Changes in resting-state brain networks after cognitive-behavioral therapy for chronic pain.

BACKGROUND: Cognitive-behavioral therapy (CBT) is thought to be useful for chronic pain, with the pathology of the latter being closely associated with cognitive-emotional components. However, there are few resting-state functional magnetic resonance imaging (R-fMRI) studies. We used the independent component analysis method to examine neural changes after CBT and to assess whether brain regions predict treatment response. METHODS: We performed R-fMRI on a group of 29 chronic pain (somatoform pain disorder) patients and 30 age-matched healthy controls (T1). Patients were enrolled in a weekly 12-session group CBT (T2). We assessed selected regions of interest that exhibited differences in intrinsic connectivity network (ICN) connectivity strength between the patients and controls at T1, and compared T1 and T2. We also examined the correlations between treatment effects and rs-fMRI data. RESULTS: Abnormal ICN connectivity of the orbitofrontal cortex (OFC) and inferior parietal lobule within the dorsal attention network (DAN) and of the paracentral lobule within the sensorimotor network in patients with chronic pain normalized after CBT. Higher ICN connectivity strength in the OFC indicated greater improvements in pain intensity. Furthermore, ICN connectivity strength in the dorsal posterior cingulate cortex (PCC) within the DAN at T1 was negatively correlated with CBT-related clinical improvements. CONCLUSIONS: We conclude that the OFC is crucial for CBT-related improvement of pain intensity, and that the dorsal PCC activation at pretreatment also plays an important role in improvement of clinical symptoms via CBT.

Intermittent pneumatic compression versus additional prophylaxis with enoxaparin for prevention of venous thromboembolism after laparoscopic surgery for gastric and colorectal malignancies: multicentre randomized clinical trial.

BACKGROUND: The role of antithrombotic chemoprophylaxis in prevention of venous thromboembolism (VTE) in laparoscopic surgery for gastric and colorectal malignancies is unknown. This study compared the addition of enoxaparin following intermittent pneumatic compression (IPC) with IPC alone in patients undergoing laparoscopic surgery for gastrointestinal malignancy. METHODS: In this multicentre RCT, eligible patients were older than 40 years and had a WHO performance status of 0 or 1. Exclusion criteria were prescription of antiplatelet or anticoagulant drugs and history of VTE. Patients were allocated to IPC or to ICP with enoxaparin in a 1 : 1 ratio. Stratification factors included sex, location of cancer, age 61 years and over, and institution. Enoxaparin was administered on days 1-7 after surgery. Primary outcome was VTE, evaluated by multidetector CT on day 7. RESULTS: Of 448 patients randomized, 208 in the IPC group and 182 in the IPC with enoxaparin group were evaluated. VTE occurred in ten patients (4·8 per cent) in the IPC group and six (3·3 per cent) in the IPC with enoxaparin group (P = 0·453). Proximal deep vein thrombosis and/or pulmonary embolism occurred in seven patients (3·4 per cent) in the IPC group and one patient (0·5 per cent) in the IPC with enoxaparin group (P = 0·050). All VTE events were asymptomatic and non-fatal. Bleeding occurred in 11 of 202 patients in the IPC with enoxaparin group, and one patient needed a transfusion. All bleeding events were managed by discontinuation of the drug. CONCLUSION: IPC with enoxaparin after laparoscopic surgery for gastric and colorectal malignancies did not reduce the rate of VTE. Registration number: UMIN000011667 ( https://www.umin.ac.jp/).

