Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats.

BACKGROUND: Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. OBJECTIVES: The study aimed to determine the protective efficacy of attenuated Salmonella strain expressing highly conserved Brucella immunogens in goats. METHODS: Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 109 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. RESULTS: Among these vaccines inoculated at 5 × 109 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Real-time polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the non-vaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. CONCLUSIONS: These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage.

Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. We demonstrate here that ubiquitin-conjugating enzyme E2S (UBE2S), a member of the ubiquitin-conjugating enzyme family (E2s), was downregulated by endoplasmic reticulum stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis.IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that the UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin-conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage, and thus UBE2S may contribute to viral oncogenesis.

Hepatitis C Virus Modulates Solute carrier family 3 member 2 for Viral Propagation.

Hepatitis C virus (HCV) exploits an extensive network of host proteins to maintain chronic infection. Using RNA-Seq technology, we identified 30 host genes that were differentially expressed in cell culture grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected solute carrier family 3 member 2 (SLC3A2) for further investigation. SLC3A2, also known as CD98hc, is a member of the solute carrier family and encodes a subunit of heterodimeric amino acid transporter. SLC3A2 and LAT1 constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. In this study, we showed that HCV upregulated both mRNA and protein expression levels of SLC3A2 and this upregulation occurred through NS3/4A-mediated oxidative stress. HCV also elevated SLC3A2/LAT1 complex level and thus mammalian target of rapamycin complex 1 (mTORC1) signaling was activated. We further showed that L-leucine transport level was significantly increased in Jc1-infected cells as compared with mock-infected cells. Using RNA interference technology, we demonstrated that SLC3A2 was specifically required for the entry step but not for other stages of the HCV life cycle. These data suggest that SLC3A2 plays an important role in regulating HCV entry. Collectively, HCV exploits SLC3A2 for viral propagation and upregulation of SLC3A2 may contribute to HCV-mediated pathogenesis.

Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection.

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 109 colony-forming unit (CFU)/mL (Group B) and 5 × 1010 CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

Humulus japonicus Prevents Dopaminergic Neuron Death in 6-Hydroxydopamine-Induced Models of Parkinson's Disease.

Humulus japonicus (HJ), popularly known as Japanese hops, is a traditional herbal medicine widely used for the treatment of pulmonary disease, skin disease, and hypertension in Korea. HJ exerts scavenging effects against reactive oxygen species (ROS), such as superoxide radical, hydroxyl radical, and hydrogen peroxide. Moreover, dysfunction and damage of mitochondria elicited by ROS are of critical importance in the pathogenesis of Parkinson's disease (PD). The present study aimed to examine neuroprotective potential of extracts of HJ using in vitro and in vivo 6-hydroxydopamine (6-OHDA) models. SH-SY5Y cells were cultured to explore the mechanisms for the neuroprotective effect of HJ in vitro. Unilateral 6-OHDA-induced mouse model of PD was established to investigate the neuroprotective effect of HJ on dopaminergic neurons in substantia nigra pars compacta (SNc) and striatum in vivo. Methanol extract of HJ (HJM) significantly attenuated cytotoxicity and the mitochondrial apoptosis pathway caused by 6-OHDA in SH-SY5Y cells. In addition, HJM significantly increased glutathione levels and decreased phosphorylation of ERK1/2 in SH-SY5Y cells exposed to 6-OHDA. In the in vivo study, the administration of methanol or ethanol extract of HJ improved the motor dysfunction and notably reduced dopaminergic cell death and fiber loss in the SNc and striatum caused by 6-OHDA. Our findings demonstrate that HJ may have therapeutic potential to protect dopaminergic neuron degeneration in Parkinson's disease.

Anti-atherogenic effect of Humulus japonicus in apolipoprotein E-deficient mice.

