Muscle wasting associated with pathologic change is a risk factor for the exacerbation of joint swelling in collagen-induced arthritis in cynomolgus monkeys.

BACKGROUND: Not only joint destruction but also muscle wasting due to rheumatoid cachexia has been problem in terms of quality of life of patients with rheumatoid arthritis (RA). In the present study, we performed histopathological examination and assessed relationships between characteristic parameters relating to muscle and joint swelling in a collagen-induced arthritis (CIA) model using cynomolgus monkeys (CMs). METHODS: Female CMs were used and CIA was induced by twice immunizations using bovine type II collagen with Freund's complete adjuvant. Arthritis level was evaluated from the degree of swelling at the peripheral joints of the fore and hind limbs. Food consumption, body weight, and serum biochemical parameters were measured sequentially. Five or 6 animals per time point were sacrificed at 2, 3, 5 and 9 weeks after the first immunization to obtain quadriceps femoris specimens for histopathology. Pimonidazole hydrochloride was intravenously administered to determine tissue hypoxia in skeletal muscle. RESULTS: Gradual joint swelling was observed and the maximum arthritis score was noted at Week 5. In histopathology, necrosis of muscle fiber in the quadriceps femoris was observed only at Week 2 and the most significant findings such as degeneration, atrophy, and regeneration of muscle fiber were mainly observed at Week 5. Food consumption was decreased up to Week 4 but recovered thereafter. Body weight decreased up to Week 5 and did not completely recover thereafter. A biphasic increase in serum cortisol was also observed at Weeks 2 and 5. Histopathology showed that muscle lesions were mainly composed of degeneration and atrophy of the muscle fibers, and ATPase staining revealed that the changes were more pronounced in type II muscle fiber than type I muscle fiber. In the pimonidazole experiment, mosaic pattern in skeletal muscle was demonstrated in the intact animal, but not the CIA animal. Increased arthritis score was accompanied by a decrease in serum creatinine, a marker that reflects muscle mass. CONCLUSIONS: Muscle wasting might exacerbate joint swelling in a collagen-induced arthritis model of cynomolgus monkeys.

Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes.

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.

Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey.

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84-86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated.

Microminipig, a non-rodent experimental animal optimized for life science research:novel atherosclerosis model induced by high fat and cholesterol diet.

Atherosclerotic lesions were observed in male and ovariectomized female Microminipig (MMP) fed a high fat and cholesterol diet with sodium cholate (HFCD/SC) for 3 months. HFCD/SC induced hypercholesterolemia accompanied by an increase in serum total cholesterol (T-Cho), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and cholesterol ester (CE). Unlike the mouse or rabbit, a dominant LDL-C fraction in the intact MMP, similar to that in humans, was observed by serum lipoprotein analysis. HFCD/SC increased body weight gain. At the end of the experiment, computed tomography scans of conscious animals showed that HFCD/SC had decreased liver attenuation values (Hounsfield unit) and increased subcutaneous and abdominal fat, suggesting the induction of fatty liver and obesity. HFCD/SC induced atherosclerotic lesions in systemic arteries, including the external and internal iliac arteries, abdominal aorta, coronary artery, and cerebral arterial circle. Atherosclerosis and pathological findings induced by HFCD/SC in MMP were similar to those in humans. The MMP is a potentially suitable tool for investigating human atherosclerosis.

A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

Microminipig, a Non-rodent Experimental Animal Optimized for Life Science Research: Novel Atherosclerosis Model Induced by High Fat and Cholesterol Diet.