ANTECEDENTES: El papel de la quimioprofilaxis para la prevención del tromboembolismo venoso (venous thromboembolism, VTE) en la cirugía laparoscópica de los tumores malignos gástricos y colorrectales se desconoce. El objetivo de este estudio fue comparar la quimioprofilaxis antitrombótica (enoxaparina) y la compresión neumática intermitente (intermittent pneumatic compression, IPC) en pacientes sometidos a cirugía laparoscópica de tumores malignos abdominales. MÉTODOS: Se efectuó un ensayo aleatorizado, controlado y multicéntrico de pacientes sometidos a cirugía laparoscópica de tumores gástricos y colorrectales en Japón. Los criterios de inclusión eran pacientes mayores de 40 años de edad y con un estado funcional de WHO de 0-1. Los criterios de exclusión fueron la prescripción al paciente de fármacos antiagregantes o anticoagulantes y la historia de VTE. Los pacientes fueron asignados a IPC y ICP con la adición de enoxaparina en una relación 1:1. Los factores de estratificación incluyeron el sexo, la localización del cáncer, la edad mayor o menor de 61 años, y la institución. La enoxaparina fue administrada en los días postoperatorios (postoperative day, POD) 1-7. El resultado primario fue la VTE evaluada mediante tomografía computarizada multidetector en el POD7. Los cálculos de la potencia determinaron que se requerían 184 pacientes en cada grupo. RESULTADOS: De los 448 pacientes aleatorizados, se evaluaron finalmente 208 pacientes en el grupo IPC y 182 pacientes en el grupo IPC más enoxaparina. La VTE ocurrió en 10 de 208 pacientes en el grupo IPC (4,8%) y 6 de 182 pacientes en el grupo IPC más enoxaparina (3,3%) (P = 0,45). La trombosis venosa profunda proximal (proximal deep vein thrombosis, DVT) y/o el embolismo pulmonar (pulmonary embolism, PE) ocurrieron en 7 de 208 pacientes en el grupo IPC (3,4%) y 1 de 182 pacientes en el grupo IPC más enoxaparina (0,55%) (riesgo relativo 0,163, i.c. del 95% 0,020-1,314, P = 0,0503). Todos los eventos de VTE fueron asintomáticos y no mortales. Se produjo una hemorragia en 11 de 202 pacientes en el grupo IPC con enoxaparina (5,4%, i.c. del 95% 3,1%-9,5%, P < 0,001), y un paciente precisó transfusión. Todos los eventos hemorrágicos pudieron ser tratados con la interrupción del fármaco. CONCLUSIÓN: La IPC con la adición de enoxaparina tras cirugía laparoscópica de los tumores malignos gástricos y colorrectales no disminuye la VTE.

Impact of technically qualified surgeons on laparoscopic colorectal resection outcomes: results of a propensity score-matching analysis.

BACKGROUND: The Endoscopic Surgical Skill Qualification System (ESSQS) was introduced in Japan to improve the quality of laparoscopic surgery. This cohort study investigated the short- and long-term postoperative outcomes of colorectal cancer laparoscopic procedures performed by or with qualified surgeons compared with outcomes for unqualified surgeons. METHODS: All laparoscopic colorectal resections performed from 2010 to 2013 in 11 Japanese hospitals were reviewed retrospectively. The procedures were categorized as performed by surgeons with or without the ESSQS qualification and patients' clinical, pathological and surgical features were used to match subgroups using propensity scoring. Outcome measures included postoperative and long-term results. RESULTS: Overall, 1428 procedures were analysed; 586 procedures were performed with ESSQS-qualified surgeons and 842 were done by ESSQS-unqualified surgeons. Upon matching, two cohorts of 426 patients were selected for comparison of short-term results. A prevalence of rectal resection (50·3 versus 40·5 per cent; P < 0·001) and shorter duration of surgery (230 versus 238 min; P = 0·045) was reported for the ESSQS group. Intraoperative and postoperative complication and reoperation rates were significantly lower in the ESSQS group than in the non-ESSQS group (1·2 versus 3·6 per cent, P = 0·014; 4·6 versus 7·5 per cent, P = 0·025; 1·9 versus 3·9 per cent, P = 0·023, respectively). These findings were confirmed after propensity score matching. Cox regression analysis found that non-attendance of ESSQS-qualified surgeons (hazard ratio 12·30, 95 per cent c.i. 1·28 to 119·10; P = 0·038) was independently associated with local recurrence in patients with stage II disease. CONCLUSION: Laparoscopic colorectal procedures performed with ESSQS-qualified surgeons showed improved postoperative results. Further studies are needed to investigate the impact of the qualification on long-term oncological outcomes.