Humulus japonicus (HJ) is used as a traditional medicine in Korea owing to its multiple properties including anti-mycobacterial, antioxidant and antihypertensive effects. The present study aimed to examine the anti­inflammatory and anti-atherogenic effects of a methanol extract of HJ. In lipopolysaccharide-stimulated RAW 264.7 cells, HJ significantly suppressed the mRNA expression and secretion of pro-inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1ß and IL-6)], and the release of inflammatory mediators such as nitrite and prostaglandin E2, together with a concomitant decrease in the mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2. To examine whether HJ is capable of inhibiting experimental atherogenesis in an animal model, we randomly divided apolipoprotein E-deficient (apoE-/-) mice into three groups: mice fed an atherogenic diet plus vehicle (0.5% carboxymethyl cellulose) as the control vehicle group, and mice fed an atherogenic diet plus either 100 (HJ100) or 500 mg/kg (HJ500) of HJ as the experimental groups. After 12 weeks of HJ administration, lipid accumulation and the formation of atherosclerotic lesions in the aorta (en face) and the aortic sinus markedly decreased in the HJ500 group compared with the corresponding values in the vehicle control group. Moreover, monocyte and macrophage infiltration in the aortic sinus was markedly reduced in the HJ500 group. Reverse transcription-quantitative polymerase chain reaction analysis of the whole aorta showed that the mRNA levels of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, CD68 and IL-18 were significantly decreased in the HJ500 group. Collectively, these findings suggest that HJ may suppress atherosclerosis by inhibiting lipid accumulation and the expression of pro-atherogenic factors, and it may be effective at preventing the development of atherosclerosis.

Inhibitory effect of withaferin A on Helicobacter pylori­induced IL­8 production and NF­κB activation in gastric epithelial cells.

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

1'-Acetoxychavicol acetate isolated from Alpinia galanga ameliorates ovalbumin-induced asthma in mice.

The World Health Organization reports that 235 million people are currently affected by asthma. This disease is associated with an imbalance of Th1 and Th2 cells, which results in the upregulation of cytokines that promote chronic inflammation of the respiratory system. The inflammatory response causes airway obstruction and can ultimately result in death. In this study we evaluated the effect of 1'-acetoxychavicol acetate (ACA) isolated from Alpinia galanga rhizomes in a mouse model of ovalbumin (OVA)-induced asthma. To generate the mouse model, BALB/c mice were sensitized by intraperitoneal injection of OVA and then challenged with OVA inhalation for 5 days. Mice in the vehicle control group were sensitized with OVA but not challenged with OVA. Treatment groups received dexamethasone, 25 mg/kg/day ACA, or 50 mg/kg/day ACA for 5 days. Asthma-related inflammation was assessed by bronchoalveolar lavage fluid cell counts and histopathological and immunohistochemical analysis of lung tissues. Our results showed that ACA reduced the infiltration of white blood cells (especially eosinophils) and the level of IgE in the lungs of mice challenged with OVA and suppressed histopathological changes such as airway remodeling, goblet-cell hyperplasia, eosinophil infiltration, and glycoprotein secretion. In addition, ACA inhibited expression of the Th2 cytokines interleukin (IL)-4 and IL-13, and Th1 cytokines IL-12α and interferon-γ. Because asthmatic reactions are mediated by diverse immune and inflammatory pathways, ACA shows promise as an antiasthmatic drug candidate.

Stephania delavayi Diels. inhibits breast carcinoma proliferation through the p38 MAPK/NF-κB/COX-2 pathway.

The nuclear factor κB (NF-κB)/inhibitor of κ kinase-ß (IKKß) signaling pathway is important in tumor promotion and progression. MDA-MB-231 human breast carcinoma cells express COX-2 and show a constitutive phosphorylation of NF-κB. Many non-specific inhibitors of IKKß and NF-κB are used to inhibit tumor promotion and progression. The Stephania delavayi Diels. (S. delavayi Diels.) extract has been reported to safely activate B cell immunity and there is evidence suggesting that it may be a promising new anticancer therapeutic agent. S. delavayi Diels. extract suppressed proliferation of the breast cancer cell lines MDA-MB-231 and MCF-7 by inducing cell death. To aid in the development of the S. delavayi Diels. extract as a therapeutic agent, its mechanisms of action were investigated, in particular its effects on p38 MAPK, NF-κB and COX-2, which play important roles in inflammation and cancer. S. delavayi Diels. stimulated p38 MAPK phosphorylation but reduced NF-κB phosphorylation and COX-2 expression in a dose- and time-dependent manner. Thus, S. delavayi Diels., which appears to act primarily through p38 MAPK/NF-κB/COX-2 signaling in breast carcinomas, may be a potent anticancer agent with target specificity and low toxicity.

Molecular markers of heavy metal toxicity--a new paradigm for health risk assessment.

Last decade has witnessed increased interest in studies dealing with molecular markers of health and disease expression of genes. Specific toxicant "signatures" have been detected using genome base technologies such as microarrays. Further toxins have been classified on the basis of these signatures. Knowledge on these signatures has helped in the identification of novel drug candidates. This review discusses the gene expression studies recently made on arsenic, cadmium, mercury, chromium, lead, copper, nickel, manganese, and other essential elements. Toxicogenomics standards and their organizations have also been briefly described. Although this information can not be considered as complete, recent reports from different laboratories on bacteria, fish, laboratory animals and humans have been summarized. It is expected that toxicogenomics data presented in this review will be helpful in planning and excretion of human health risk assessment programs.

