrome P450 enzymes (CYPs). KUC-7322 was the only metabolite observed. Hydrolysis of ritobegron to KUC-7322 was likely catalyzed by carboxylesterases in human liver microsomes. The maximum velocity of the reaction (V(max))/Michaelis-Menten constant (K(m)) for hydrolysis of ritobegron to KUC-7322 was much higher in rat serum than those in other species. There were also species differences in the conjugation of KUC-7322. Sulfate conjugates of ritobegron were detected in all species, whereas glucuronide and glutathione conjugates of KUC-7322 were only observed in rat liver subcellular fractions. Ritobegron and KUC-7322 did not affect the CYP-mediated metabolism of probe substrates in human liver microsomes and organic anion transporter 1 (OAT1)-, OAT2-, OAT3-, organic cation transporter 2 (OCT-2)-, OCT3-, or organic cation/carnitine transporter 1 (OCTN1)-mediated uptake of probe substrates in S2 cells. Ritobegron, but not KUC-7322, inhibited P-glycoprotein-mediated digoxin transport in Caco-2 cells. Significant uptake of KUC-7322 was observed in OAT3-expressing S2 cells. Therefore, CYP-mediated drug-drug interactions are not likely when ritobegron is administered with CYP substrates or inhibitors. Ritobegron may increase the plasma concentrations of P-glycoprotein substrates, such as digoxin, and the plasma concentration of KUC-7322 may increase when it is administered in combination with OAT inhibitors such as probenecid.

Anomalous connection of the left posterior renal vein with the left ascending lumbar vein in a Japanese cadaver.

A rare variation was found in one of the two left renal veins in a 94-year-old male cadaver undergoing routine dissection. The characteristic findings in the cadaver included, in addition to the primary left renal vein, the presence of a posterior left renal vein draining to the left ascending lumbar vein without communicating with the inferior vena cava and other renal veins. Variations in the number and arrangement of the vessels terminating in the renal veins are common, but to our knowledge, variation similar to our findings has not been previously reported. This variation may represent an immature form of the complicated development of the renal vessels.

Sphenoparietal sinus and superficial middle cerebral vein thrombosis: A case report and review of literature.

BACKGROUND: Cerebral venous sinus thrombosis is rare and might be overlooked by healthcare providers. It often occurs in the transverse sinuses, superior sagittal sinus, and the vein of Trolard. Sphenoparietal sinus (SPS) and/or superficial middle cerebral vein (SMCV) thrombosis is rare and only 12 cases reported in the literature. CASE DESCRIPTION: We report a 47-year-old woman with iron deficiency anemia associated with myoma uteri who developed left SPS and SMCV thrombosis. She presented with sudden unconsciousness, right hemiplegia, and aphasia. Brain computed tomography showed subcortical hemorrhages in the left frontal and temporal lobes. Magnetic resonance imaging did not reveal the cause of the bleeding. Although antihypertensive treatment with nicardipine was initiated, she deteriorated into coma the next day and underwent emergency decompressive craniectomy. Thrombosis of the SMCV was identified during surgery. Re-examination of preoperative T2 star-weighted imaging revealed thrombosis of the SPS and SMCV. CONCLUSION: All but one of the reviewed cases had the thrombosis develop on the left side, which may be attributed to anatomical and brain functional laterality. When an edematous change or cortical hemorrhage of unknown cause is encountered within the perisylvian region, especially on the left side, the possibility of SPS and SMCV thrombosis should be considered.

Analysis of circulating microRNA during early gestation in Japanese black cattle.

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Unmyelinated axons are more vulnerable to degeneration than myelinated axons of the cardiac nerve in Parkinson's disease.

AIMS: We recently demonstrated accumulation of α-synuclein aggregates of the cardiac sympathetic nerve in Parkinson's disease (PD) and a possible relationship between degeneration of the cardiac sympathetic nerve and α-synuclein aggregates. The aim of this study is to determine whether there is a difference in the degenerative process between unmyelinated and myelinated axons of the cardiac nerve. METHODS: We immunohistochemically examined cardiac tissues from four pathologically verified PD patients, nine patients with incidental Lewy body disease (ILBD) and five control subjects, using antibodies against neurofilament, myelin basic protein (MBP) and α-synuclein. First, we counted the number of neurofilament-immunoreactive axons not surrounded by MBP (unmyelinated axons) and those surrounded by MBP (myelinated axons). Next, we counted the number of unmyelinated and myelinated axons with α-synuclein aggregates. RESULTS: (i) The percentage of unmyelinated axons in PD (77.5 ± 9.14%) was significantly lower compared to that in control subjects (92.2 ± 2.40%). (ii) The ratio of unmyelinated axons with α-synuclein aggregates to total axons with α-synuclein aggregates in ILBD ranged from 94.4 to 100 (98.2 ± 2.18%). Among axons with α-synuclein aggregates, unmyelinated axons were the overwhelming majority, comprising 98.2%. CONCLUSION: These findings suggest that in PD unmyelinated axons are more vulnerable to degeneration than myelinated axons of the cardiac nerve, because α-synuclein aggregates accumulate much more abundantly in unmyelinated axons.

