Investigation of the cytotoxic effect of flavopiridol in canine lymphoma cell lines.

The cyclin-dependent kinase (CDK) inhibitor, flavopiridol, was tested as a potential new cancer therapeutic agent to treat canine lymphoma by examining its effect on cell growth of canine lymphoma cell lines in vitro. Flavopiridol induced profound cell death in all eight lymphoma cell lines at 400 nM, and in all cases cell death was due to apoptosis. Apoptosis was inhibited by caspase inhibitor, despite the variable sensitivities between cell lines. Analysis of the mechanism of flavopiridol-induced apoptosis showed that Rb phosphorylation was inhibited, possibly due to CDK4 or CDK6 inhibition. There was also decreased expression of Rb protein and anti-apoptotic proteins, Mcl-1 and XIAP, possibly through transcriptional regulation by inhibition of CDK7 or CDK9 activation. Canine lymphoma cell line-xenotransplanted mice were then treated with flavopiridol and profound tumour shrinkage was observed. This study describes a new therapeutic approach using flavopiridol for canine lymphoma treatment.

Terrestrial gamma radiation dose rate in Ryukyu Islands, subtropical region of Japan.

In order to explain the distribution of natural radiation level in the Asia, in situ measurements of dose rate in air due to terrestrial gamma radiation have been conducted in a total of 21 islands that belong to Ryukyu Islands (Ryukyu Archipelago), subtropical rejoin of southwest Japan. Car-borne surveys have also been carried out in Okinawa-jima, the biggest island of the archipelago. Based on the results for these measurements, arithmetic mean, the maximum and the minimum of the dose rates at 1 m in height from the unpaved soil ground in the archipelago were estimated to be 47, 165 and 8 nGy h(-1), respectively. A comparative study of car-borne data obtained prior to and subsequent to the 2011 Fukushima nuclear accident, as for Okinawa-jima, indicated that the nuclear accident has no impact on the environmental radiation at the present time.

High-normal blood pressure is associated with new-onset electrocardiographic left ventricular hypertrophy.

Whether high-normal blood pressure (BP) is a predictor of new-onset electrocardiographic (ECG)-left ventricular hypertrophy (LVH) is not known. A total of 4112 subjects who underwent physical examinations were enrolled in this study. BP was measured on entry. Standard 12-lead ECG was recorded at initial evaluation and 3 years later. BP categories were defined on the basis of the 2013 European Society of Hypertension/European Society of Cardiology guidelines. Of the 4112 subjects, 133 developed ECG-LVH 3 years later. Crude cumulative prevalence rates of new-onset ECG-LVH were 2.0% for the optimal BP group, 3.2% for the normal BP group, 5.1% for the high-normal BP group and 5.0% for the hypertension group. After adjustment for age, gender, body mass index, smoking, low-density lipoprotein cholesterol, log-transformed high-density lipoprotein cholesterol, log-transformed triglycerides, estimated glomerular filtration rate, haemoglobin A1c, haemoglobin, uric acid and antihypertensive medication use, compared with the optimal BP group, the odds ratios of new-onset ECG-LVH for the normal BP, high-normal BP and hypertension groups were 1.52 (95% confidence interval (CI): 0.93-2.49, P=0.094), 2.38 (95% CI: 1.40-4.03, P=0.001) and 2.44 (95% CI: 1.43-4.18, P=0.001), respectively. Even high-normal BP was significantly associated with the presence of new-onset ECG-LVH.

The effect of ephedrine and phenylephrine on BIS values during propofol anaesthesia.

BACKGROUND: The purpose of this study was to evaluate the effect of ephedrine and phenylephrine on propofol concentrations and bispectral index during propofol anesthesia. METHODS: General anaesthesia was induced with propofol and was maintained with propofol (4 mg kg-1 h-1) and fentanyl. Vecuronium was used to facilitate the artificial ventilation of the lungs. Patients with systolic blood pressure > 90 mmHg were defined as the control group (n = 16). Patients who had to be treated for larger decreases in arterial blood pressure (systolic blood pressure 60, whereas no patient in the control or phenylephrine groups had bispectral index >60. There were no significant differences in propofol concentrations or cardiac output relative to baseline at 3 or 10 min after the administration of ephedrine or phenylephrine. CONCLUSIONS: Ephedrine increases bispectral index values without decreasing propofol concentrations during general anesthesia.

Changes in the effect of propofol in response to altered plasma protein binding during normothermic cardiopulmonary bypass.

