Gallbladder Agenesis in 17 Dogs: 2006-2016.

BACKGROUND: Gallbladder agenesis (GBA) is extremely rare in dogs. HYPOTHESIS/OBJECTIVES: To describe the history, clinical signs, diagnosis, treatment, and outcomes of dogs with GBA. ANIMALS: Seventeen client-owned dogs with GBA. METHODS: Medical records from 2006 through 2016 were retrospectively reviewed. Dogs were included when GBA was suspected on abdominal ultrasonography and confirmed by gross evaluation. Signalment, clinical signs, clinicopathological data, diagnostic imaging, histopathology, treatment, and outcome were recorded. RESULTS: Dogs were of 6 different breeds, and Chihuahuas (10 of 17) were most common. Median age at presentation was 1.9 (range, 0.7-7.4) years. Clinical signs included vomiting (5 of 17), anorexia (2 of 17), ascites (2 of 17), diarrhea (1 of 17), lethargy (1 of 17), and seizures (1 of 17). All dogs had increased serum activity of at least 1 liver enzyme, most commonly alanine aminotransferase (15 of 17). Fifteen dogs underwent computed tomography (CT) cholangiography; common bile duct (CBD) dilatation was confirmed in 12, without evidence of bile duct obstruction. Gross evaluation confirmed malformation of the liver lobes in 14 of 17 dogs and acquired portosystemic collaterals in 5 of 17. Ductal plate malformation was confirmed histologically in 16 of 17 dogs. During follow-up (range, 4-3,379 days), 16 of 17 dogs remained alive. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with GBA exhibit clinicopathological signs of hepatobiliary injury and hepatic histopathological changes consistent with a ductal plate abnormality. Computed tomography cholangiography was superior to ultrasound examination in identifying accompanying nonobstructive CBD distention. Computed tomography cholangiography combined with laparoscopic liver biopsy is the preferable approach to characterize the full disease spectrum accompanying GBA in dogs.

Endoscopic photodynamic therapy using talaporfin sodium for recurrent intranasal carcinomas after radiotherapy in three dogs.

Radiation is the treatment of choice for canine nasal tumours but, in almost all cases, there is local recurrence associated with poor prognosis. This report describes the effect of endoscopic photodynamic therapy using talaporfin sodium for canine intranasal carcinoma recurring after radiation therapy. Rhinoscopic photodynamic therapy was administered after radiation therapy in three dogs with recurrent intranasal carcinoma. Two to 24 illuminations of a 665-nm diode laser were performed two hours after intravenous bolus injection of 5·0 mg/kg of talaporfin sodium. Photodynamic therapy induced almost complete remission and prolonged survival time in all cases suggesting that it might be a useful treatment for intranasal carcinomas that recur after radiation.

Pharmacokinetics of TAK-475, a Squalene Synthase Inhibitor, in Rats and Dogs.

The pharmacokinetics of TAK-475 (lapaquistat acetate), a squalene synthase inhibitor, was investigated in rats and dogs. After oral administration of (14)C-labeled TAK-475 ([(14)C]TAK-475) to rats and dogs at a dose of 10 mg/kg, the bioavailability (BA) was relatively low at 3.5 and 8.2%, respectively. The main component of the radioactivity in the plasma was M-I, which has a comparable pharmacological activity to TAK-475 in vitro. The radioactivity in the portal plasma after intraduodenal administration of [(14)C]TAK-475 to portal vein-cannulated rat was also mainly M-I, suggesting that most of the TAK-475 was hydrolyzed to M-I during the permeable process in the intestine. The concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration of [(14)C]TAK-475 to rats. The main elimination route of the radioactivity was fecal excretion after oral administration of [(14)C]TAK-475 to rats and dogs, and the absorbed radioactivity was mainly excreted via the bile as M-I in rats. M-I excreted into the bile was partially subjected to enterohepatic circulation. These results suggest that although the BA values of TAK-475 are low, M-I can exert compensatory pharmacological effects in the animals. These pharmacokinetic characteristics in animals were also confirmed in the clinical studies. The evaluation of M-I disposition is important for the pharmacokinetics, pharmacodynamics and toxicity of TAK-475 in animals and humans.

MicroRNA-16 mediates the regulation of a senescence-apoptosis switch in cutaneous T-cell and other non-Hodgkin lymphomas.

