Light Microscopic and Ultrastructural Characteristics of Heterophil Toxicity and Left-shifting in Green Sea Turtles (Chelonia mydas) from Taiwan.

Heterophil toxic change (TC) and left-shifting (LS) are widely used as indicators of accelerated granulopoiesis. However, the ultrastructure of heterophil TC and LS in sea turtles remain poorly understood. This study aimed to describe the ultrastructural characteristics of sea turtle TC and LS heterophils, compare the staining quality of accessible staining methods, and provide a better understanding of the clinical applications and limitations of heterophil TC and LS examinations. Blood samples were collected from 21 rescued sea turtles from January 2017 to September 2018. Morphologic (n = 22) and ultrastructural (n = 15) examination of TC and LS heterophils were performed, and the qualities of three staining methods (Wright-Giemsa stain, Diff-Quik stain and Liu's stain) were analyzed to diagnose TC and LS heterophils. In addition, the diagnostic values of TC and LS heterophils were examined. Diff-Quik stain was significantly inferior in the assessment of heterophil TC and/or LS comparing to the Wright-Giemsa stain and Liu's stain (Mann-Whitney test, P < 0.001). Microscopic examinations of heterophil TC and/or LS were comparable to transmission electron microscopy examinations (Cohen's kappa coefficient, κ = 1). The correlation between the presence of heterophil TC and/or LS and clinical inflammatory state was weak (Spearman's rank correlation coefficient, rs = 0.171, p = 0.445). In conclusion, this is the first study to describe the ultrastructural characteristics of reptile TC and LS heterophils. Wright-Giemsa stain and Liu's stain were suitable staining methods for the microscopic observations of TC and LS heterophil in sea turtles. Given the poor correlation between TC and/or LS and clinical findings, TC and LS are not a suitable diagnostic indicator of green sea turtles' inflammation status.

Overexpression of the Chromosome Partitioning Gene parA in Azorhizobium caulinodans ORS571 Alters the Bacteroid Morphotype in Sesbania rostrata Stem Nodules.

Azorhizobium caulinodans ORS571 is a diazotroph that forms N2-fixing nodules on the roots and stems of the tropical legume Sesbania rostrata. Deletion of the parA gene of this bacterium results in cell cycle defects, pleiomorphic cell shape, and formation of immature stem nodules on its host plant. In this study, we constructed a parA overexpression mutant (PnptII-parA) to complement a previous study and provide new insights into bacteroid formation. We found that overproduction of ParA did not affect growth, cell morphology, chromosome partitioning, or vegetative nitrogen fixation in the free-living state. Under symbiosis, however, distinctive features, such as a single swollen bacteroid in one symbiosome, relatively narrow symbiosome space, and polyploid cells were observed. The morphotype of the PnptII-parA bacteroid is reminiscent of terminal differentiation in some IRLC indeterminate nodules, but S. rostrata is not thought to produce the NCR peptides that induce terminal differentiation in rhizobia. In addition, the transcript patterns of many symbiosis-related genes elicited by PnptII-parA were different from those elicited by the wild type. Accordingly, we propose that the particular symbiosome formation in PnptII-parA stem-nodules is due to cell cycle disruption caused by excess ParA protein in the symbiotic cells during nodulation.

Ultrastructural changes of goat corpus luteum during the estrous cycle.

The present study was designed to study the ultrastructure of goat corpora lutea (CL, n=10) and structural changes as related to steroidogenic functions during the estrous cycle. The reproduction status of goats was estimated by analyzing serum progesterone concentrations. The CL at various stages was surgically collected. To characterize ultrastructural features associated with steroidogenesis, tissue and cellular structures were studied. Blood supplies were examined based on features of the endothelial cells and capillary structures in the CL. Activated endothelial cells and developing vessels were observed in the early stage, whereas mature endothelial cells, accumulating extracellular matrix fibers, and stabilized vessels were observed in the middle and late stages of assessment. In the late stage of assessment, shrunken goat luteal cells scattered around the capillaries were detected and formed circular regression areas. Features of autophagy and luteal cell apoptosis were noted. In large luteal cells, steroidogenic organelles were present, including microvillar channels, endoplasmic reticulum, and mitochondria. Conformational changes in the endoplasmic reticulum and increased mitochondria with tubular cristae were observed in the early-middle CL transitions. In contrast, mitochondria swelled and the cristae transformed to the lamellar type in the late stage, suggesting that organelle plasticity could contribute to steroidogenesis in goat CL. In conclusion, results suggest angiogenesis occurs in early developing CL and programmed cell death occurred in the late stage of CL assessment in the present study. Structures and quantiles of steroidogenic organelles are correlated with the steroidogenic functions in goats.