Atherosclerotic lesions were observed in male and ovariectomized female Microminipig (MMP) fed a high fat and cholesterol diet with sodium cholate (HFCD/SC) for 3 months. HFCD/SC induced hypercholesterolemia accompanied by an increase in serum total cholesterol (T-Cho), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and cholesterol ester (CE). Unlike the mouse or rabbit, a dominant LDL-C fraction in the intact MMP, similar to that in humans, was observed by serum lipoprotein analysis. HFCD/SC increased body weight gain. At the end of the experiment, computed tomography scans of conscious animals showed that HFCD/SC had decreased liver attenuation values (Hounsfield unit) and increased subcutaneous and abdominal fat, suggesting the induction of fatty liver and obesity. HFCD/SC induced atherosclerotic lesions in systemic arteries, including the external and internal iliac arteries, abdominal aorta, coronary artery, and cerebral arterial circle. Atherosclerosis and pathological findings induced by HFCD/SC in MMP were similar to those in humans. The MMP is a potentially suitable tool for investigating human atherosclerosis.

Novel microminipig model of atherosclerosis by high fat and high cholesterol diet, established in Japan.

Novel atherosclerotic lesions were induced in the Microminipig (MMP, registered with the Japanese Ministry of Agriculture, Forestry and Fisheries as a novel variety of swine), the smallest pig available for experimental use, by feeding a high fat (12%) and high cholesterol (5%) diet (HFCD) with sodium cholate (SC, 0.7%) (HFCD/SC) for three months. Three MMPs were used: a male fed with normal diet (M-ND), and a male and an ovariectomized female fed with HFCD/SC (M-HFCD/SC and Fx-HFCD/SC). HFCD/SC induced hypercholesterolemia accompanied by an increase in serum total cholesterol (T-Cho), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and cholesterol ester (CE) from the first week. Serum levels of T-Cho, LDL-C and CE reached a maximum in two to three weeks, and HDL-C gradually increased during the experimental period (duration). Serum lipoprotein analysis showed a dominant LDL-C fraction, as seen in humans, in all three MMPs. Body weight gain in the MMPs fed with HFCD/SC was greater than in the animal fed with M-ND. At the end of the experiment, computed tomography scans of conscious animals showed increases in subcutaneous and abdominal fat in those fed with HFCD/SC, suggesting the induction of obesity. Atherosclerotic lesions in systemic arteries (including external and internal iliac arteries, abdominal aorta, coronary artery, cerebral arterial circle), fatty changes, and foamy cell infiltration in the liver and spleen were histopathologically observed in the MMPs fed with HFCD/SC. Atherosclerosis and the pathological findings induced by HFCD/SC in MMPs were similar to the pathological changes associated with human atherosclerosis, suggesting that the MMP has the potential to be a suitable animal model for human atherosclerosis.

Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques.

Cynomolgus and rhesus macaques are frequently used in preclinical trials due to their close evolutionary relationships to humans. We conducted an initial screening for genetic variants in cynomolgus and rhesus macaque genes orthologous to human CYP3A4 and CYP3A5. Genetic screening of 78 Indochinese and Indonesian cynomolgus macaques and 34 Chinese rhesus macaques revealed a combined total of 42 CYP3A4 genetic variants, including 12 nonsynonymous variants, and 34 CYP3A5 genetic variants, including nine nonsynonymous variants. Four of these nonsynonymous variants were located at substrate recognition sites or the heme-binding region, domains essential for protein function, including c.886G>A (V296M) and c.1310G>A (S437N) in CYP3A4 and c.1437C>G (N479K) and c.1310G>C (T437S) in CYP3A5. The mutant proteins of these genetic variants were expressed in Escherichia coli and purified. Metabolic activity of these proteins measured using midazolam and nifedipine as substrates showed that none of these protein variants substantially influences the drug-metabolizing capacity of CYP3A4 or CYP3A5 protein. In Indonesian cynomolgus macaques, we also found IVS3+1delG in CYP3A4 and c.625A>T in CYP3A5, with which an intact protein cannot be produced due to a frameshift generated. Screening additional genomes revealed that two of 239 animals and three of 258 animals were heterozygous for IVS3+1delG of CYP3A4 and c.625A>T of CYP3A5, respectively. Some genetic variants were unevenly distributed between Indochinese and Indonesian cynomolgus macaques and between cynomolgus and rhesus macaques. Information on genetic diversity of macaque CYP3A4 and CYP3A5 presented here could be useful for successful drug metabolism studies conducted in macaques.

Characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys.

Recently, troponin T (TnT) and troponin I (TnI) have been reported as suitable biomarkers of myocardial injury for pre-clinical toxicity studies. The purpose of the present study was to investigate the characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys. Initially, tissue distribution of biomarkers was investigated in nine organs (including the heart, liver, and kidneys) collected from naive cynomolgus monkeys. The results showed that TnT and TnI were distributed specifically in the heart, and were not detected in other tissues. Secondly, changes in blood biomarker levels and histopathological changes in cardiac tissue were investigated following myocardial injury induced by concomitant administration of isoproterenol (ISO) and vasopressin (VASO). Compared with pre-dosing, TnT and TnI were markedly increased in the ISO + VASO groups, in which severe histopathological changes including necrosis and vacuolation of muscle fibers were observed. In order to investigate the relationship of biomarker levels with the severity of myocardial injury, Spearman's correlation coefficient was calculated between C(max) and AUC and necrosis and vacuolation scores in the heart. A high correlation between necrosis and vacuolation in the heart and TnT and TnI levels was noted. These results suggest that TnT and TnI possess high sensitivity and specificity for myocardial injury in cynomolgus monkeys, and are useful biomarkers for detection of drug-induced myocardial injury in cynomolgus monkeys.

A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism.

The monkey CYP2C76 gene does not correspond to any of the human CYP2C genes, and its enzyme is at least partly responsible for the species difference occasionally seen in drug metabolism between monkeys and humans. To establish a line and/or lines of monkeys that are expected to show metabolic patterns highly similar to humans, we set out to find monkeys that lacked CYP2C76 activity. By genetic screening of 73 monkeys and a database search of expressed sequence tags, we found a total of 10 nonsynonymous genetic variants in the coding region of CYP2C76, including a null genotype (c.449TG>A). Some of the variants were differently distributed between two animal groups originating from different geographical regions (Indochina and Indonesia). After screening 170 additional genomic samples, we identified a total of eight animals (six males and two females) that were heterozygous for c.449TG>A, which could be used for establishing a homozygous line. If the homozygotes show drug-metabolizing properties more similar to humans than wild-type monkeys, the homozygotes may serve as a better animal model for drug metabolism. The data presented in this article provide the essential genetic information to perform a successful study by using cynomolgus monkeys and present a possible tool to generate a better animal model for drug metabolism.

Sex-related differences in the expression of mfGSTA2, a novel GST identified in cynomolgus monkey (Macaca fascicularis).

Glutathione S-transferase (GST) is one of the most important phase II drug-metabolizing enzymes, catalyzing the conjugation of electrophilic substrates to glutathione. Unlike in humans, a surprisingly limited number of GST genes have been identified in monkeys. The identification of additional GST genes in this model system would prove to be advantageous, because monkeys remain an important predictor of drug effects and toxicities in humans during preclinical studies. In this study, we report the identification and characterization of the following six cDNAs in cynomolgus monkeys: mfGSTA1, mfGSTA2, mfGSTM5, mfMGST1, mfGSTO1, and mfGSTZ1. These cDNAs encode GSTs highly homologous (approximately 95%) to human GST cDNAs. Among these, the mfGSTA1, mfGSTM5, mfMGST1, mfGSTO1, and mfGSTZ1 cDNAs correspond to a single human GST counterpart, whereas the mfGSTA2 cDNA is highly similar to human GSTA1 and GSTA2 cDNAs. An analysis of tissue samples indicates that these GST genes are predominantly expressed in the liver along with some extrahepatic expression as determined by real-time reverse transcriptase-polymerase chain reaction. It is interesting to note that mfGSTA2 is significantly differentially expressed between males and females in the jejunum, where a striking 8-fold higher expression level is observed in males. These results suggest that a potential sex difference in the metabolism of drugs may be mediated by mfGSTA2. This also provides a basis for the investigation of sex-dependent drug metabolism in monkeys.

Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis.

OBJECTIVE: Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. METHODS: Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. RESULTS: In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. CONCLUSION: These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.