Multiple sequential genetic and epigenetic alterations underlie cancer development and progression. Overcoming cellular senescence is an early step in cancer pathogenesis. Here, we demonstrate that a noncoding regulatory RNA, microRNA-16 (miR-16), has the potential to induce cellular senescence. First, we examined the expression of miR-16 in primary cutaneous T-cell lymphoma (CTCL) and other non-Hodgkin T/natural killer (NK)-cell lymphomas and found that miR-16 was downregulated than that in the corresponding normal cells. Notably, miR-16 expression was reduced as the primary CTCL progressed from the early stage to the advanced stage. Next, we transduced CTCL cells with miR-16 to examine whether this miRNA exhibited tumor-suppressive effects in CTCL cells. In CTCL cells expressing wild-type p53, forced expression of miR-16 enhanced p21 expression via downregulation of the polycomb group protein Bmi1, thereby inducing cellular senescence. Alternatively, in CTCL cells lacking functional p53, miR-16 induced compensatory apoptosis. The miR-16 transfection significantly decreased senescent cells and increased apoptotic cells in p21-knockdown CTCL cells expressing wild-type p53, suggesting that the presence or absence of p21 may be the most important condition in the senescence-apoptosis switch in CTCL lymphomagenesis. Furthermore, we found that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) restored the expression of miR-16 and its essential targets, induced senescence in CTCL cells expressing wild-type p53 and promoted apoptosis in cells with nonfunctional p53. Moreover, we found that other T/NK-cell lymphoma cell lines showed similar tumor-suppressive effects in response to miR-16 and SAHA and that these effects were dependent on p53 status. These results suggested that epigenetic silencing of miR-16 may be a key step during lymphoma development. Elucidation of the essential targets of miR-16 and SAHA provides a basis for the clinical application of SAHA in the treatment of CTCL and other non-Hodgkin T/NK-cell lymphomas.

Clinical efficacy and treatment persistence of monthly minodronate for osteoporotic patients unsatisfied with, and shifted from, daily or weekly bisphosphonates: the BP-MUSASHI study.

UNLABELLED: This multi-center, prospective, open-label, observational study evaluated the effects of once-monthly minodronate (50 mg) on treatment persistence, bone turnover markers, bone mineral density, low back pain, and upper gastrointestinal symptoms in outpatients with osteoporosis previously treated with daily or weekly bisphosphonate products. INTRODUCTION: The purposes of this study were to investigate the effects of once-monthly oral minodronate (MIN 50 mg) on bone turnover markers and bone mineral density, low back pain, and upper gastrointestinal symptoms, as well as preference for and treatment persistence of MIN 50 mg among Japanese osteoporosis patients currently treated with daily or weekly bisphosphonates. METHODS: Study patients were allocated based on their preference to either the Switch group (patients willing to switch over to MIN 50 mg) or the Continue group (patients wanting to continue their current therapies). Patients' treatment persistence and satisfaction levels with the therapies were assessed using a self-administered questionnaire. The study endpoints were serum TRACP-5b, serum P1NP, bone mineral density, upper gastrointestinal symptoms, and low back pain. RESULTS: In total, 264 and 133 patients were allocated into the Switch and Continue groups, respectively. Approximately, 65 % of patients were willing to switch to MIN 50 mg, with the predominant reason being "less frequent dosing more convenient." Treatment persistence was significantly higher in the Switch group (MIN 50 mg) than the Continue group. Almost all patients with abnormal bone metabolism markers demonstrated normalization after switchover. MIN 50 mg alleviated low back pain and upper gastrointestinal symptoms induced by prior bisphosphonate use. CONCLUSIONS: MIN 50 mg alleviates low back pain, reduces bone turnover markers and increases bone density, and induces fewer upper gastrointestinal symptoms after switchover from prior bisphosphonate products, and therefore, it may provide patients with a more convenient treatment option and enhance long-term treatment persistence.

Dysregulation of BMI1 and microRNA-16 collaborate to enhance an anti-apoptotic potential in the side population of refractory mantle cell lymphoma.

