Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Distribution of diaphragm blood flow during sevoflurane anaesthesia in dogs

Purpose

The purpose of this study was to determine the effect of increasing the concentrations of sévoflurane anaesthesia on the distribution of diaphragm blood flow (Qdi) in ten dogs during mechanical ventilation.

Methods

Animals were divided into two groups, sévoflurane (n = 6) and time control (n = 4) groups. Blood flow to the crural and the costal diaphragm (Qcru, Qcost) was determined by the hydrogen clearance technique at 0, 0.5, 1.0 and 1.5 minimum alveolar concentration (MAC) of sévoflurane after a 30 min period of steady-state conditions. Cardiac output (CO) and the mean arterial blood pressure (MBP) were also measured.

Results

Sevoflurane anaesthesia caused a reduction in CO (L · min^?1) from a control value of 1.51 ± 0.21 to 1.38 ±0.1 (0.5 MAC), 1.09 ± 0.15 (1.0 MAC) and 0.98 ± 0.12 (1.5 MAC) (Mean ± SD). Mean blood pressure, Qcru and Qcost also decreased with increasing depth of anaesthesia. In addition, the decrease of Qcru was greater than that of Qcost at all levels of MBP and CO. No change occurred in these variables in the lime control group.

Conclusion

Sevoflurane anaesthesia changes the distribution of Qdi with a greater reduction occurring in Qcru than in Qcost.     

Repair of a ruptured aortic arch aneurysm complicated by postoperative paraplegia: Report of a case

We report herein the rare case of a 79-year-old man who suffered permanent paraplegia after undergoing an otherwise successful total arch replacement for a ruptured aortic arch aneurysm. During cardiopulmonary bypass, perfusion to the distal aorta was maintained from the femoral artery, and postoperative aortography showed intact tributaries from the aorta including the intercostal arteries. Postoperative paraplegia is an extremely rare complication of operations on the aortic arch; however, we speculate that the paraplegia in this patient could be attributed either to a steal phenomenon involving the radicular artery, or to the anatomical particularity of the spinal cord artery described by Cole and Gutelius as the segmental system.     

Myonephropathic-metabolic syndrome as a complication of cardiopulmonary bypass

We encountered eight rare cases of myonephropathic metabolic syndrome (MNMS) which developed as a complication of the femoral arterial cannulation (FAC) during cardiopulmonary bypass (CPB). Seven were boys ranging in age from 4–17 years, and all had undergone open heart surgery using CPB with a hemodilution technique. These eight corresponded to 1.9 per cent of the 420 patients treated with CPB before June, 1974. The pump priming fluid used was either Ringer’s lactate solution alone or that containing a small amount of colloidal solution. Duration of CPB ranged from 52 min to 2 hrs and 42 min, but the FAC period was more than 3 hrs in each case. Acute renal failure occurred in 3 and 2 required peritoneal dialysis. Severe respiratory insufficiency occurred in 2 and one died 3 months after the operation. The most effective means to prevent the development of MNMS seems to be the local cooling of the cannulated limb during FAC. MNMS did not occur in 444 cases of CPB with FAC after July 1974, and here local cooling was applied in all cases.     

Combined effects of caffeine and malnutrition on the newborn rat's myocardium.

We hypothesized that the pathological effects on the neonatal rat heart could be aggravated by Cu deficiency due to the combined effects of caffeine exposure and malnutrition. Upon birth, pups were mixed and randomly picked; 8 pups were assigned to each dam and then divided into 4 groups. Group 1 dams received a normal diet containing 20% protein. Group 2 dams were fed 20% protein diet supplemented with caffeine (4 mg/100 g BW). Group 3 dams received 6% protein diet as a malnourished group, and group 4 dams received 6% protein diet supplemented with caffeine (4 mg/100 g BW). On postnatal day 10, dams and pups were killed. Group 2 tended to have a decrease in the Cu levels of dams' plasma and milk and in pups' plasma and heart tissue compared to those of group 1. This pattern was not observed consistently between groups 3 and 4. Transmission electron microscopy of group 2 pups' hearts revealed a degree of disruption in the mitochondria compared to normal mitochondria seen in group 1. There was no consistent change in the mitochondria of group 4 compared to group 3. The caffeine level observed in all categories of group 4 (dams' plasma and milk, pups' plasma and heart tissue) was lower than those in group 2. Although malnutrition affected body weight and heart weight, combined effects of caffeine and malnutrition on Cu content in the neonatal heart was relatively minor compared to the well nourished group. This well nourished group showed that the effects of caffeine on Cu were more consistent, resulting the changes of mitochondria.     

Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin.

Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.     

Chemopreventive and therapeutic efficacy of orally active tyrosine kinase inhibitors in a transgenic mouse model of gallbladder carcinoma.

Biliary tract cancer (BTC) is the second most common primary hepatobiliary cancer after hepatocellular cancer. At the time of diagnosis, most BTC are at an advanced stage and are unresectable. There is presently no effective curative treatment of the advanced disease nor is there any effective clinical therapy that will prevent the development of BTC. All of these factors render gallbladder cancer nearly incurable with a poor survival rate. The aim of our study was to provide a better understanding of the mechanisms involved in the development of gallbladder carcinoma as the advancement of more effective treatment options would significantly improve prognosis. In the present study, we examined the effect of gefitinib, a selective epidermal growth factor receptor/tyrosine kinase inhibitor (EGFR/TKI), on the development of gallbladder carcinoma in BK5.erbB2 mice. In addition, we examined the effect of another quinazoline derivative, GW2974, which is able to block the activation of both the EGFR and erbB2, in this model. Animals were treated with either 400 ppm gefitinib or 200 ppm GW2974 as a supplement in the diet using either a chemopreventive or therapeutic protocol. The results show that both compounds were potent chemopreventive and therapeutic agents in this mouse model of human BTC. The results also suggest that activation of the EGFR plays an important role in development of BTC in this model and that targeting both the EGFR and erbB2 may be an effective strategy for treatment of this disease.     

Reduced anxious behavior in mice lacking the CCK2 receptor gene.

Cholecystokinin 2 (CCK2) receptors have been implicated as mediators of anxiety in standard mouse models such as exploratory behavior both in black and white test boxes and in elevated plus-mazes. We investigated the role of the CCK2 receptor in anxiety by evaluating the behavior of mice lacking the gene for this receptor in these standard anxiety models (i.e., exploratory behavior in a black and white test box and exploratory behavior in an elevated plus-maze). In the black and white test box, mice lacking the CCK2 receptor gene showed significantly increased numbers of transitions between the boxes compared to control mice. In the elevated plus-maze, mice lacking the CCK2 receptor gene displayed significantly more head dips than control mice. These results suggest that mice lacking the CCK2 receptor gene are less anxious than normal mice.     

Cytomegalovirus reactivation in patients with multiple myeloma

The introduction of novel antimyeloma agents has improved the outcome of multiple myeloma (MM) dramatically. However, it has also led to an increasing incidence of Herpesviridae family virus infections, including a high incidence of post‐transplant cytomegalovirus (CMV) reactivation after treatment with novel agents. We herein retrospectively assessed the CMV reactivation in all 120 newly diagnosed patients with MM consecutively seen and treated at our hospital. CMV antigenemia tests were ordered in 58 patients depending on the clinical context, and the incidence of CMV reactivation and proven/suspected CMV disease requiring antiviral therapy was 20% (24 of 120) and 11% (13 of 120) respectively, including those without stem cell transplantation (SCT). The clinical and laboratory characteristics of these patients were compared with those in 34 CMV antigenemia‐negative (CMV‐negative) patients. Patients with extramedullary disease or a low absolute lymphocyte count (ALC) had a higher risk of developing CMV reactivation. In addition, the median duration from the time of MM diagnosis to CMV reactivation was 5.0 months. These results suggest that, regardless of whether or not undergoing SCT, elderly patients with MM receiving novel agents should be monitored for CMV reactivation to allow for the timely diagnosis and treatment, especially for those with extramedullary disease.