Pacific oyster-derived polysaccharides attenuate allergen-induced intestinal inflammation in a murine model of food allergy.

Oyster-derived polysaccharides (OPS) have been shown to modulate the T helper (Th)1/Th2 immunobalance toward the Th1-dominant direction in antigen-primed splenocytes. In the present study, we hypothesized that OPS might attenuate intestinal inflammation associated with food allergy, a Th2-dominant immune disorder. BALB/c mice were sensitized twice with ovalbumin (OVA) absorbed to alum and then repeatedly challenged with intragastric OVA to induce intestinal allergic responses. The mice were administered by gavage with OPS and/or vehicle (distilled water) once/d during the two sensitization phases, and once every other day during the challenge phase. Administration with OPS attenuated OVA challenge-elicited diarrhea, and the infiltration of mast cells in the intestine. OPS demonstrated a protective effect on the reduced ratio of villus length over crypt depth of the intestine in allergic mice. Furthermore, OPS administration markedly attenuated the intestinal expression of the Th2 signature cytokine interleukin-4 (IL-4). Collectively, these results demonstrated the in vivo antiallergic activity of OPS, which is associated with the suppression of allergen-induced intestinal Th2 responses and mast cell activation.

Differentiation of lung stem/progenitor cells into alveolar pneumocytes and induction of angiogenesis within a 3D gelatin--microbubble scaffold.

The inability to adequately vascularize tissues in vitro or in vivo is a major challenge in lung tissue engineering. A method that integrates stem cell research with 3D-scaffold engineering may provide a solution. We have successfully isolated mouse pulmonary stem/progenitor cells (mPSCs) by a two-step procedure and fabricated mPSC-compatible gelatin/microbubble-scaffolds using a 2-channel fluid jacket microfluidic device. We then integrated the cells and the scaffold to construct alveoli-like structures. The mPSCs expressed pro-angiogenic factors (e.g., b-FGF and VEGF) and induced angiogenesis in vitro in an endothelial cell tube formation assay. In addition, the mPSCs were able to proliferate along the inside of the scaffolds and differentiate into type-II and type-I pneumocytes The mPSC-seeded microbubble-scaffolds showed the potential for blood vessel formation in both a chick chorioallantoic membrane (CAM) assay and in experiments for subcutaneous implantation in severe combined immunodeficient (SCID) mice. Our results demonstrate that lung stem/progenitor cells together with gelatin microbubble-scaffolds promote angiogenesis as well as the differentiation of alveolar pneumocytes, resulting in an alveoli-like structure. These findings may help advance lung tissue engineering.

Amygdala opioid receptors mediate the electroacupuncture-induced deterioration of sleep disruptions in epilepsy rats.

BACKGROUND: Clinical and experimental evidence demonstrates that sleep and epilepsy reciprocally affect each other. Previous studies indicated that epilepsy alters sleep homeostasis; in contrast, sleep disturbance deteriorates epilepsy. If a therapy possesses both epilepsy suppression and sleep improvement, it would be the priority choice for seizure control. Effects of acupuncture of Feng-Chi (GB20) acupoints on epilepsy suppression and insomnia treatment have been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot). Therefore, this study was designed to investigate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi acupoints on sleep disruptions in rats with focal epilepsy. RESULTS: Our result indicates that administration of pilocarpine into the left central nucleus of amygdala (CeA) induced focal epilepsy and decreased both rapid eye movement (REM) sleep and non-REM (NREM) sleep. High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints, in which a 30-min EA stimulation was performed before the dark period of the light:dark cycle in three consecutive days, further deteriorated pilocarpine-induced sleep disruptions. The EA-induced exacerbation of sleep disruption was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting the involvement of amygdaloid opioid receptors. CONCLUSION: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints exhibits no benefit in improving pilocarpine-induced sleep disruptions; in contrast, EA further deteriorated sleep disturbances. Opioid receptors in the CeA mediated EA-induced exacerbation of sleep disruptions in epileptic rats.

Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy.