BACKGROUND: During normothermic cardiopulmonary bypass (CPB), the effect on propofol pharmacokinetics of changes in its binding to plasma proteins is consistent with the predictions of the well-stirred model of hepatic elimination for nonrestrictively cleared drug. However, whether changes in binding lead to clinically significant changes in the drug effect remains unclear. The purpose of this study was to assess changes in the drug effect of propofol in response to altered plasma binding using quantitative EEG measurements. METHODS: Thirty patients undergoing cardiac surgery were assigned randomly to receive propofol infusions at 4 (Group P-4) or 6 (Group P-6) mg kg(-1) h(-1) during surgery. The concentration of propofol in blood samples, collected from the radial artery at predetermined intervals, was determined by HPLC. The unbound fraction of drug in plasma was estimated using equilibrium dialysis. Bispectral index (BIS) and burst suppression ratio (BSR) were measured at the time blood samples were collected. RESULTS: The total concentration of propofol in blood was unchanged during CPB relative to the pre-CPB value in both groups. However, the fraction of unbound propofol in blood increased by 2-fold during CPB. While BIS values were unchanged during CPB in Group P-4, there was a slight, but significant, decrease in Group P-6. In both groups, BSR significantly increased during CPB. BIS values showed a weak correlation with the concentration of unbound propofol (r(2)=0.19, P<0.001). BSR showed a moderate correlation with the concentration of unbound propofol (r(2)=0.56, P<0.001). CONCLUSIONS: The anaesthetic effect of propofol significantly increased during CPB without any alteration in the total drug concentration. The enhanced efficacy may be caused by a reduction in plasma binding of the drug.

Changes in apparent systemic clearance of propofol during transplantation of living related donor liver.

BACKGROUND: Propofol is used during living-related donor liver transplantation because its metabolism is not greatly affected by liver failure. However, the pharmacokinetics of propofol during liver transplantation have not been fully defined. The purpose of this study was to evaluate the apparent systemic clearance of propofol during the dissection, anhepatic and reperfusion phases of living-related donor liver transplantation, and to estimate the role of the small intestine and lung as extrahepatic sites for propofol disposition. METHODS: Ten patients scheduled for living-related donor liver transplantation were enrolled in the study. Anaesthesia was induced with vecuronium 0.1 mg kg(-1) and propofol 2 mg kg(-1), and then maintained by 60% air, 0.5-1.5% isoflurane in oxygen and a constant infusion of propofol at 2 mg kg(-1) h(-1). Apparent systemic clearance during the dissection, anhepatic and reperfusion phases was calculated from the pseudo-steady-state concentration for each phase. Disposition in the small intestine was determined by measuring arteriovenous blood concentration in 10 liver transplantation donors. Pulmonary disposition was determined by measuring the arteriovenous blood concentration in 10 recipients during the anhepatic phase. The data are expressed as mean (sd). RESULTS: Apparent systemic clearances in the dissection, anhepatic and reperfusion phases were 1.89 (sd 0.48) litre min(-1), 1.08 (0.25) litre min(-1) and 1.53 (0.51) litre min(-1), respectively. The concentration of propofol in the portal vein was lower than in the radial artery. The intestinal extraction ratio calculated from the concentration in the radial artery and portal vein was 0.24 (0.12). There were no significant differences in propofol concentrations between the radial and pulmonary arteries. CONCLUSION: Apparent systemic clearance was decreased by approximately 42 (10)% during the anhepatic phase compared with the dissection phase. After reperfusion, liver allografts rapidly began to metabolize propofol. The small intestine also participates in the metabolism of propofol.

A modified system of stress radiography for patellofemoral instability.

Axial radiographs were obtained under valgus and external rotation stress at 45 degrees of knee flexion with and without contraction of the quadriceps muscle in order to assess the dynamics of patellar subluxation or dislocation. The radiography was performed on 82 knees in 61 patients with patellofemoral instability, and on 44 normal knees. The lateral patellofemoral angle and the congruence angle were measured and compared with the conventional Merchant views. Both parameters showed greater differences between symptomatic and normal knees on the stress radiographs obtained without quadriceps contraction. There was a major difference in the lateral patellofemoral angles between the groups, which clearly distinguished symptomatic knees from normal controls. Congruence angles on stress radiography had a significant correlation with the functional scores obtained after a period of conservative treatment and a positive correlation with the frequency of patellar subluxation. When the quadriceps contracted, two patterns of patellar shift were observed. While the patella reduced into the trochlear groove in all normal knees and about 70% of the symptomatic knees, contraction of the quadriceps caused further subluxation of the patella in the remaining symptomatic knees. All the knee joints which showed this displacement failed to respond to conservative treatment and eventually required surgical treatment. Thus, this technique of stress radiography is a simple, cost-effective and useful method of evaluating patellar instability and predicting the prognosis.

Novel LPL mutation (L303F) found in a patient associated with coronary artery disease and severe systemic atherosclerosis.

