The supernatants were collected for the assays Activation of cas

The supernatants were collected for the assays. Activation of caspase-9 is based on hydrolysis of the substrate n-Acetyl-Leu-Glu-His-Asp7-amido-4-trifluoromethylcoumarin

(Ac-LEHD-AFC) by caspase-9, resulting in the release of fluorescent 7-amino-4-trifluromethyl coumarin (AFC) moiety, while hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3, resulted in the release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety. Reaction were performed AZD0530 in vivo in buffer containing supernatant proteins (50 μg/sample for caspase-9 and 25 μg/Sample for caspase-3) and caspase substrates, at 37 °C for 2 h, followed by fluorimetric detection using the excitation and emission wavelengths of 400/505 nm and 360/460 nm for caspase-9 and caspase-3, respectively. The experimental data were evaluated using the analysis of variance (ANOVA), followed by the Dunnet test for the

comparison of the various treated groups with their controls, using the GraphPrism program, version 5.1 for Windows. The results were considered statistically significant at p < 0.05. The results showed that the congener BDE-99 inhibited cell proliferation after 24 and 48 h CT99021 supplier of incubation, showing significant effects at the higher concentrations tested (18.22 ± 6.42% and 41.77 ± 10.5% for 10 μM and 25 μM, respectively) after 24 h of exposure. A significant effect was observed for concentrations as low as 0.5 μM when the cells were exposed to the compound for 48 h (Fig. 1). Moreover, it was also demonstrated that the congener BDE-99 was able to induce a decrease in cell viability during both incubation periods for almost all the concentrations that lead to an inhibition of HepG2 cell proliferation (Fig. 2). These results demonstrated that there is a correlation between the effects observed in the first two experiments. Fig. 3 shows

FAD the effect of BDE-99 on the mitochondrial membrane potential (MMP). The MMP also changed after exposure to 10 and 25 μM of the compound for 24 h. This effect was intensified after 48 h of incubation, showing significant effects in concentrations as low as 0.5 μM. Similar results to those of the MMP assay were observed in the ROS accumulation test. Fig. 4 shows a significant increase in ROS accumulation after 24 h of incubation with BDE-99 at the highest concentration tested (25 μM). However when the effect was evaluated for 48 h, the exposure to 5 μM of the compound was sufficient to significantly increase ROS accumulation in the HepG2 cells. To better understand the mechanism by which BDE-99 induces cell death, we evaluated the exposure of phosphatidyl serine on the outer cell membrane by assessing the FITC-annexin-V positive cells. Fig.

While this model revealed distance to active gas wells as exhibit

While this model revealed distance to active gas wells as exhibiting a negative control on methane concentrations, this does not indicate that gas wells are definitively causing higher methane concentrations; since these gas wells are inherently

producing from methane-rich strata this may indicate that methane concentrations are higher in close proximity to these particular formations, but it is not possible to discern the cause of the relationship without further investigation. Crizotinib Sulfate was also found to be negatively correlated to methane in this model, providing further evidence for some biologically driven methane production. This follows thermodynamic principles given that sulfate reduction yields more energy than methanogenesis; thus methane is produced when sulfate concentrations are reduced ( Schlesinger, 1997).

The three most significant variables in the model (p < 0.001) – hardness, sodium, and barium – together could explain 77% of the observed variation in dissolved methane. We acknowledge that including both sodium and hardness could introduce some multicollinearity into the model since sodium and hardness (as the sum of magnesium and calcium) tend to be negatively correlated; however, we find that removing either sodium or hardness from the model strongly reduces its predictive power, indicating that they are both contributing to it. These results are informative for better understanding the drivers of observed methane patterns. Sodium was positively correlated with methane concentrations

and hardness was negatively correlated with methane. This is consistent with previously described geochemical patterns that indicated that methane likely resulted from bedrock-groundwater interactions and lengthy residence times. The positive correlation between barium and methane concentrations also indicates that there is a geologic relationship with methane patterns. While barium can be present Tau-protein kinase due to human activities, including use in gas well drilling mud, it also is naturally present in geologic formations. Barium has been found in western New York to be primarily sourced from the mineral barite (BaSO4) ( Moore and Staubitz, 1984), which may also be present in formations underlying this study region. Using measured environmental variables, regression models for methane were developed with high explanatory power. While these models were developed using data from Chenango County, New York, they could have similar predictive power in nearby areas of New York and Pennsylvania with similar shale-dominated bedrock geology. With other studies in New York observing some higher methane concentrations than here (Kappel and Nystrom, 2012 and Heisig and Scott, 2013), it will be important to refine this model to try to better capture these patterns. In the future, it would also be beneficial to work toward creating improved regression models based on more easily quantified parameters (e.g.