ANTECEDENTES: El Sistema de Certificación de Habilidades Quirúrgicas Endoscópicas (Endoscopic Surgical Skill Qualification System, ESSQS) fue introducido en Japón para mejorar la calidad de la cirugía laparoscópica. En este estudio de cohortes se investigaron los resultados postoperatorios a corto y a largo plazo de las intervenciones laparoscópicas de cáncer colorrectal realizadas por o con la asistencia de cirujanos con certificación en comparación con cirujanos no certificados. MÉTODOS: Todas las resecciones colorrectales laparoscópicas realizadas entre 2010 y 2013 en 11 hospitales japoneses fueron revisadas retrospectivamente. Los procedimientos se clasificaron en función de si habían sido realizados por cirujanos con o sin certificación del ESSQS, y las características clínicas, patológicas y quirúrgicas de los pacientes se utilizaron para emparejar los subgrupos mediante puntuaciones de propensión. Las variables de resultado incluyeron los resultados postoperatorios y a largo plazo RESULTADOS: En total se analizaron 1.428 procedimientos, incluyendo 586 y 842 procedimientos realizados con y sin cirujanos certificados por ESSQS, respectivamente. Tras el emparejamiento, se seleccionaron dos cohortes de 426 pacientes para la comparación de resultados a corto plazo. Se observó una mayor prevalencia de resecciones rectales (50,3% versus 40,1%, P = 0,0001) y un tiempo quirúrgico más corto (230 versus 238 min, P = 0,04) en el grupo ESSQS. Las tasas de complicaciones intra- y postoperatorias y de reoperaciones fueron significativamente más bajas en el grupo ESSQS que en el grupo no ESSQS (1,2%, 4,6% y 1,9% versus 3,6%, 7,5% y 3,9%, P = 0,01; 0,03, y 0,02, respectivamente). Estos hallazgos se confirmaron tras el análisis de emparejamiento por puntaje de propensión. El análisis de regresión de Cox mostró que la no participación de cirujanos certificados con ESSQS (razón de oportunidades, odds ratio, OR 12,3; i.c. del 95%, 1,28-119,1; P = 0,03) se asoció independientemente con la recidiva local en los casos en estadio II. CONCLUSIÓN: Los procedimientos colorrectales laparoscópicos realizados por cirujanos certificados por ESSQS presentaron mejores resultados postoperatorios. Son necesarios más estudios para determinar el impacto de la certificación en los resultados oncológicos a largo plazo.

Anti-glucose-6-phosphate isomerase, anti-cyclic citrullinated peptide antibodies and HLA-DRB1 genotypes in Japanese patients with early rheumatoid arthritis.

OBJECTIVE: Our goal was to evaluate the associations of antibodies (Abs) to glucose-6-phosphate isomerase (GPI) with Abs to cyclic citrullinated peptide (CCP) and HLA-DRB1 genotypes in Japanese patients with early rheumatoid arthritis (RA). METHODS: One hundred and eight patients with early RA (85 female, 23 male) who visited our clinic within 1 year of symptom onset were examined for anti-GPI and anti-CCP Ab levels, and HLA-DRB1 genotype. Anti-GPI and anti-CCP Ab levels, and HLA-DRB1 genotypes were also determined in 63 controls and 265 healthy controls, respectively. RESULTS: Of the 108 patients with early RA and the 63 controls, 20 (18.5%) and 3 (4.8%) were anti-GPI Ab-positive, respectively. Of the 20 patients with anti-GPI Abs, 17 (85%) were positive for anti-CCP Abs. HLA-DRB1*0405 and shared epitope (SE) carrier frequencies were significantly increased not only in anti-GPI Ab-positive patients (p=0.00057, odds ratio [OR] 4.6, 95% CI 1.8-11.8; p=0.0011, OR 5.0, 95% CI 1.7-14.0), but also in anti-GPI Ab-negative patients (p=0.0017, OR 2.2, 95% CI 1.3-3.7; p=0.00011, OR 2.6, 95% CI 1.6-4.3), when compared with controls. In addition, the carrier frequency of HLA-DRB1*1201 was significantly increased in anti-GPI Ab-positive patients compared with controls (p=0.0056, OR 4.3, 95% CI 1.4-13.2). CONCLUSIONS: The majority of anti-GPI Ab-positive RA patients constitute a subset of HLA-DRB1* SE-associated, anti-CCP Ab-positive RA patients.

Differential association of HLA-DRB1 alleles in Japanese patients with early rheumatoid arthritis in relationship to autoantibodies to cyclic citrullinated peptide.