Evaluation of the toxic potentials of a new camptothecin anticancer agent CKD-602 on fertility and early embryonic development in rats.

This study examined the potential adverse effects of a new camptothecin anticancer agent, CKD-602, on the fertility and early embryonic development of Sprague-Dawley rats. Ninety-six rats of each gender were divided into four groups: three treatment groups and a control group. CKD-602 was administered intravenously to male rats at 0, 4.7, 14, and 42 microg/kg from 63 days prior to mating until the end of the mating period, and to female rats from 14 days before mating until day 6 of gestation. All the males were sacrificed after the end of the 14-day mating period, while all the females were subjected to a caesarean section on day 15 of gestation. In the high dose group, a high incidence of hair loss was observed in both genders. A decrease in the level of food consumption, followed by a decrease in body weight gain was also observed in both genders. At the scheduled necropsy, the gross postmortem examinations revealed an increase in the incidence of thymic atrophy, paleness of the thoracic and abdominal organs in both genders and an increase in the serum testosterone concentration. In addition, there was a decrease in the thymus weight of the males and an increase in the liver, spleen, kidneys, lung, and heart weights of the females. There was an increase in the number of fetal deaths and post-implantation losses as well as a decrease in the litter size found at the caesarean section of the dams. No treatment-related effect on the histopathological findings, sexual cycle, pre-coital time, mating index, fertility index, pregnancy index, and sperm parameters was observed. There were no adverse effects on the general findings and reproductive performance of the parent animals and early embryonic development in the low and medium dose groups. Overall, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are believed to be 14 microg/kg for both general toxicity and early embryonic development, and more than 42 microg/kg for the reproductive performance of the parent animals.

A novel missense mutation in the mouse hairless gene causes irreversible hair loss: genetic and molecular analyses of Hr m1Enu.

A novel autosomal recessive mutant was produced using N-ethyl-N-nitrosourea mutagenesis. The characteristics of the mutant mice included progressive irreversible hair loss within a month of birth, wrinkled skin, and long curved nails. Linkage analysis revealed that the causative gene is linked to D14Mit193 on chromosome 14. Sequence analysis of the complete cDNA of the candidate gene, hairless (Hr), identified a homozygous G-to-T transition at nucleotide 3572, leading to the substitution of glycine by tryptophan, designated Gly960Trp. This missense mutation occurs in the vicinity of repression domain 3 of the hairless protein (HR). This allele was named Hr(m1Enu). The relative amounts of Hr mRNA and HR protein determined by real-time PCR and Western blot analyses, respectively, were slightly elevated in the mutant mice. Quantitative real-time PCR analysis revealed the increased expression of Kc1 and Vdr in the mutant mice, whereas the expression of Nrs1 and Krtap16-6 was decreased. These results suggest that the Gly960Trp substitution in HR protein in Hr(m1Enu) mice may alter the function of HR as a transcriptional corepressor.

Characteristics of home cage locomotion in cynomolgus monkeys.

The profiles of home cage locomotor activity and its rhythmicity were investigated using a camera system for 4 consecutive days in cynomolgus monkeys. Nine male and nine female cynomolgus monkeys were used in this study, and were found to have the same profile in nocturnal behavior in that they were mostly inactive during the night. The locomotor activity of both sexes showed a normal activity and inactivity rhythm, which had an inverse relationship between normal activity and inactivity, showing a fluctuation profile of being higher in the light cycle and lower in the dark cycle. The four-day locomotion time of the females was higher than that of the males in the dark period. The females also showed a higher locomotion count and D/L ratio in the dark period than the males during the 4 consecutive days. These findings indicate that the females have greater locomotor activity in the dark period than that of the males. The present findings may serve as useful basic data for the behavioral assessment of overall animal movements in toxicological studies using monkeys.

Indirect indicator of transport stress in hematological values in newly acquired cynomolgus monkeys.