The proto-oncogene BMI1 and its product, Bmi1, is overexpressed in various types of tumors, particularly in aggressive tumors and tumors resistant to conventional chemotherapy. BMI1/Bmi1 is also crucially involved in cancer-initiating cell maintenance, and is recurrently upregulated in mantle cell lymphoma (MCL), especially aggressive variants. Recently, side population (SP) cells were shown to exhibit tumor-initiating characteristics in various types of tumors. In this study, we show that recurrent MCL cases significantly exhibit upregulation of BMI1/Bmi1. We further demonstrate that clonogenic MCL SP shows such tumor-initiating characteristics as high tumorigenicity and self-renewal capability, and that BMI1 was upregulated in the SP from recurrent MCL cases and MCL cell lines. On screening for upstream regulators of BMI1, we found that expression of microRNA-16 (miR-16) was downregulated in MCL SP cells by regulating Bmi1 in the SPs, leading to reductions in tumor size following lymphoma xenografts. Moreover, to investigate downstream targets of BMI1 in MCL, we performed cross-linking/chromatin immunoprecipitation assay against MCL cell lines and demonstrated that Bmi1 directly regulated pro-apoptotic genes such as BCL2L11/Bim and PMAIP1/Noxa, leading to enhance anti-apoptotic potential of MCL. Finally, we found that a proteasome inhibitor bortezomib, which has been recently used for relapsed MCL, effectively induced apoptosis among MCL cells while reducing expression of Bmi1 and increasing miR-16 in MCL SP. These results suggest that upregulation of BMI1 and downregulation of miR-16 in MCL SP has a key role in the disease's progression by reducing MCL cell apoptosis. Our results provide important new insight into the pathogenesis of MCL and strongly suggest that targeting BMI1/Bmi1 might be an effective approach to treating MCL, particularly refractory and recurrent cases.

Intraoperative identification of canine hepatocellular carcinoma with indocyanine green fluorescent imaging.

OBJECTIVES: To evaluate the feasibility of high-sensitivity near-infrared fluorescence imaging with indocyanine green for intraoperative identification of hepatocellular carcinoma in dogs. METHODS: Twelve hepatic nodules were surgically resected from six dogs. In each dog, 0 · 5 mg/kg indocyanine green was intravenously injected for 12 to 18 hours preoperatively. The hepatic nodules were investigated under laparotomy using a near-infrared fluorescence imaging light camera system prior to resection. Resected nodules were histopathologically diagnosed and their fluorescence images were evaluated. RESULTS: Of the 12 hepatic nodules, 6 were diagnosed as hepatocellular carcinoma and 6 as nodular hyperplasia. Indocyanine green-fluorescence was observed in four large hepatocellular carcinoma nodules and one case of nodular hyperplasia, whereas it was absent in the remaining nodules. The sensitivity and positive predictive values of indocyanine green fluorescent imaging for hepatocellular carcinoma was 71 · 4 and 80 · 0%, respectively. Complete resection of the hepatic masses was achieved in all dogs. CLINICAL SIGNIFICANCE: Near-infrared fluorescence imaging may be feasible for intraoperative mapping of hepatocellular carcinomas in hepatic lobes and may help increase the chance of complete resection of hepatocellular carcinoma in dogs.

The role of microRNA-150 as a tumor suppressor in malignant lymphoma.

MicroRNA (miRNA; miR) is a class of small regulatory RNA molecules, the aberrant expression of which can lead to the development of cancer. We recently reported that overexpression of miR-21 and/or miR-155 leads to activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway in malignant lymphomas expressing CD3(-)CD56(+) natural killer (NK) cell antigen. Through expression analysis, we show in this study that in both NK/T-cell lymphoma lines and samples of primary lymphoma, levels of miR-150 expression are significantly lower than in normal NK cells. To examine its role in lymphomagenesis, we transduced miR-150 into NK/T-cell lymphoma cells, which increased the incidence of apoptosis and reduced cell proliferation. Moreover, the miR-150 transductants appeared senescent and showed lower telomerase activity, resulting in shortened telomeric DNA. We also found that miR-150 directly downregulated expression of DKC1 and AKT2, reduced levels of phosphorylated AKT(ser473/4) and increased levels of tumor suppressors such as Bim and p53. Collectively, these results suggest that miR-150 functions as a tumor suppressor, and that its aberrant downregulation induces continuous activation of the PI3K-AKT pathway, leading to telomerase activation and immortalization of cancer cells. These findings provide new insight into the pathogenesis of malignant lymphoma.

Serum hyaluronic acid in dogs with congenital portosystemic shunts.