OBJECTIVE: To evaluate the role of HLA-DRB1 genotypes and antibodies to cyclic citrullinated peptides (anti-CCP antibodies) in the development and radiographic progression of Japanese patients with rheumatoid arthritis (RA). METHODS: One hundred and ten patients with early RA (88 female, 22 male) who visited our clinic within 1 year of symptom onset were examined for anti-CCP antibody levels and HLA-DRB1 genotypes. HLA-DRB1 genotypes were also determined in 265 healthy controls. Radiographic progression over a 2-year interval was evaluated using the Larsen's method in 66 patients. RESULTS: Among the 110 patients with early RA, 82 patients (74.5%) were anti-CCP positive. Carrier frequency of HLA-DRB1*0405 was significantly increased in RA patients with anti-CCP antibodies compared with controls and RA patients without anti-CCP antibodies (odds ratio [OR] 3.4, 95% confidence interval [95% CI] 2.0-5.7 and OR 3.3, 95% CI 1.3-8.6, respectively). Carriership of one or two SE alleles was significantly associated with production of anti-CCP antibodies (OR 2.7, 95% CI 1.1-6.7 and OR 9.3, 95% CI 1.1-78.2, respectively). On the other hand, allele frequency of HLA-DRB1*0901 was significantly increased in RA patients without anti-CCP antibodies compared with controls and RA patients with anti-CCP antibodies (OR 2.2, 95% CI 1.1-4.1 and OR 3.0, 95% CI 1.4-6.4, respectively). CONCLUSION: In Japanese patients with RA, HLA-DRB1 SE alleles are associated with production of anti-CCP antibodies and HLA-DRB1 alleles appear to be differently associated with early RA depending on anti-CCP positivity as in Caucasian patients with RA.

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus.

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.

Dendritic cells/chimerism/alleviation of chronic allograft rejection.

Chronic rejection (CR) is the major obstacle of long-term successful organ transplantation. Using a recently developed rat model of CR, we found that heart allografts susceptible to development of CR showed an early (< 10 days) dramatic disappearance of donor MHC class II+ cells, including ED2+ tissue macrophages, and an influx of recipient ED1+ macrophages intermixed with small numbers of recipient ED2+ and OX62+ cells. In contrast, donor MHC class II+ cells persisted in allografts resistant to CR with a small influx of recipient macrophages. MHC class II+ cells function as potent modulators of the immune system and may mediate both stimulatory and tolerogenic immune reactions after transplantation. Persistence of donor MHC class II+ antigen-presenting cells (APC) in CR-free graft acceptance suggests that transplantation tolerance is an active immune response requiring antigen presentation to the recipient immune system in the proper context by dendritic cells and other APC.

Immunomodulation for intestinal transplantation by allograft irradiation, adjunct donor bone marrow infusion, or both.

BACKGROUND: The passenger leukocytes in the intestine have a lineage profile that predisposes to graft-versus-host disease (GVHD) in some animal models and have inferior tolerogenic qualities compared with the leukocytes in the liver, other solid organs, and bone marrow. Elimination by ex vivo irradiation of mature lymphoid elements from the bowel allografts is known to eliminate the GVHD risk. We hypothesized that infusion of donor bone marrow cells (BMC) in recipients of irradiated intestine would improve tolerogenesis without increasing the risk of GVHD. METHODS: Orthotopic small intestine transplantation was performed with the GVHD-prone Lewis (LEW)-to-Brown Norway (BN) combination and the reverse GVHD-resistant BN-to-LEW model under a short course of tacrolimus treatment (1 mg/kg/day, days 0-13, 20, 27). Grafts were irradiated ex vivo, using a 137Cs source. In selected experimental groups, donor BMC (2.5 x 10(8)) were infused on the day of small intestine transplantation. RESULTS: The unmodified LEW intestine remained intact, whether transplanted alone or with adjunct donor BMC infusion, but all of the BN recipients died of GVHD after approximately 2 months. Intestinal graft irradiation (10 Gy) effectively prevented the GVHD and prolonged survival to 92.5 days, but all of the BN recipients died with chronic rejection of the LEW grafts, which was prevented by infusion of adjunct donor BMC without causing GVHD. In the GVHD-resistant reverse strain direction (BN-->LEW), all intestinal recipients treated for 27 days with tacrolimus survived > or =150 days without regard for graft irradiation or adjunct BMC, but chronic rejection was severe in the irradiated intestine, moderate in the unaltered graft, and least in the irradiated intestine transplanted with adjunct BMC. Mild arteritis in the 150 day allografts of both strain combinations (i.e., LEW--> BN and BN-->LEW) may have been irradiation associated, but this was prevented when weekly doses of tacrolimus were continued for the duration of the experiment rather than being stopped at 27 days. CONCLUSIONS: Recipients are protected from GVHD by irradiating intestinal allografts, but the resulting leukocyte depletion leads to chronic rejection of the transplanted bowel. The chronic rejection is prevented with adjunct donor BMC without causing GVHD. Although application of the strategy may be limited by the possibility of radiation injury, the results are consistent with the paradigm that we have proposed to explain organ-induced graft acceptance, tolerance, and chronic rejection.