Family-based association testing strongly implicates DRD2 as a risk gene for schizophrenia in Han Chinese from Taiwan.

The gene that codes for dopamine receptor D2 (DRD2 on chromosome 11q23) has long been a prime functional and positional candidate risk gene for schizophrenia. Collectively, prior case-control studies found a reliable effect of the Ser311Cys DRD2 polymorphism (rs1801028) on risk for schizophrenia, but few other polymorphisms in the gene had ever been evaluated and no adequately powered family-based association study has been performed to date. Our objective was to test 21 haplotype-tagging and all three known nonsynonymous single-nucleotide polymorphisms (SNPs) in DRD2 for association with schizophrenia in a family-based study of 2408 Han Chinese, including 1214 affected individuals from 616 families. We did not find a significant effect of rs1801028, but we did find significant evidence for association of schizophrenia with two multi-marker haplotypes spanning blocks of strong linkage disequilibrium (LD) and nine individual SNPs (Ps<0.05). Importantly, two SNPs (rs1079727 and rs2283265) and both multi-marker haplotypes spanning entire LD blocks (including one that contained rs1801028) remained significant after correcting for multiple testing. These results further add to the body of data implicating DRD2 as a schizophrenia risk gene; however, a causal variant(s) in DRD2 remains to be elucidated by further fine mapping of the gene, with particular attention given to the area surrounding the third through fifth exons.

Inhibition of cytokinesis by a lipid metabolite, psychosine.

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

Galanin and galanin receptor type 1 suppress proliferation in squamous carcinoma cells: activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors.

Galanin receptor 1 (GALR1) maps to a common region of 18q loss in head and neck squamous cell carcinomas and is frequently inactivated by methylation. To investigate effects of GALR1 and its signaling pathways, we stably expressed hemaglutinin-tagged GALR1 in a human oral carcinoma cell line (UM-SCC-1-GALR1) that expresses no endogenous GALR1. In transfected cells, galanin induced activation of the extracellular-regulated protein kinase-1/2 (ERK1/2) and suppressed proliferation. Galanin stimulation mediated decreased expression of cyclin D1 and increased expression of the cyclin-dependent kinase inhibitors (CKI), p27(Kip1) and p57(Kip2). Pretreatment with the ERK1/2-specific inhibitor U0126 prevented these galanin-induced effects. Phosphatidylinositol 3-kinase (PI3K) pathway activation did not differ in UM-SCC-1-GALR1 and UM-SCC-1-mock cells after galanin treatment. Pertussis toxin and LY294002 inhibition demonstrated that galanin and GALR1 induce ERK1/2 activation via Galphai, not the PI3K pathway-linked to the Gbetagamma subunit. Galanin and GALR1 also inhibit colony formation and tumor growth in vivo. Our results implicate GALR1, a Gi protein-coupled receptor, as a tumor suppressor gene that inhibits cell proliferation via ERK1/2 activation.

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine.

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.

Application of portable near-infrared spectrometer to Heliotron J plasma diagnostics.

A simple near-infrared (NIR) spectrometer with a wavelength range of 898-2130 nm has recently been applied to diagnose Heliotron J plasmas. It adopts a symmetrical crossed Czerny-Turner mount equipped with a thermoelectrically cooled 512 channel InGaAs linear sensor. Reciprocal linear dispersion was deduced to 96.37 nm/mm at the center of the detector. External filters can be inserted into the path of the collection optics to reject second-order spectra, as needed. Absolute intensity calibration was performed together with a visible spectrometer using a tungsten halogen lamp, and the effect of the transmittance fringe in the visible region of the applied long-pass filter on the NIR calibration was investigated. The intended application of the NIR spectrometer includes extending the wavelength region of a spectral monitor to less contaminated regions for Heliotron J plasma studies. In preliminary measurements, we observed the Paschen series for the hydrogen pellet injection plasma and two atomic helium lines, i.e., 2S-2P singlet and triplet lines, in helium gas puffing experiments. A continuum spectrum in this regime that is attributable to black-body radiation from hot spots on the plasma-facing components was identified. In addition, this may also be used to monitor background radiation in the YAG-Thomson scattering signals near 1064 nm.