OBJECTIVES: To compare the serum level of hyaluronic acid in dogs with congenital portosystemic shunt with that in healthy dogs and to investigate the perioperative change in serum hyaluronic acid following shunt attenuation. METHODS: Blood samples were obtained from 29 congenital portosystemic shunt dogs before the operation, and 2 and 4 weeks after the operation from 17 and 7 dogs, respectively. The serum hyaluronic acid level of these dogs was measured and compared with that of 10 healthy beagles. RESULTS: The median preoperative hyaluronic acid level in dogs with congenital portosystemic shunt was significantly elevated compared with that in healthy dogs. Furthermore, the median postoperative hyaluronic acid level significantly decreased compared with the median preoperative levels in congenital portosystemic shunt dogs. CLINICAL SIGNIFICANCE: In the case of dogs with congenital portosystemic shunt, the reduction of intrahepatic portal blood flow might lower the clearance rate of hyaluronic acid in hepatic sinusoidal endothelial cells, so hyaluronic acid clearance could be improved by attenuation of a shunt vessel. Hence, serum hyaluronic acid levels might be useful to evaluate liver function and also have the potential to evaluate successful attenuation of a shunt vessel in dogs with congenital portosystemic shunt. Further investigations are required to clarify whether serum hyaluronic acid offers significant benefits over existing markers such as serum bile acid or ammonia concentrations.

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours.

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.

T-shaped re-anastomosis of graft for outlet obstruction after free jejunal graft.

We experienced three patients with persistent outlet obstruction after free jejunal graft and performed T-shaped re-anastomosis for relief of this symptom. Two patients underwent a laryngopharyngectomy for hypopharyngeal cancer and the other patient underwent a laryngopharyngectomy and total esophagectomy for concurrent hypopharyngeal cancer and esophageal cancer. We reconstructed alimentary conduit by a free jejunal reconstruction without using surgical microscopes. In brief, a graft vein and the internal jugular vein were anastomosed and a graft artery and the carotid artery were anastomosed. Then, the anastomosis of pharyngojejunostomy was carried out in a side-to- end fashion, followed by an end-to- end jejunesophagostomy. In a T-shaped re-anastomosis, the flexure of the transplanted jejunum was separated by GIA (US Surgical Corporation, Norwalk, CT, USA). In cases where the efferent part was redundant, the proximal or distal site was resected and straightened in order to avoid outlet stasis. After this, the end-to-side anastomosis between the efferent part and the bottom of proximal horizontal portion of the graft was performed by CDH (Ethicon, Somerville, NJ, USA) or Olsen's one layer method. These three patients received this operation and were relieved from persistent dysphagia. This method is a safe and easy procedure for relief from dysphagia and for recovery of quality of life for patients with this complication. However, it is of utmost importance to perform a reconstruction followed by profluent passage at the first operation.

Effect of moderate exercise on excess post-exercise oxygen consumption and catecholamines in young women.

AIM: The purpose of this study was to examine the effects of moderate exercise on the magnitude and duration of excess post-exercise oxygen consumption (EPOC) and catecholamine concentrations in 7 young women. METHODS: The subjects performed 30 or 60 minutes of exercise (30-ME and 60-ME, respectively) on separate days at an intensity of 60% of maximal oxygen uptake on a cycle ergometer. The glucose, epinephrine and norepinephrine in the plasma, and free fatty acids and glycerol in the serum were measured at the end of the 60 min of the rest period before each exercise, immediately after the performance of each exercise and 30 min, 1, 2 and 24 h time points after each exercise. RESULTS: EPOC lasted for 46.1+/-25.4 and 116.1+/-79.8 min after the 30- and 60-ME, respectively. Corresponding total EPOC were 2636+/-1125 and 5208+/-1880 ml, respectively. The mean differences were statistically significant (p<0.05 each). The serum free fatty acids significantly increased immediately after the 60-ME. The serum glycerol was significantly increased immediately after the 30-ME and was also significantly increased immediately after and 30 min time point after the 60-ME. The plasma epinephrine and norepinephrine significantly increased immediately after the 30- and 60-ME, respectively. The plasma epinephrine at the 30 min time point after the 60-ME significantly correlated with the magnitude (r=0.955) and duration (r=0.897) of EPOC. Norepinephrine at the 30 min time point after the 60-ME also significantly correlated with the magnitude (r=0.774) of EPOC. CONCLUSION: The results indicate that longer duration exercise results in a greater and longer EPOC, and the plasma catecholamines may possibly contribute to EPOC in young women. Although, the overall magnitude of EPOC is small, if exercise frequency and compliance are strong, some long term benefits may accrue.

A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.

BACKGROUND AND OBJECTIVES: Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone. MATERIALS AND METHODS: The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model. RESULTS: The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA. CONCLUSIONS: The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons.