Ginsenoside Rb1 reduces neurodegeneration in the peri-infarct area of a thromboembolic stroke model in non-human primates.

Ginsenoside Rb1 (GRb1), a major component of the traditional herb ginseng, has been reported to show a neuroprotective effect in a rodent ischemic model. The purpose of this study was to investigate effects of GRb1 on early and delayed brain injuries in a non-human primate thromboembolic stroke model. Thromboembolic stroke was induced by occlusion of the middle cerebral artery by injection of an autologous blood clot into the left internal carotid artery. GRb1 (300 microg/kg per day, i.v.) and vehicle were administered from 7 days before embolization to the day following embolization (total: 8 times). Neurological deficits were observed at 1, 6, and 24 h and at 2, 4, and 7 days after embolization. At 7 days after embolization, neuron damage in the peri-infarct area and core region were assessed by NeuN, TUNEL, and GFAP staining. GRb1 improved the skeletal muscle coordination score of the neurologic deficits (median: GRb1 vs vehicle = 10 vs 12, P<0.05). In the GRb1 group, positive neurons expressed by NeuN staining were noted in the ischemic peri-infarct area, and TUNEL- and GFAP-positive cells significantly decreased, when compared with vehicle. These results demonstrated that GRb1 ameliorated both early and delayed injuries in the thromboembolic stroke model in non-human primates.

Effects of neonatally administered low-dose diethylstilbestrol on the induction of mammary carcinomas and dysplasias induced by 7,12-dimethylbenz [a] anthracene in female rats.

The aim of this study was to investigate the effects of neonatal administration of diethylstilbestrol (DES) on the induction of mammary carcinomas (MCs) and dysplasias (MDs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female rats. Three different methods of continuous administration of DES (1 microg) were used: 0-14, 0-5 and 6-14 days after birth, and all rats were given DMBA (10 mg) at 50 days after birth. All rats administered DES showed persistent estrus and anovulatory ovaries. In rats administered DES from 0-14 days after birth, neither MCs nor MDs were observed, and serum levels of both estrogen and progesterone were significantly lower than in controls at 100 days after birth. In rats administered DES from 0-5 days after birth, the incidence and number of MCs were significantly lower while the number ofMDs was slightly higher than in controls. In rats administered DES from 6-14 days after birth, the incidence of MCs was equal to that of the controls while the incidence and number ofMDs were significantly higher. These results suggest that neonatal periods of exposure and doses of endocrine disruptors, such as DES, could affect the incidence and progression of MCs and MDs.

Characterization of cynomolgus monkey cytochrome P450 (CYP) cDNAs: is CYP2C76 the only monkey-specific CYP gene responsible for species differences in drug metabolism?

Cynomolgus monkey CYP2C76 does not have a corresponding ortholog in humans, and it is at least partly responsible for differences in drug metabolism between monkeys and humans. To determine if CYP2C76 is the only monkey-specific CYP gene, we identified cynomolgus monkey cDNAs for CYP2A23, CYP2A24, CYP2E1, CYP2J2, CYP3A5, CYP3A8, CYP4A11, CYP4F3, CYP4F11, CYP4F12, and CYP4F45. These CYP cDNAs showed a high sequence identity (93-96%) to the homologous human CYP cDNAs. The monkey CYPs were preferentially expressed in liver among the analyzed tissues. Moreover, all five analyzed monkey CYPs (CYP2A23, CYP2A24, CYP2E1, CYP3A5, and CYP3A8) metabolized typical substrates for human CYPs in the corresponding subfamilies. These results suggest that these 11 monkey CYP cDNAs are closely related to the human CYP cDNAs and thus, unlike CYP2C76, are not apparent monkey-specific cDNAs.

Increased bone mineral density in aged rats with spontaneous mammary dysplasia.