, 2001a) For most study catchments, 210Pb-based background lake

, 2001a). For most study catchments, 210Pb-based background lake sedimentation rates (1900–1952 medians) ranged from about 20–200 g m−2 a−1 (Fig. 2). Only the mountainous catchment regions, excluding the Vancouver Island-Insular Mountains, contained a significant number of lakes with background rates exceeding 200 g m−2 a−1. A few lakes in the Coast and Skeena mountains exhibited very high background

rates (>1000 g m−2 a−1). Relatively low rates (<20 g m−2 a−1) were observed for most of the Insular Mountain lake catchments. Environmental changes experienced by the lake catchments in the study are described by our suite of land use and climate change variables p38 MAPK inhibitors clinical trials (Table 1). Cumulative intensities of land use increased steadily for study catchments overall, especially shown by the trends in road density (Fig. 3). For buy AUY-922 the

late 20th century, averaged road densities were highest for the Insular Mountains (up to 1.90 km km−2) and lowest for the Coast Mountains (up to 0.26 km km−2). By the end of the century, other region catchments had intermediate road densities ranging between 0.46 and 0.80 km km−2. Land use histories for individual study catchments were temporarily variable. The percentage of unroaded catchments over the period of analysis ranged from 0 to 44% for the Insular and Coast mountain regions, respectively. Road densities in excess of 2 km km−2 were observed for several Insular Edoxaban Mountain catchments, one Nechako Plateau catchment, and one Nass Basin catchment. Land use variables are all positively correlated,

with highest correlations occurring between road and cut density and between seismic cutline and hydrocarbon well density (Foothills-Alberta Plateau region only). Temperature and precipitation differences among regions and individual lake catchments are related to elevation, continentality, and orographic setting. Temperature data show interdecadal fluctuations and an increasing trend since the mid 20th century for all regions (Fig. 3). Precipitation has increased slightly over the same period and high correlations are observed among temperature and precipitation change variables. Minor regional differences in climate fluctuations include reduced interdecadal variability in highly continental (i.e. Foothills and Alberta Plateau) temperatures during the open-water season and in coastal (i.e. Insular and Coast mountain) temperatures during the closed-water season, as well as greater interdecadal variability in coastal precipitation between seasons and regions. Sedimentation trends during the second half of the 20th century are highly variable between lake catchments (Fig.

e , the Alpine Space projects ALPFFIRS (fire danger rating and pr

e., the Alpine Space projects ALPFFIRS (fire danger rating and prediction; and MANFRED (management adaptation strategies to climate change; This recent interest for the fire issue has been arising from new evidences

observed in fire regime dynamics; for example, the extremely hot summer 2003 and other hotspots occurring during 2006, demonstrated that under suitable fire weather conditions it can burn in Austrian forests nearly everywhere (Gossow et al., 2007), and gave rise to a systematic data collection still not addressed (Arpaci et al., 2013). Furthermore, regional and national fire organizations are providing costly fire fighting Everolimus concentration services and must provide a safe work environment to fire-fighters. In this key, important steps have been also moved in the direction of cooperation at the national, or regional, boundaries. In fact, fire management

in the Alpine region is fragmented in many different fire organizations; only in Italy, seven regional authorities share 100,000 km2 of buy C646 land to manage, what makes also challenging to get harmonized forest fire datasets as to provide an exhaustive picture at Alpine level. Global change, i.e., current changes in land-use, climate and society, poses several new issues and challenges to fire management in Europe, including the Alpine area (Fernandes et al., 2013). In addition to the long-term ongoing land-use change, pronounced climatic shifts are predicted for mountainous areas of Europe (Reinhard et al., 2005 and Moriondo et al., 2006). Climate warming is likely to Chlormezanone interact with land-use changes and alter fire regimes in the Alpine region in unpredicted ways (Schumacher and Bugmann, 2006 and Wastl et al., 2012), with potentially serious consequences on ecosystem services, including economic losses and social