BACKGROUND: The effect of seizure suppression by acupuncture of Feng-Chi (GB20) acupoints has been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot), however, there is a lack of scientific evidence to prove it. This current study was designed to elucidate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi (GB20) acupoints on the epileptic activity by employing an animal model of focal epilepsy. METHODS: Administration of pilocarpine into the left central nucleus of amygdala (CeA) induced the focal epilepsy in rats. Rats received a 30-min 100 Hz EA stimulation of bilateral Feng-Chi acupoints per day, beginning at 30 minutes before the dark period and performing in three consecutive days. The broad-spectrum opioid receptor antagonist (naloxone), µ-receptor antagonist (naloxonazine), δ-receptor antagonist (naltrindole) and κ-receptor antagonist (nor-binaltorphimine) were administered directly into the CeA to elucidate the involvement of CeA opioid receptors in the EA effect. RESULTS: High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints did not suppress the pilocarpine-induced epileptiform electroencephalograms (EEGs), whereas it further increased the duration of epileptiform EEGs. We also observed that epilepsy occurred while 100 Hz EA stimulation of Feng-Chi acupoints was delivered into naïve rats. EA-induced augmentation of epileptic activity was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting that activation of opioid receptors in the CeA mediates EA-exacerbated epilepsy. CONCLUSIONS: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints has no effect to protect against pilocarpine-induced focal epilepsy; in contrast, EA further exacerbated focal epilepsy induced by pilocarpine. Opioid receptors in the CeA mediated EA-induced exacerbation of focal epilepsy.

Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats.

Theta rhythms generated in the hippocampus are controlled by the pacemaker in the medial septum-diagonal band of Broca (MS-DBB). The median raphe nucleus (MRN) transmits serotonergic signals to the MS-DBB, which suppresses the septo-hippocampus-produced theta waves, whereas GABAergic interneurons in the MRN facilitate the generation of theta oscillations. Animal studies have indicated that fear increases theta oscillations. Moreover, anxiolytics reduce reticular formation-elicited theta rhythms and theta blockade decreases anxiety. In this study, we hypothesized that the MRN mediates anxiety reduction caused by the theta blockade. Our results demonstrated that inescapable-footshock stimulation significantly increased the power of low-frequency theta oscillations (4-7 Hz) in rats. Both the electrical stimulation of MRN and administration of bicuculline into the MRN successfully desynchronized footshock-induced theta oscillations. Compared to the naïve rats, inescapable-footshock stimulation diminished the entry percentage and time spent in the open arms of the elevated plus maze (EPM), behavioral indicators of anxiety. Rats treated with either MRN stimulation or bicuculline administration to desynchronize theta oscillations reduced anxiety caused by the inescapable-footshock stimulation. Our results demonstrated that the electrical stimulation of MRN or blockade of the GABAergic pathways in the MRN interferes with theta oscillations and reduces anxiety, implicating the role of MRN.

Acrylamide-induced mitochondria collapse and apoptosis in human astrocytoma cells.

Acrylamide (ACR) can be produced during food processing and has neurotoxic effects in humans. This study aims to determine ACR induced apoptotic responses in human astrocytoma U-1240 MG cells to realize the incurred toxic mechanisms. Under 1 and 2mM ACR exposure, cell viability decreased as time increased. The increments in sub-G(1) phase were 87.5-fold, and pro-caspase 3 and PARP protein expressions decreased 35% and 54.5% respectively relative to the control after 2mM ACR treatment. Molecular evidence of Bax/bcl-2 ratio and cytochrome c expression increased 8.86-fold and 6.81-fold as well as pro-caspase 9 decreased 67.8% relative to the control respectively under 2mM ACR exposure. Trolox, an ROS scavenging agent, attenuated cell death and induced ROS production by 2mM ACR. The ultrastructure alterations of mitochondria showed marked vesicular matrix compartmentalization and cytoplasmic vacuole formation after 2mM ACR was treated for 48h, whereas those treated for 72h showed chromatin condensation, pyknosis, and swelling. These results indicate long-term exposure to ACR induced mitochondria collapse and finally led to apoptosis. Although 2mM ACR is higher than average daily intake dosage, workers in chemical industries may be exposed to sufficient doses to entail health risks.

Plastidial starch phosphorylase in sweet potato roots is proteolytically modified by protein-protein interaction with the 20S proteasome.

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.

Kappa-opioid receptors in the caudal nucleus tractus solitarius mediate 100 hz electroacupuncture-induced sleep activities in rats.

Previous results demonstrated that 10 Hz electroacupuncture (EA) of Anmian acupoints in rats during the dark period enhances slow wave sleep (SWS), which involves the induction of cholinergic activity in the caudal nucleus tractus solitarius (NTS) and subsequent activation of opioidergic neurons and µ-receptors. Studies have shown that different kinds of endogenous opiate peptides and receptors may mediate the consequences of EA with different frequencies. Herein, we further elucidated that high-frequency (100 Hz)-EA of Anmian enhanced SWS during the dark period but exhibited no direct effect on rapid eye movement (REM) sleep. High-frequency EA-induced SWS enhancement was dose-dependently blocked by microinjection of naloxone or κ-receptor antagonist (nor-binaltorphimine) into the caudal NTS, but was affected neither by µ- (naloxonazine) nor δ-receptor antagonists (natatrindole), suggesting the role of NTS κ-receptors in the high-frequency EA-induced SWS enhancement. Current and previous results depict the opioid mechanisms of EA-induced sleep.