Spontaneous mammary tumors were seen in seven of the 12 breeding female rats aged 2 years. All mammary tumors were diagnosed as mammary dysplasia (MD). Bone mineral contents (BMC) and bone mineral density (BMD) of their lumbar vertebrae and femur were determined using dual energy X-ray absorptiometry (DXA). In rats with MD, body weight (BW), BMD of the lumbar vertebrae and BMC of the femur were significantly higher than in the rats without MD. Although corpus luteum (CL) and follicles were seen in the ovaries of all animals, the number of CL in rats with MD was significantly lower than the rats without MD. It was suggested that high BMD, BW and decreased CL promoted mammary tumors.

Left ventricular volume and function in cynomolgus monkeys using real-time three-dimensional echocardiography.

BACKGROUND: The purpose of this study was to investigate the feasibility of evaluating cardiac function by real time three-dimensional (RT3D) echocardiography in isoflurane-anesthetized male cynomolgus monkeys. Additionally differences between inhibitory effects of beta-blockers and a Ca channel blocker on left ventricular (LV) function were examined. METHODS AND RESULTS: End-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF) in the control (without any drug effect) were not significantly changed by repetitive measurement at a 30-day interval. Propranolol and metoprolol (0.1 and 0.3 mg/kg/10 minutes, i.v.) caused a dose-dependent increase in ESV, but little effect on EDV, resulting in a decrease in EF. Verapamil (0.1 and 0.3 mg/kg/10 minutes, i.v.) increased both EDV and ESV, but decreased EF was noted at 0.3 mg/kg. CONCLUSIONS: These results demonstrate the feasibility of RT3D echocardiography in providing reproducible estimations of LV volume and EF in monkeys when evaluating drugs that may affect cardiac function.

Effects of phosphodiesterase (PDE) inhibitors on human ether-a-go-go related gene (hERG) channel activity.

It is presumed that phosphodiesterase (PDE) inhibitors have two mechanisms for inhibition of hERG currents in the acute applications to cells: direct channel block, and downregulation of human ether-a-go-go related gene (hERG) activities by PKA-dependent pathway mediated phosphorylation through their inhibitory effects against PDE enzymes. However, it is unknown whether PDE inhibition contributes to the inhibitory effects of PDE inhibitors on hERG currents. This study examined the effects of various PDE inhibitors on hERG currents using both the whole-cell and perforated patch-clamp techniques in hERG transfected CHO-K1 cells. The study also investigated the contribution of the PKA-dependent pathway to the inhibitory effects of PDE inhibitors on hERG currents. Of the PDE inhibitors tested, vinpocetine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), vesnarinone, rolipram and dipyridamole decreased hERG currents in a concentration-dependent manner. Vinpocetine and vesnarinone markedly decreased the hERG current with an IC (50)of 0.13 and 20.6 microm, respectively, at comparatively low concentrations. Furthermore, vinpocetine caused a cumulative block of hERG currents. Milrinone, amrinone and zaprinast had no effect on the hERG current up to 100 microm. Of the PDE3 inhibitors (vesnarinone, amrinone and milrinone), only vesnarinone showed an hERG inhibitory effect. The inhibitory effects of vinpocetine and vesnarinone were not significantly affected by the co-application of protein kinase inhibitors. Furthermore, the protein kinase activators had no effect on hERG currents. It is concluded that vinpocetine and vesnarinone block the hERG channel directly, and that the inhibitory effect on intracellular PDE in the PKA-dependent pathway may not be involved in the inhibition of hERG currents in hERG transfected CHO-K1 cells.

Students' ability to organize biochemical and biochemistry-related terms correlates with their performance in a biochemical examination.

Organization is believed to be related to understanding and memory. Whether this belief was applicable in biochemical education was examined about two years after students had experienced biochemistry classes in their first year. The ability of organizing information in biochemistry was judged from the number of correct links of 886 biochemical and biochemistry-related terms to 14 headings, and the level of understanding and memory on biochemical material was determined from the number of correct answers to biochemical items. The result showed a statistically significant positive correlation between the ability of organization and the level of understanding and memory. Thus, biochemistry teachers need to show the organization of what they teach for the help of the students who have a low level of understanding and memory.