Indicators of transport stress were investigated in blood parameters of five male cynomolgus monkeys obtained from abroad. They underwent air and ground travel-related stress in transport cages for a 15-hour transit time. On arrival, hematological parameters of white blood cell count, red blood cell count, hemoglobin concentration, and hematocrit values were within the limits of reference range, indicating that these parameters were not typical changes derived from transport stress loading. An increase in neutrophil-to-lymphocyte (N/L) ratio with a marked increase in neutrophils and a decrease in lymphocytes was observed on arrival, and the increased N/L ratio returned approximately to the normal level 1 week after arrival. The serum cortisol level markedly increased on the day of arrival and it returned to normal 1 week after arrival. These findings indicate that the transport process was stressful for animals, showing increases in N/L ratio as well as cortisol level. Thus, it is possible that an increase in N/L ratio may be utilized as an indirect indicator of transport stress in newly acquired cynomolgus monkeys, as it has the similar pattern of change in cortisol with an increased cortisol level on the day of arrival.

Effects of combined treatment with sildenafil and itraconazole on the cardiovascular system in telemetered conscious dogs.

Sildenafil, a potent PDE5 inhibitor, is widely prescribed as a treatment of erectile dysfunction. Itraconazole is an inhibitor of CYP3A4, a metabolic enzyme of sildenafil. In the current study, we investigated the effects of single treatment with sildenafil and combined treatment with sildenafil and itraconazole on blood pressure, heart rate, and QT interval in conscious beagle dogs. After a transmitter was implanted to beagle dogs for conscious state experiments, a single oral dose of sildenafil was administered to the beagle dogs at dose levels of 3, 15, and 30 mg/kg. Blood pressure, heart rate, and lead II ECG were measured prior to dosing and at 0.5, 1, 2, 4, 6, and 24 h postdosing. In the study of combined treatment with sildenafil and itraconazole, the 100 mg/kg dose of itraconazole was orally administered 1 h prior to oral administration of sildenafil. No changes in blood pressure were observed at any doses in animals receiving either single treatment with sildenafil or combined treatment with sildenafil and itraconazole. Increased heart rate from 0.5 h to 6 h postdosing and decreased QT interval were observed in animals receiving single treatment with sildenafil at 15 or 30 mg/kg. When 30 mg/kg of sildenafil was coadministered with 100 mg/kg of itraconazole, drug-related effects such as increased heart rate and decreased QT interval were significantly enhanced as compared to sildenafil-alone administration at 6 h postadministration. These results demonstrated that increased heart rate and decreased QT interval, the adverse effects of sildenafil, were enhanced and prolonged when sildenafil was coadministered with itraconazole. Therefore, caution should be taken when sildenafil is coadministered with itraconazole, a CYP3A4 inhibitor, or when administered to elderly patients or patients with hepatic or renal impairment who cannot metabolize and excrete sildenafil normally.

Embryotoxic effects of CKD-602, a new camptothecin anticancer agent, in rats.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce embryotoxicity was investigated in the Sprague-Dawley rat. One hundred mated females (sperm in vaginal lavage=day 0) were distributed among three treatment groups and a control group. CKD-602 was administered intravenously at dose levels of 0, 5, 20 and 80 microg/kg/d to pregnant rats from days 6 to 15 of gestation. The vehicle control rats received an equivalent volume of 1 ml distilled water with d-mannitol 50mg and tartaric acid 0.06 mg. All dams were subjected to the caesarean section on day 20 of gestation. There were no signs of maternal toxicity or embryotoxicity at 5 microg/kg/d, but at 20 microg/kg/d, there was an increase in relative brain weight. At 80 microg/kg/d, reduced food intake, suppressed body weight and increased weight of spleen were observed in dams. An increase in the resorptions and dead fetuses, a decrease in litter size, fetal and placental weights were also found. In addition, various types of external, visceral and skeletal malformations occurred. Characteristic malformations included absent eye bulge, agnathia, dilated cerebral ventricle, anophthalmia, absent thoracic centrum, fused vertebral arch, fused rib, among others. Visceral and skeletal variations were observed. Retarded ossification of several skeletal districts and delayed ossification of sternebrae, metatarsals and sacrocaudal vertebrae were also observed. The results show that CKD-602 is embryotoxic and teratogenic at a minimally maternally toxic dose, i.e. at 80 microg/kg/d in rats. The no-observed-adverse-effect level (NOAEL) of CKD-602 for developmental toxicity was considered to be 20 microg/kg/d, however, the NOAEL for maternal toxicity was 5 microg/kg/d.

Hematological and serum biochemical values in cynomolgus monkeys anesthetized with ketamine hydrochloride.