impacts. Higher frequency of exceptional droughts and heat waves in the Alps may increase the occurrence of high intensity fires of relatively large size, particularly on southern slopes (Moser et al., 2010, Ascoli et al., 2013a and Vacchiano et al., 2014a). Unlike in other regions, for instance the Mediterranean basin, the future scenario of large wildfires in the Alps is more likely to be similar to the third generation (sensu Castellnou and Miralles, 2009) than to the fourth and fifth ones. The reason lies in the relatively milder fire-weather, also in a climate change scenario, less flammable fuels and the lower extent and different structure of the wildland–urban interface. Despite this, a change towards the third generation might entail negative consequences on soil stability ( Conedera et al., 2003) and timber quality ( Beghin et al., 2010 and Ascoli et al.

The authors effectively balance between these two endpoints of hi

The authors effectively balance between these two endpoints of historical ignorance. The text conveys a great deal of information, but is quite accessible to a non-specialist reader interested in natural history and environmental change. The scholarship is thorough, balanced, and impeccable, and the writing is engaging. The text is nicely illustrated with diagrams, historic maps, and matched

historic and contemporary photographs. The matched photographs are particularly effective because juxtaposed on the same page, facilitating visual comparison of changes through time. The title refers to irreversible changes to the river through the Tucson Basin, mainly from urbanization and groundwater overdrafts. The authors conclude the book by noting that, although “the Santa Cruz River of old can be neither CB-839 in vivo restored nor revived” (p. 182), the river can be managed to minimize flood risk and maximize ecosystem services. This “will require both an acknowledgement of history and fresh perspectives on how to manage rivers and floodplains in urban areas of the Southwest” (p. 182). This

book provides a firm foundation for such a path forward. “
“Lagoons are widely distributed throughout the world ocean coasts. They constitute about 13 percent of the total world coastline (Barnes, 1980). They represent 5.3 percent of European coastlines (Razinkovas et al., 2008), with more than 600 lagoons in the Mediterranean area alone (Gaertner-Mazouni and De Wit, 2012). From geological and geomorphological viewpoints, coastal lagoons are ephemeral systems that can change in time (becoming estuaries or infilled; Davies, 1980). The nature of this change depends on the main factors controlling their evolution, such as mean sea level, hydrodynamic setting, river sediment supply and pre-existing topography. As observed by Duck and da Silva (2012), however, these coastal forms are seldom if ever allowed to evolve naturally. They are often modified by Dichloromethane dehalogenase human intervention typically

to improve navigability or in attempts to maintain the environmental status quo. By controlling their depth and topography, humans have exploited them for many centuries for food production (fisheries, gathering of plants and algae, salt extraction, aquaculture, etc.) (Chapman, 2012). These modifications can transform radically the lagoon ecosystem. Human activities have also influenced the evolution of the Lagoon of Venice (Italy) over the centuries (Gatto and Carbognin, 1981, Favero, 1985, Carbognin, 1992, Ravera, 2000, Brambati et al., 2003 and Tosi et al., 2009). Together with the historical city of Venice, the Venice Lagoon is a UNESCO World Cultural and Natural Heritage Site. The first human remains in the lagoon area date back to the upper Paleolithic age (50,000–10,000 BC). The lithic remains found in Altino (Fig.

2D) Both the pharmacological AMPK inhibitor compound C (Figs  3A

2D). Both the pharmacological AMPK inhibitor compound C (Figs. 3A, B)

and transfection with AMPK shRNA (Figs. 3C, D) also suppressed osteogenic differentiation of hDP-MSC. The shRNA silencing of AMPK early during hDP-MSC activation (day 1) prevented activation of AMPK/Raptor and restored the activity of the negative autophagy regulators mTOR/S6K, resulting in the inhibition of LC3-II increase (Fig. 3E). On the other hand, late inhibition of AMPK at day 3 by compound C completely failed to block osteogenic differentiation (day 7 ALP values: 2.07 ± 0.10 and 2.11 ± 0.06 in control and compound C-treated hDP-MSC, respectively; n = 3, p > 0.05). Similarly, autophagy inhibitors bafilomycin and chloroquine were also ineffective in preventing hDP-MSC differentiation if added at day GDC0199 Bcl-2 phosphorylation 3 (ALP values: 1.82 ± 0.15, 1.76 ± 0.10 and 1.74 ± 0.08 in control, bafilomycin and chloroquine-treated hDP-MSC; n = 3, p > 0.05). Therefore, it appears that early AMPK-dependent autophagy is required for optimal differentiation of hDP-MSC to osteoblasts. Finally, we explored the role of Akt/mTOR activation in AMPK-dependent osteogenic differentiation of hDP-MSC. The selective Akt antagonist DEBC (Figs. 4A, B), as well as pharmacological mTOR inhibitor rapamycin (Figs. 4C, D) or