Regulation of serum thrombopoietin levels by platelets and megakaryocytes in patients with aplastic anaemia and idiopathic thrombocytopenic purpura.

To clarify the regulatory mechanism of thrombopoietin (TPO, c-Mpl ligand) in chronic thrombocytopenic conditions, we determined TPO levels in the sera of patients with aplastic anaemia (AA; n = 26) and idiopathic thrombocytopenic purpura (ITP; n = 32) by an enzyme-linked immunosorbent assay. Despite a similarity in platelet counts, serum TPO levels in the AA group were markedly higher than those in the ITP group: 20.41 +/- 9.71 f mol/ml (mean +/- SD) and 1.66 +/- 0.55 f mol/ml, respectively, both of which were significantly elevated compared to normal subjects (n = 41; 1.22 +/- 0.37). In both groups, serum TPO level showed an inverse correlation with the platelet count. We determined the megakaryocyte volume using bone marrow clot section and found that it was markedly small in the AA group; while in the ITP group it was augmented with a correlation to serum TPO level. Our findings suggest that TPO levels may be regulated not only by platelets but also megakaryocytes in AA and ITP.

Plasma concentration of von Willebrand factor in acute myocardial infarction.

Recent investigations have revealed the crucial role of von Willebrand factor (vWF) in platelet thrombus formation under flow conditions. The plasma concentrations of vWF were measured together with various hemodynamic and hemostatic parameters in 51 cases of acute myocardial infarction. In 10 randomly selected cases, the plasma concentrations and distribution of multimers vWF were serially determined after reperfusion therapy by percutaneous transluminal coronary angioplasty (PTCA). The vWF concentration at the onset of the acute myocardial infarction was significantly higher than in an age-matched control group (vWF AG: 18.7 +/- 1.2 microg/ml vs. 10.3 +/- 0.5 microg/ml, p = 8.43 x 10-(12), mean +/- SE). Simultaneous determination of hemodynamic and hemostatic parameters revealed that the only two parameters that were significantly correlated with the patients' plasma vWF concentrations were their pulmonary capillary wedge pressure (PCWP) and heart rate, suggesting a relationship between hemodynamic changes induced by the onset of myocardial infarction and the vWF plasma concentrations. Serial determinations revealed that the vWF concentrations had not changed 1 h after reperfusion therapy, but that they significantly increased by 24 to 72 h. The distribution of the larger multimers of vWF also increased in the acute and subacute phase. The vWF concentration and multimer distribution normalized 14 days after the onset of the myocardial infarction. Our findings suggest that the vWF concentration increased in acute myocardial infarction patients, possibly in association with the hemodynamic deterioration that occurs in acute myocardial infarction.

Serum thrombopoietin (c-Mpl ligand) levels in patients with liver cirrhosis.

To clarify the role of c-Mpl ligand (thrombopoietin: TPO) in liver cirrhosis (LC), we examined serum TPO levels (sTPO) in patients with LC (N = 44), chronic hepatitis (CH; N = 13) and healthy controls (N = 41) by an enzyme-linked immunosorbent assay. Although platelet counts of all LC patients (89 +/- 59 x 10(9)/l; mean +/- SD) were lower than those of controls and CH patients, sTPO levels in LC patients (1.23 +/- 0.51 fmol/ml) were the same as those in controls (1.22 +/- 0.37) and CH patients (1.18 +/- 0.36). Platelet counts were significantly higher in splenectomized patients than in unsplenectomized patients, but the sTPO level did not differ between these two groups. In LC patients, the sTPO level was not correlated with the platelet count, but was correlated with prothrombin time, activated partial thromboplastin time, and total bilirubin, indicating that production of TPO in the liver decreases slightly with the development of liver dysfunction. Our findings suggest that production of TPO is maintained in LC patients and their thrombocytopenia is not due to a defect in platelet production.

Hairy cell leukemia with translocation (11;20)(q13;q11) and overexpression of cyclin D1.

We report on a male Japanese patient with hairy cell leukemia (HCL). A cytogenetic study with lipopolysaccharide stimuli showed a novel translocation (11;20)(q13;q11) in 10% of the analyzed cells. Northern blot analysis and RT-PCR analysis for cyclin D1 revealed the overexpression of cyclin D1, although the southern blot analysis of PRAD1 gene showed no rearrangement. In this particular case, the t(11;20)(q13;q11) might play some role in the oncogenesis of HCL and the overexpression of cyclin D1.