The effects of ketamine anesthesia on both hematological and serum biochemical variables were investigated in 19 male and 15 female cynomolgus monkeys. Blood samples were obtained from the cephalic vein within 30 minutes of an intramuscular injection of ketamine hydrochloride (10 mg/kg). Ketamine anesthesia caused a reduction in leukocyte counts and a significant reduction in lymphocytes percentages. Ketamine anesthesia also increased the serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine phosphokinase (CPK), but reduced the serum concentrations of glucose, inorganic phosphate, sodium and potassium. The alterations of hematological and serum biochemical values will be discussed. These alterations should be considered when designing studies for and interpreting data from cynomolgus monkeys.

Effects of CKD-602, a new camptothecin anticancer agent, on pregnant does and embryo-fetal development in rabbits.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce developmental toxicity was investigated in the New Zealand White rabbit. Seventy-two artificially inseminated females (artificial insemination=day 0) were distributed among three treatment groups and a control group. CKD-602 was at dose levels of 0, 0.024, 0.048, or 0.096 mg x kg(-1) x day(-1) administered intravenously to pregnant does from days 6 to 18 of gestation. All does were subjected to caesarean section on day 28 of gestation. At 0.096 mg x kg(-1) x day(-1), 2 cases of abortion and 3 cases of death in pregnant rabbits were found in late gestation. In addition, an increase in the embryonic resorptions and a decrease in the litter size were found. At 0.048 mg x kg(-1) x day(-1), a single doe aborted on gestational day 26. An increase in the embryonic resorptions and fetal morphological alterations and a decrease in the litter size were also found. There were no signs of maternal toxicity or developmental toxicity at 0.024 mg x kg(-1) x day(-1). The results show that 13-day repeated intravenous dose of CKD-602 during the major organogenetic period in rabbits produces increased incidence of abortion and death, increased number of embryonic resorptions and fetal morphological alterations, and decreased litter size at dose levels of above 0.048 mg x kg(-1) x day(-1). In the current experimental conditions, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are considered to be 0.048 mg x kg(-1) x day(-1) for does and 0.024 mg x kg(-1) x day(-1) for embryo-fetal development.

Safety pharmacology of DW-224a, a novel fluoroquinolone antibiotic agent.

To investigate the safety pharmacology of a novel fluoroquinolone antibiotic agent, DW-224a, on the vital functions, we studied its effects on the central nervous system, cardiovascular system and respiratory system. To determine the effects on the central nervous system, we used a modified Irwin's test at each time point after oral administration of DW-224a to mice. In this test, we found that the treatment of test article had no effects on motor activity, behavioral changes, coordination, and sensory/motor reflex responses. The effects of DW-224a on the cardiovascular system were evaluated by the use of a telemetry system in beagle dogs. At 360 min post-DW-224a (100 mg/kg) administration, QT interval prolongation was observed. However, there were no changes in heart rate, blood pressure, and electrocardiogram at all doses and each time points with the exception of QT-interval prolongation as compared to the vehicle treated group. In experiments designed to determine the changes of respiratory function in rats, we found no changes at all doses and time points. We investigated the effects of DW-224a on the human ether-a-go-go-related gene (hERG) mediated potassium currents to evaluate its potential to induce QT interval prolongation. When whole cell patch-clamp electrophysiology was used, DW-224a inhibited hERG currents with IC50 of 218.12 +/- 39.51 microM though its effect was less potent than that of E-4031, a positive control drug. Our data suggested that DW-224a showed no adverse effects on the central nervous system, cardiovascular system, and respiratory system, with the exception of the effect on the QT interval prolongation.

Fine localization of Nefl and Nef3 and its exclusion as candidate gene for lens rupture 2(lr2).

Cataract causing lr2 gene is found in the CXSD mouse, which is a recombinant inbred strain of BALB/c and STS mice. For the process of positional cloning of lr2, several candidate genes were selected in the middle region of chromosome 14, but most of them were excluded by combination of recombination and homozygosity mapping. Components of neurofilament proteins, neurofilament light polypeptide (Nefl) and neurofilament3 medium (Nef3), were linked to D14Mit87 which was not separated from the lr2 locus in the homozygosity mapping. When the expression levels of Nefl and Nef3 in eyes were compared in CXSD and BALB/c mice, there were no differences in expression levels. The cDNA sequences of the two genes from CXSD, BALB/c and STS mice were subsequently compared. Several nucleotide differences in cDNA sequences were detected between the mice strains but the majority of the changes were silent mutations that did not alter the amino acids. The sole amino acid difference, E567K in the glutamate rich region of Nfm, between BALB/c and CXSD was found to be a simple genetic polymorphism because the same substitution existed in STS, a non-cataract mouse strain. Therefore we excluded Nefl and Nef3 from the candidate genes for lr2 based on expression and mutation analyses.