transfection with mTOR siRNA (Fig. 4E), inhibited hDP-MSC differentiation to osteoblasts, as confirmed by alkaline phosphatase assay and RT-PCR/immunoblot analysis of osteocalcin, Runx2 and BMP2. Similar effect, although somewhat second less pronounced, was observed even if DEBC or Akt were added at day 3 (day 7 ALP values: 1.47 ± 0.09, 1.20 ± 0.05 and 1.28 ± 0.01 in control, DEBC- or rapamycin-treated hDP-MSC; n = 3, p < 0.05) or even day 5 of differentiation (data not shown). The suppression of Akt phosphorylation

in DEBC-treated hDP-MSC prevented activation of mTOR/S6K at day 5 of differentiation, while AMPK activation remained largely unaffected ( Fig. 5A). Both the mTOR siRNA and rapamycin reduced the phosphorylation of mTOR/S6K without affecting the activation of either Akt or AMPK ( Figs. 5A, B). Finally, AMPK downregulation with compound C or shRNA mimicked the inhibitory effects of DEBC on the activation status of Akt and mTOR/S6K in differentiating hDP-MSC at day 5 ( Figs. 5A, C), indicating AMPK as an upstream signal for Akt activation and subsequent increase in mTOR/S6K activity. These data demonstrate that the optimal osteogenic transformation of hDP-MSC requires AMPK-dependent phosphorylation of Akt and consequent activation of mTOR at the latter stages of differentiation. The present study demonstrates a central role of the intracellular energy sensor AMPK in the osteogenic differentiation program of hDP-MSC.

In all instances the significance level was set at 5% (p < 0 05)

In all instances the significance level was set at 5% (p < 0.05). The treatment with LASSBio 596 per os significantly avoided the influx of PMN cells, airspaces collapse ( Table 1), as well as the rising of TNF-α, IL-6 and IL-1β levels in lung and liver tissues ( Fig. 1). Additionally, the elevated pulmonary mechanical parameters ( Fig. 2),

the presence of alveolar collapse, edema and alveolar septum thickness present in TOX Afatinib in vitro ( Fig. 3) were not observed in the LASS group. LASS and CTRL did not differ in any parameters studied. MCYST-LR was not detected in lung tissue, but free MCYST-LR was similarly detected in liver tissue in both LASS and TOX groups (Fig. 4), but not in CTRL. The disarray in liver architecture expressed by necrosis, inflammation, high degree of binucleated hepatocytes, cytoplasmatic vacuolization, dilated sinusoidal Epacadostat ic50 spaces and steatosis were less evident in the LASS than TOX group (Fig. 5). The main findings of the present study were: 1) the treatment with LASSBio 596 per os avoided lung and liver inflammation and pulmonary mechanical dysfunction found in TOX mice; 2) in addition a qualitative

improvement in liver structure was observed. It is known that MCYST-LR contamination leads to a direct liver insult followed by damage on several organs such as lung, kidney and intestine (Ito et al., 2001). However, acute lung injury related to MCYST-LR exposure is scantly assessed. Our group previously reported that respiratory system can be injured even by sub-lethal doses of MCYST-LR administered by pulmonary or extrapulmonary routes (Picanço et al., 2004 and Soares et al., 2007). This suggests that these toxins even Cediranib (AZD2171) when administered at low concentrations may be present in the circulation and directly trigger a network of inflammatory responses mediated by immune cells in many organs (Wang et al., 2008). MCYST-LR inhibits PP1 and 2A, yielding an unusual cellular protein phosphorylation, and, thus, possibly activates protein kinase C. The latter activates phospholipase