Local magnetic order vs superconductivity in a layered cuprate

We report on the phase diagram for charge-stripe order in La1.6-xNd0. 4SrxCuO4, determined by neutron and x-ray scattering studies and resistivity measurements. From an analysis of the in-plane resistivity motivated by recent nuclear-quadrupole-resonance studies, we conclude that the transition temperature for local charge ordering decreases monotonically with x, and hence that local antiferromagnetic order is uniquely correlated with the anomalous depression of superconductivity at x approximately 1 / 8. This result is consistent with theories in which superconductivity depends on the existence of charge-stripe correlations.

An intrinsic ATPase inhibitor binds near the active site of yeast mitochondrial F1-ATPase.

An ATPase inhibitor and its stabilizing factor, the 9K protein, are regulatory factors of F1F0-ATPase. The binding sites for these factors on F1 were examined using the zero length cross-linkers, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. The cross-linked products were analyzed by immunoblotting after SDS-polyacrylamide gel electrophoresis. The inhibitor and the 9K protein cross-linked to the alpha and beta subunits of F1, indicating that they interacted with both subunits. Peptide mapping and amino acid sequence analysis of the cross-linked products after weak acid hydrolysis showed that the inhibitor cross-linked to the Pro334-Asp363 region of the beta subunit. Amino acid sequence analysis of the cross-linked peptide showed that the inhibitor binds to Asp363 of the beta subunit. As this region contains the amino acid residues, including Tyr359, that are modified by nucleotide analogs and form the active site, the inhibitor probably binds to the catalytic site of F1.

The carboxyl-terminal region of the yeast ATPase inhibitor is indispensable for the stability of the protein in mitochondria.

The role of the carboxyl-terminal region of the yeast mitochondrial ATPase inhibitor was investigated. Three progressive C-terminal deletion mutants of the inhibitor were constructed: (i) Ile58-->end; (ii) Ile51-->end; and (iii) Gln43-->end. The truncated inhibitor was detected in extracts of Ile58-->end mutant yeast cells. For the Ile51-->end mutant, the truncated inhibitor was only detected when the cells were grown on medium containing the membrane-permeable metal chelator, o-phenanthroline, which inhibits mitochondrial proteases. The most greatly truncated inhibitor protein, Gln43-->end, was never detected even in the cells grown in the presence of the metal chelator. The rates of ATP synthesis and hydrolysis in the mutant mitochondria containing the Ile51-->end inhibitor were similar to those in wild type control cells, while the Ile51-->end inhibitor protein was degraded in the cells unless they were incubated in the presence of the chelator. These results indicate that the carboxyl-terminal region of the ATPase inhibitor is not involved in the its inhibitory action on the F1Fo-ATPase, but is required for the stable conformation of the protein which is protected against degradation by proteases.

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria.

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.

Binding of an intrinsic ATPase inhibitor to the interface between alpha- and beta-subunits of F1FoATPase upon de-energization of mitochondria.

Yeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9K protein, and 15K protein. Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption [Ichikawa, N. et al. (1990) J. Biol. Chem. 265, 6274-6278; Yoshida, Y. et al. (1990) Eur. J. Biochem. 193, 49-53]. Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells. However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9K or 15K protein, the inactivation of ATPase was slow and incomplete. Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the alpha- and beta-subunits. This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits.

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.

Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.

Liver cirrhosis with marked thrombocytopenia and highly elevated serum thrombopoietin levels.

Three patients with liver cirrhosis (LC) and a bleeding tendency due to marked thrombocytopenia of less than 20 x 10(9)/l were admitted to our hospital for further examination. Bone marrow examination revealed megakaryocytic hypoplasia in all three patients. All patients exhibited amegakaryocytic thrombocytopenic purpura, myelodysplastic syndrome, or bone marrow hypoplasia. 111In-labeled platelet kinetic studies revealed decreased platelet production in all patients. Although serum thrombopoietin (sTPO) levels are usually within the normal level in patients with LC, the sTPO levels of our patients were about 10 times higher than the levels of normal subjects (1.22 +/- 0.37 fmol/ml): 13.34, 16.79, and 10.46 fmol/ml, respectively. These sTPO data supported our findings of decreased megakaryopoiesis. Our findings suggest that examination of sTPO levels is useful in determining the etiology of marked thrombocytopenia in LC patients.