Frequency ranges of heart rate variability related to autonomic nerve activity in the mouse.

Mice have gained more and more attention in recent years and been widely used in transgenic experiments. Although the number of researches on the heart rate variability (HRV) of mice has been gradually increasing, a consensus on the frequency ranges of autonomic modulation has not been established. Therefore, the main purpose of this study was to find a HRV "prototype" for conscious mice in the state of being motionless and breathing regularly (called "genuinely resting"), and to determine the frequency ranges corresponding to the autonomic modulation. Further, whether these frequencies will change when the mice move freely was studied to evaluate the feasibility of the HRV spectrum as an index of the autonomic modulation of mice. The recording sites were specially arranged to simultaneously obtain the electrocardiography and electromyography data to be provided for the use of HRV analysis and motion monitoring, respectively. The states of being motionless and breathing regularly as judged from the electromyography results were selected as a genuine resting state of a conscious mouse. The frequencies related to autonomic modulation of HRV were determined by comparing the spectrum changes before and after blockades of the autonomic tone by different pharmaceutical agents in both the genuine resting state and freely moving states. Our results showed that the HRV of mice is not suitable for indexing sympathetic modulation; however, it is possible to use the spectral power in the frequency range between 0.1 and 1 Hz as an index of parasympathetic modulation.

Biphasic effects of baicalin, an active constituent of Scutellaria baicalensis Georgi, in the spontaneous sleep-wake regulation.

AIM OF THE STUDY: Baicalin is an active compound originating from the root of Scutellaria baicalensis Georgi, which has been used for anti-inflammation, anti-bacteria, anti-hypertension, anti-allergy and sedation since ancient China, though the neuronal mechanisms involved in the sedative effect is still unclear. Baicalin possesses the ability to decrease the expression of pro-inflammatory cytokines and nuclear factor (NF)-κB activity. Furthermore, baicalin has demonstrated an anxiolytic-like effect via activation of γ-aminobutyric acid-A (GABA(A)) receptors. Pro-inflammatory cytokines (e.g. interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α) and the GABAergic system promote sleep. This study was designed to determine whether the GABA(A) receptor activation and/or the suppression of pro-inflammatory cytokines mediate(s) baicalin-induced sleep alterations. MATERIALS AND METHODS: Baicalin was intracerebroventricularly (ICV) administered 20 min either prior to the beginning of the light period or before the onset of the dark period. Electroencephalogram (EEG) and gross body movement were acquired for sleep analysis. Pharmacological blockade of IL-1 and GABA(A) receptors were employed to elucidate the involvements of IL-1 and GABA(A) receptors in baicalin-induced sleep alterations. IL-1ß concentrations obtained after baicalin administration in several distinct brain regions were determined by ELISA. RESULTS: ICV administration of baicalin decreased slow wave sleep (SWS) during the first 2h of the light period. Rapid eye movement sleep (REMS) was not altered. The blockade of IL-1ß-induced SWS enhancement by baicalin suggests that the antagonism of IL-1 receptors is involved in baicalin-induced SWS decrement during the light period. However, IL-1ß concentrations during the light period were not altered after baicalin administration. In contrast, baicalin increased both SWS and REMS during hours 8-10 of the dark (active) period when baicalin was administered at the beginning of the dark period, and its effects were blocked by the GABA(A) receptor antagonist bicuculline. CONCLUSION: Baicalin exhibits biphasic effects on sleep-wake regulation; the decrease of SWS during the light period and increases of SWS and REMS during the dark period. Inhibition of IL-1 action and enhancement of GABA(A) receptor activity may mediate baicalin's effects during the light and dark period, respectively.

Toxicity assessments of nanoscale zerovalent iron and its oxidation products in medaka (Oryzias latipes) fish.

Iron-based nanotechnologies are increasingly used for environmental remediation; however, toxicologic impacts of iron nanoparticles on the aquatic ecosystem remain poorly understood. We treated larvae of medaka fish (Oryzias latipes) with thoroughly characterized solutions containing carboxymethyl cellulose (CMC)-stabilized nanoscale zerovalent iron (nZVI), aged nanoscale iron oxides (nFe-oxides) or ferrous ion (Fe[II]) for 12-14 days' aqueous exposure to assess the causal toxic effect(s) of iron NPs on the fish. With the CMC-nZVI solution, the dissolved oxygen level decreased, and a burst of reactive oxygen species (ROS) was generated as Fe(II) oxidized to ferric ion (Fe[III]); with the other two iron solutions, these parameters did not significantly change. CMC-nZVI and Fe(II) solutions caused acute lethally and sublethally toxic effects in medaka larvae, with nFe-oxide-containing solutions causing the least toxic effects. We discuss modes of toxic action of iron NPs and chronic toxic effects in terms of hypoxia, Fe(II) toxicity and ROS-mediated oxidative damage.