Three-dimensional finite element model to predict patterns of pterygomaxillary dysjunction during Le Fort I osteotomy.

The aim of this study was to determine whether non-linear three-dimensional finite element analysis (3D-FEA) can be applied to simulate pterygomaxillary dysjunction during Le Fort I osteotomy (LFI) not involving a curved osteotome (LFI-non-COSep), and to predict potential changes in the fracture pattern associated with extending the cutting line. Computed tomography (CT) image data (100 snapshots) after LFI were converted to 3D-CT images. 3D-FEA models were built using preoperative CT matrix data and used to simulate pterygomaxillary dysjunction. The pterygomaxillary dysjunction patterns predicted by the 3D-FEA models of pterygomaxillary dysjunction were classified into three categories and compared to the pterygomaxillary dysjunction patterns observed in the postoperative 3D-CT images. Extension of the cutting line was also simulated using the 3D-FEA models to predict the risk and position of pterygoid process fracture. The rate of agreement between the predicted pterygomaxillary dysjunction patterns and those observed in the postoperative 3D-CT images was 87.0% (κ coefficient 0.79). The predicted incidence of pterygoid process fracture was higher for cutting lines that extended to the pterygomaxillary junction than for conventional cutting lines (odds ratio 4.75; P<0.0001). 3D-FEA can be used to predict pterygomaxillary dysjunction patterns during LFI-non-COSep and provides useful information for selecting safer procedures during LFI-non-COSep.

An immunohistological study of the integration at the bone-tendon interface after reconstruction of the anterior cruciate ligament in rabbits.

We studied bone-tendon healing using immunohistochemical methods in a rabbit model. Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification.

Characterization of the phosphoenzyme that is involved in the Ca2+ -Ca2+ exchange catalyzed by the Ca2+ -ATPase of sarcoplasmic reticulum vesicles.

The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange.

Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site.

The sarcoplasmic reticulum Ca(2+)-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg2+ and absence of Ca2+. This inactivation was Mg(2+)-dependent, and prevented by high-affinity Ca2+ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg2+ in this inactivation was in agreement with that for Mg2+ in phosphorylation of the enzyme with Pi. Mg.ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg2+. The enzyme was completely protected by Pi against the inactivation with an affinity consistent with that of the catalytic site for Pi. The inactivated enzyme showed neither phosphorylation with Pi nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F-. The contents of tightly-bound Mg2+ and F- in the inactivated enzyme were determined after unbound Mg2+ and F- were removed by gel filtration. 2.3 mol of Mg2+ and 3.7 mol of F- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg2+ and F- were entirely released by low-affinity Ca2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg2+ and F- to the catalytic site.

Characterization of the substrate-induced conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2(+)-ATPase by using different kinds of substrate.

Cys-674 of the sarcoplasmic reticulum Ca2(+)-ATPase was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. When Ca2+ was present, the fluorescence intensity and anisotropy decreased greatly upon addition of any substrate used. The observed affinity for each substrate agreed with the previously observed affinity of the catalytic site. The fluorescence drop induced by the adenine nucleotides, ATP and adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable ATP analog), was much faster than that induced by other substrates. The ATP-induced fluorescence drop preceded phosphoenzyme formation when the ATP concentration was high, but the fluorescence drop coincided with phosphoenzyme formation when it was slowed by reducing ATP concentrations. The fluorescence drop induced by ITP or acetyl phosphate was slow even at high concentrations of the substrate, and it coincided with phosphoenzyme formation. When Ca2+ was absent, the fluorescence intensity and anisotropy decreased only slightly upon addition of any substrate other than the adenine nucleotides. They decreased substantially upon addition of the adenine nucleotides, but the kinetics of this fluorescence drop were quite different from that of the fluorescence drop induced by any substrate in the presence of Ca2+. These results show that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca2(+)-activated enzyme. This change precedes phosphoenzyme formation in the catalytic cycle and is greatly accelerated by the adenine moiety of the substrate.