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.

Physiological responses during and following karate training in women.

BACKGROUND: The purpose of this study was to examine whether each exercise and an entire karate training session can achieve: 1). accepted training intensity thresholds for effective aerobic capacity training; 2). energy expenditure (EE) thresholds for total body mass and fat weight loss; and, 3). elevation in excess postexercise oxygen consumption (EPOC). METHODS: We investigated physiological responses during 5 types of karate training in female karate practitioners: basic techniques without (S-Basics) and with (M-Basics) movements, sparring techniques without (TECH I) and with (TECH II) an opponent, and kata. RESULTS: The mean percent of maximum oxygen uptake reserve (%VO2R), percent of maximum heart rate (%HRmax), and maximum heart rate reserve (%HRR) for S-Basics were below the accepted threshold and for M-Basics were marginal or above the threshold for increasing VO2max. For TECH I, TECH II, kata, and the entire 70-min practice, the mean %HRmax and %HRR were well above the threshold, however, %VO2R was below the threshold. Although the mean EPOC measured for 5 min immediately following the entire 70-min karate training did not differ from resting VO2. The blood lactate responses to the 5 types of karate exercises ranged from 1.2+/-0.3 to 2.2+/-0.8 mmol L(-1). The mean EE for each karate exercise ranged from 157+/-10 kJ to 314+/-16 kJ. The mean EE for the entire 70 min practice and EPOC were 1120+/-64 kJ and 28+/-2 kJ, respectively. CONCLUSIONS: The training intensities of karate exercises studied in women were light to moderate, effects of karate training on EPOC was minimal, and the mean EE was marginal to the accepted threshold for total body mass and fat weight loss.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Cigarette smoking, blood pressure and serum lipids and lipoproteins in middle-aged women.

The relationship of cigarette smoking with blood pressure and serum lipids and lipoproteins was studied in the 3934 middle-aged women aged 40 to 59 years. After adjusting age, body mass index (BMI), alcohol intake and physical activity scores, the mean systolic and diastolic blood pressures (SBP and DEP, respectively) did not indicate dose-dependent relationships. The largest significant mean differences in SBP (4.6 mmHg), DBP (3.9 mmHg), high density lipoprotein cholesterol (HDL-C) (9.6 mg/dL), ratio of total cholesterol to HDL-C (TC/HDL-C) (0.8), triglycerides (TG) (22.9 mg/dL) and the logarithmic transformation of TG (Log TG) (0.26) were found between the non-smokers and smokers. When age, BMI, alcohol intake and physical activity scores were included in the forward stepwise multiple regression analyses, there were negative relationships found for cigarette smoking and SBP, DBP and HDL-C and positive relationships for cigarette smoking and TC/HDL-C, TG, Log TG and low density lipoprotein cholesterol. Although the results are somewhat variable, the present study shows cigarette smoking is negatively associated with SBP and DBP and unfavorably associated with serum lipids and lipoproteins in middle-aged women.

Investigation of patients' demand for community pharmacies: relationship between pharmacy services and patient satisfaction.

We performed an investigation on the patients' demand for community pharmacy based on the analysis of questionnaire responses on community pharmacy services from the patients at 32 pharmacies in Tokyo and Osaka. In the previous study, we developed seven evaluation indices for pharmacy services, and showed that the functions most sought by patients in the "ideal pharmacies" were "Attitude of pharmacy/pharmacist", "Convenient hours" and "Information management". The objective of this study is to determine the relationship between these pharmacy functions and patient satisfaction by analyzing responses from the same questionnaire survey. Overall satisfaction score with the "pharmacy used today" was employed as the dependent variable, while the six factors derived from the 26-item evaluation scale in the questionnaire by factor analysis were used as the independent variables. As a result of analysis, it was found that four variables had a significant positive correlation with patient satisfaction, one had a significant inverse correlation, and one showed no significant correlation (p < 0.05). These results suggest that: attitude of the pharmacists such as general attitude and specialized activities of pharmacy/pharmacist such as providing information and explanations, and convenience of hours are not only judged to be important by patients, but also influence their satisfaction; comfortable facilities and availability of OTC drugs, while rated relatively low by patients in terms of importance, do influence their satisfaction; and convenience of location does not influence patient satisfaction. It was also indicated that insufficient inventories of prescribed medications have an impact upon patient satisfaction. This investigation offers evidence to provide patient-based pharmacy services.

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase.

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.