A2 and cyclooxygenase, triggering inflammation (Nobre et al., 2001, Nobre et al., 2003 and Kujibida et al., 2006). Moreover, the influx of PMN also yields to the release of pro-inflammatory cytokines and reactive oxygen species (ROS) that adds to the development of tissue injury (Moreno et al., 2005). When injected intraperitoneally LASSBio 596 seems effective in different models of acute lung injury, such as endotoxin model induced by lipopolysaccharide of E. coli, allergic sensitization to ovalbumin, ischemia and reperfusion, and also in acute lung injury induced by MCYST-LR ( Rocco et al., 2003, Campos et al., 2006, Morad et al., 2006 and Carvalho et al., 2010). In order to circumvent MCYST-LR undesirable effects, we have recently reported a possible treatment of pulmonary damage induced by acute exposure to MCYST-LR by the intraperitoneal administration of LASSBio 596 or dexamethasone ( Carvalho et al., 2010).

These results suggest that the differences between both BCG-treat

These results suggest that the differences between both BCG-treated groups selleck kinase inhibitor were driven by difference in sickness indicators, and in particular the capability to recover lost weight and to display horizontal locomotor activity. The difference between BCG-treatment groups in weight change was detected in the univariate analysis meanwhile the difference in horizontal locomotor activity was highlighted by linear discriminant analysis. These results confirm the additional insight offered by complementary approaches. Furthermore, mouse number 22 pertaining to group BCG10 was classified in the correct group. The

tail suspension test measurement of mouse number 22 was the lowest of the

group; however the value was not distant from the second lowest measurement. Using the nearest neighbor mouse and the seven sickness and depression-like indicators, all mice were correctly assigned to the correct BCG-treatment group. Using the information on all seven sickness and depression-like indicators from the two most proximal neighbor mice, all BCG0 mice and all BCG10 mice were discriminated into the corresponding groups. Among the BCG5 group, four mice were assigned to the correct group and two mice were assigned to the BCG10 group. This result speaks to the mouse-to-mouse variability within BCG-treatment group and the between learn more within group variation. The two miss-classified BCG5 mice exhibited profiles similar to BCG10 mice. This result supports previous Metabolism inhibitor reports of varying levels of susceptibility of mice to BCG-challenge Laboratory effects on behavioral indicators including apparatus, test procedure, order of tests, and experimenter error have been widely recognized (Chesler et al., 2002a, Chesler et al., 2002b and Brown, 2007). Behaviors measured by a number of tests appear to be more sensitive to the previous testing experience than others (McIlwain et al., 2001). Alternative tests to measure sickness and depression-like

indicators could offer complementary information on the association between BCG-treatment and behavior. Supporting this, multivariate approaches are well suited to handle additional behavioral indicators. However, care must be exercised to ensure that the order of a larger number of tests on the same subjects does not influence the measurements. Also, consideration of multiple mouse strains would enable the testing of synergistic or antagonistic relationships between strain and BCG-treatment on behavioral indicators in addition to the detection of treatment effects that are common to all strains. Recommendations for supervised and unsupervised analyses include the availability of at least five observations per variable (Stevens, 2009).

Specific murine IgG2a isotype controls were used to monitor non-s

Specific murine IgG2a isotype controls were used to monitor non-specific binding. Stained peritoneal cells were washed

with PBS containing 2% fetal bovine serum (FBS), pelleted by centrifugation at 400 × g and fixed with PBS containing 1% (w/v) paraformaldehyde. A total of 30,000 events were acquired (FACSCanto™; Becton Dickinson, CA, USA) using the FACS Diva software (version 6.1.3) for data acquisition and analysis. Data were expressed as the mean ± SEM. Statistical variations were analyzed using multi-factorial ANOVA. P < 0.05 was considered statistically EPZ015666 research buy significant. The response of mice to the i.p. injection of Ts2 or Ts6 was studied by evaluating the influx of leukocytes into the peritoneal cavity. Ts2 or Ts6 i.p. inoculation in mice induced an increase of total leukocyte (Fig. 1A) and neutrophil (Fig. 1B) numbers Crizotinib molecular weight in the peritoneal cavity throughout the experimental time course (4, 24, 48 and 96 h). The mononuclear cells were increased after 4 and 96 h post Ts2 injection compared to mice inoculated with PBS. Ts6 increased