BACKGROUND: Patients with lipoprotein lipase (LPL) deficiency had been generally thought to be spared accelerated atherosclerosis in spite of a marked elevation of plasma triglyceride levels. However, it has been recently reported that some heterozygous and homozygous LPL-deficient patients are associated with premature atherosclerosis. In this paper, we report a 55-year-old type I hyperlipidaemic patient with a novel missense mutation in the LPL gene. PATIENT AND RESULTS: The patient had suffered from coronary artery disease, abdominal aortic aneurysm, and stenoses of the bilateral renal arteries and superficial femoral arteries. Sequencing of the genomic DNA revealed that the patient was a homozygote for the mutation, a G to C transition at nucleotide position 1069 in the exon 6, resulting in an amino acid substitution of Phe for Leu303 (L303F). Approximately 6% and approximately 40% of normal LPL activity and LPL mass, respectively, were detected in the patient's postheparin plasma. An in vitro expression study demonstrated that COS7 cells transfected with L303F mutant cDNA produced a 40% amount of LPL protein in cell lysates compared with normal cDNA, but no protein was detected in the media. Lipoprotein lipase activity was completely absent in both lysates and media of the cells transfected with the mutant cDNA, suggesting that this mutation in the LPL gene results in the production of a functionally inactive protein. CONCLUSION: This case suggests that the LPL missense mutation (L303F), which impairs lipolysis but preserves the LPL mass, is proatherogenic.

Hepatosplanchnic oxygenation is better preserved during mild hypothermic than during normothermic cardiopulmonary bypass.

PURPOSE: To assess and compare the effects of normothermic and mild hypothermic cardiopulmonary bypass (CPB) on hepatosplanchnic oxygenation. METHODS: We studied 14 patients scheduled for elective coronary artery bypass graft surgery who underwent normothermic (>35 degrees C; group I, n=7) or mild hypothermic (32 degrees C; group II, n=7) CPB. After induction of anesthesia, a hepatic venous catheter was inserted into the right hepatic vein to monitor hepatic venous oxygen saturation (ShvO(2)) and hepatosplanchnic blood flow by a constant infusion technique that uses indocyanine green. RESULTS: The ShvO(2) decreased from a baseline value in both groups during CPB and was significantly lower at ten minutes and 60 min after the onset of CPB in group I (39.5 +/- 16.2% and 40.1 +/- 9.8%, respectively) than in group II (61.1 +/- 16.2% and 61.0 +/- 17.9%, respectively; P <0.05). During CPB, the hepatosplanchnic oxygen extraction ratio was significantly higher in group I than in group II (44.0 +/- 7.2% vs 28.7 +/- 13.1%; P <0.05). CONCLUSION: Hepatosplanchnic oxygenation was better preserved during mild hypothermic CPB than during normothermic CPB.

Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance.

Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G-->A). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NO(x) concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.

Identification of sequence polymorphisms in two sulfation-related genes, PAPSS2 and SLC26A2, and an association analysis with knee osteoarthritis.

Osteoarthritis (OA) is one of the most common musculoskeletal disorders and is characterized by degeneration of articular cartilage. Sulfation of extracellular matrix proteins in articular cartilage is an important step in maintaining normal cartilage metabolism. Two sulfation-related genes have been reported as the causal genes of severe chondrodysplasias: mutations in PAPSS2 (3'-phosphoadenosine 5'-phosphosulfate synthase 2) cause spondylo-epimetaphyseal dysplasia (SEMD), and mutations in SLC26A2 (solute carrier family 26, member 2) cause diastrophic dysplasia. Given their critical roles in cartilage metabolism and the severe phenotypes that result from mutations in these genes, we examined PAPSS2 and SLC26A2 as candidate susceptibility loci for OA. We identified sequence polymorphisms in the coding and core promoter regions of these genes and analyzed their potential association with knee OA within the Japanese population. Ten sequence polymorphisms were detected in PAPSS2 and five in SLC26A2. An association analysis showed suggestive association of one minor polymorphism in the promoter region of SLC26A2. This 4-bp adenine deletion allele, del4A, was over-represented in knee OA (P = 0.043, odds ratio = 3.43) and is thought to confer a minor susceptibility to knee OA within the Japanese population. Haplotype analysis showed no evidence of association with the two genes, however, excluding them as major susceptibility loci for knee OA.

Identification of sequence polymorphisms of the COMP (cartilage oligomeric matrix protein) gene and association study in osteoarthrosis of the knee and hip joints.