mononuclear cells only after 96 h (Fig. 1C). We also determined the acute phase protein levels in the peritoneal fluid of mice injected with Ts2 or Ts6 (Fig. 2). Compared to control, the total protein levels of the peritoneal fluid peaked between 24 and 48 h and then decreased at 96 h after injection with Ts2 or Ts6 (Fig. 2). Taken together, these results demonstrated that Ts2 or Ts6 induced an inflammatory response in the peritoneal cavity, mainly during the first 24 h. Fig. 3 shows the profile of inflammatory cytokines released in the peritoneal cavity after the injection with Ts2 or Ts6. We demonstrated that Ts2 and Ts6 altered the release of specific cytokines in a time-dependent manner. Ts2 augmented the release of IL-6, IFN-γ and the regulatory cytokine IL-10 at 4 h (Fig. 3A, D and E, respectively); Carbohydrate at 24 h, however, only IL-10 was increased (Fig. 3E). At 48 h post Ts2 injection, there was an increase in the levels of TNF-α, IFN-γ, IL-10 and IL-4 (Fig. 3B, D, E and F, respectively), while at 96 h, we only observed an increase in TNF-α, IL-1β and IL-10 (Fig. 3B,

C and E, respectively). Additionally, we observed a mild difference in the cytokine profile following the Ts6 injection compared to Ts2. After 4 h, Ts6 increased the release of IL-6, TNF-α, IL-1β and IFN-γ (Fig. 3A–D, respectively), while only the release of IFN-γ was increased at 24 h (Fig. 3D). At 48 h, TNF-α and IFN-γ levels were increased (Fig. 3B and D), while only TNF-α was increased after 96 h (Fig. 3B). For comparison, the changes in the corresponding cytokine release were also measured in a control group of mice that received PBS injection. To determine whether LTB4 and PGE2 are produced as a result of the toxin injection, groups of mice were i.p. inoculated with Ts2 or Ts6 and the peritoneal fluid was collected after 4, 24, 48 and 96 h.

1) and in vivo ( Fig 2) We selected rabbits as the subject anim

1) and in vivo ( Fig. 2). We selected rabbits as the subject animals for testing the safety of PFCs because they are known to have high sensitivity to the effects of i.v. injection of PFCs [9]. The experimental animal protocol was approved by the animal research committees of Jikei University School of Medicine (Tokyo, Japan). Twenty male Japanese white rabbits (2.59 ± 0.14 kg) were divided into three groups: the Control group (n = 6), 2.2 mL/kg of physiological saline i.v. into the auricular vein; the PL

group (n = 8), 25 mg/kg of phospholipid-coated SPNs i.v.; and the AA group (n = 6), 25 mg/kg of SPNs coated with poly aspartic acid derivative i.v. The administered dosage was determined in a previous investigation of rabbit VX tumors in which 30 mg/kg of phospholipid-coated SPNs FK506 order was injected i.v., selleck chemical revealing

severe respiratory side effects in three of seven rabbits, including two animals that did not survive. In the present study, saline and SPNs were injected i.v. via a 22-G catheter (Angiocath, BD Japan, Fukushima, Japan). Anesthesia was maintained by i.m. injection of midazolam (0.04 mg/kg) and medetomidine (0.08 mg/kg). In a clinical study, Krafft et al. reported that flu-like symptoms with light fever and myalgia had occurred when PFC was excreted from the respiratory

system into the air [10]. DOK2 In our study, animals were placed on a temperature-controlled plate and their homeostatic thermal condition was maintained by measuring rectal temperature (mean ± standard deviation = 39.08 ± 0.98 °C) with a rectal digital thermometer (AW-601H and AW-650H; Nihon Koden, Tokyo, Japan). Animals were supplied pure oxygen via a face mask (1 L/min). Measured parameters included arterial blood pressure (ABP) by cuff and SpO2 with pulse rate (PR) by pulse oximeter (BSM-2301; Nihon Koden). Animals awakened spontaneously and were returned to their cages with free access to water and food on a 12-h light–dark cycle in the animal research facility at Jikei University School of Medicine. Neurological evaluation was performed according to a previous experimental report in which rabbits were injected with PFC, the neurological check points were the occurrence of paresis, convulsion, anisocoria, and nystagmus [9]. Biochemical blood plasma examination including hepatobiliary and renal functions, blood lipid were performed at pre-injection, and 1, 4, and 7 days after injection of SPN. Blood samples were taken from the auricular marginal vein.