Osteoarthrosis (OA) is a common cause of musculoskeletal disability characterized by late-onset degeneration of articular cartilage. Although several candidate genes have been reported, susceptibility genes for OA remain to be determined. Hereditary osteochondral dysplasias produce severe, early-onset OA and hence are models for common idiopathic OA. Among them are pseudoachondroplasia and multiple epiphyseal dysplasia, both of which are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. Therefore, COMP may be a susceptibility gene for OA. We screened for polymorphisms by direct sequencing of all exons of the COMP gene with their flanking intron sequences and the promoter region. We identified 16 polymorphisms, of which 12 were novel. Using six polymorphisms spanning the entire COMP gene, we examined the association of COMP in Japanese patients with OA of the knee and hip joints. Genotype and allele frequencies of the polymorphisms were not significantly different between OA and control groups, and there was no significant difference in haplotypes. These results do not support an association between COMP and OA in the Japanese population.

Localization of CD36 and scavenger receptor class A in human coronary arteries--a possible difference in the contribution of both receptors to plaque formation.

CD36 and scavenger receptor class A types I and II (SR-AI/II) are major receptors for oxidized low density lipoproteins (OxLDL) expressed on macrophages. To elucidate the role of these two macrophage scavenger receptors in the development of coronary atherosclerosis, we examined the localization of CD36 and SR-AI/II in human coronary atherosclerotic lesions. Serial cryostat sections of 49 coronary arteries obtained from 43 autopsied cases were examined immunohistochemically. Regarding the relationship between the severity of atherosclerosis and immunoreactivities to CD36, there was almost no immunoreactivity to CD36 in regions with diffuse intimal thickening, while the expression of CD36 was accelerated in parallel with the progression of atherosclerosis. In contrast, SR-AI/II was expressed persistently from regions with diffuse intimal thickening to atherosclerotic plaques. We also clarified the differential distribution of CD36 and SR-AI/II in atheromatous plaques. Close to the luminal surface of the intima, macrophages were relatively small in size, contained lesser lipids, and expressed SR-AI/II more abundantly than CD36. In contrast, macrophages in the core region were larger in size, contained more lipids, were strongly positive for CD36 and showed a weaker immunoreactivity to SR-AI/II than those in the luminal surface of the intima. In conclusion, the expression of CD36 and SR-AI/II on macrophages may be regulated differently in the process of coronary atherogenesis.

Reduced adhesion of monocyte-derived macrophages from CD36-deficient patients to type I collagen.

CD36 is an 88-kDa glycoprotein expressed on platelets and monocyte/macrophages (Mphi). CD36 is a multifunctional receptor for collagen, thrombospondin, oxidized low density lipoproteins (LDL), and long-chain fatty acids. The present study was performed to investigate whether CD36 can function as an adhesion molecule which is involved in mediating human macrophages (Mphi) adhesion to type I collagen in vitro. The Mphi of human CD36-deficient as well as normal control subjects were isolated and cultured on the multi-well plates coated with type I collagen, a natural ligand for CD36. Up to 2 h of incubation, the Mphi from CD36-deficient patients showed almost a approximately 55% decrease in adhesion to type I collagen in comparison to those from controls (P < 0.01). However, there was no significant difference in the adhesion thereafter. Furthermore, the addition of antibody against CD36 into the media of control Mphi significantly inhibited the adhesion by approximately 50% (P < 0.05). The addition of oxidized LDL (OxLDL) did not alter adhesion of Mphi from both CD36-deficient and controls. These data suggest that CD36 is involved in the adhesion of Mphi to type I collagen, especially in the early stage of adhesion.

Rho-associated kinase phosphorylates MARCKS in human neuronal cells.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous actin bundling protein and has multiple sites for phosphorylation, by which the biochemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functions, is not well understood. Using a phosphorylation-site specific antibody, we detected the phosphorylation of MARCKS at Ser159 by various protein kinases. Rho-kinase, protein kinase A, and protein kinase C, could introduce (32)P into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cells, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and time-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor HA1077. However, the phosphorylation induced by PDBu was lesser sensitive. In a skinned NTera-2 cell system, Ca(2+)-independent and GTP gamma S/ATP-stimulated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells.

(4E)-dehydrocitrals [(2E,4E)- and (2Z,4E )-3,7-dimethyl-2,4,6-octatrienals] from acarid mite Histiogaster sp. A096 (Acari: Acaridae).

A mixture of two monoterpenes was obtained as the opisthonotal gland secretion from unidentified Histiogaster sp. A096 (Acari: Acaridae), and their structures were elucidated to be (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals] by GC/MS, GC/FT-IR, UV and 1H-NMR spectra. Both isomers of (4E)-dehydrocitral prepared by syntheses in 4 steps from 3-methyl-2-butenal with 34.2% yields (based on the ylide) were separated by column chromatography into the (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. Mass spectra together with GC retention times of the purified natural (4E)-dehydrocitrals were identical with those of synthetic (2E,4E)-3,7-dimethyl-2,4,6-octatrienal and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. The geometry at the 2-C position of both synthetic (4E)-dehydrocitrals was confirmed by NOESY analyses. This is the first identification of (4E)-dehydrocitrals from the animal kingdom.