Endogenous opiates in the nucleus tractus solitarius mediate electroacupuncture-induced sleep activities in rats.

Electroacupuncture (EA) possesses various therapeutic effects, including alleviation of pain, reduction of inflammation and improvement of sleep disturbance. The mechanisms of EA on sleep improvement, however, remain to be determined. It has been stated in ancient Chinese literature that the Anmian (EX17) acupoint is one of the trigger points that alleviates insomnia. We previously demonstrated that EA stimulation of Anmian acupoints in rats during the dark period enhances non-rapid eye movement (NREM) sleep, which involves the induction of cholinergic activity in the nucleus tractus solitarius (NTS). In addition to cholinergic activation of the NTS, activation of the endogenous opioidergic system may also be a mechanism by which acupuncture affects sleep. Therefore, this study was designed to investigate the involvement of the NTS opioidergic system in EA-induced alterations in sleep. Our present results indicate that EA of Anmian acupoints increased NREM sleep, but not rapid eye movement sleep, during the dark period in rats. This enhancement in NREM sleep was dose-dependently blocked by microinjection of opioid receptor antagonist, naloxone, and the µ-opioid receptor antagonist, naloxonazine, into the NTS; administrations of δ-receptor antagonist, natrindole, and the κ-receptor antagonist, nor-binaltrophimine, however, did not affect EA-induced alterations in sleep. Furthermore, ß-endorphin was significantly increased in both the brainstem and hippocampus after the EA stimuli, an effect blocked by administration of the muscarinic antagonist scopolamine into the NTS. Our findings suggest that mechanisms of EA-induced NREM sleep enhancement may be mediated, in part, by cholinergic activation, stimulation of the opiodergic neurons to increase the concentrations of ß-endorphin and the involvement of the µ-opioid receptors.

A simple method for fabricating microwire tetrode with sufficient rigidity and integrity without a heat-fusing process.

Heat-fusing is a common process for fabricating microwire tetrodes. However, it is time-consuming, and the high-temperature treatment can easily cause the insulation of the microwire to overheat leading to short circuits. We herein provide a simple, fast method to fabricate microwire tetrodes without the heat-fusion process. By increasing the twisting density, we were able to fabricate tetrodes with good rigidity and integrity. This kind of tetrode showed good recording quality, penetrated the brain surface easily, and remained intact after chronic implantation. This method requires only general laboratory tools and is relatively simple even for inexperienced workers.

TNF-NF-kappaB signaling mediates excessive somnolence in hemiparkinsonian rats.

Daytime somnolence is common in patients with Parkinson's disease (PD); however there is a lack of understanding of the cellular mechanisms involved in mediating these effects. It has been hypothesized that microglial activation and the subsequent increase of pro-inflammatory cytokines play an important role in the pathogenesis of PD. Because some cytokines are involved in the regulation of sleep, this study was designed to determine if tumor necrosis factor (TNF) and interleukin-1beta (IL-1beta), mediate daytime somnolence in the proteasome inhibitor (MG-132)-induced hemiparkinsonian rat model. Our results indicated that microglial activation caused the loss of dopaminergic neurons in the substantia nigra, and the expression of TNF-alpha, but not IL-1beta, increased in the midbrain and hypothalamus in MG-132-induced hemiparkinsonian rats. Slow-wave sleep (SWS) increased after the induction of hemiparkinsonism, but rapid eye movement (REM) sleep was not consistently altered. Application of the TNF receptor fragment (TNFRF) blocked hemiparkinsonism-induced SWS alteration, whereas the IL-1 receptor antagonist (IL-1ra) exhibited no effect. Increased nuclear translocation of NF-kappaB in the midbrain, and the blockade of SWS enhancement in MG-132-induced hemiparkinsonian rats by an inhibitor of NF-kappaB activation indicate that the TNF-NF-kappaB cascade is a critical mediator of MG-132 hemiparkinsonian-induced sleep alteration. This observation suggests potential therapeutic interventions to target the excessive daytime somnolence in patients with PD.

Involvement of the mitochondrion-dependent pathway and oxidative stress in the apoptosis of murine splenocytes induced by areca nut extract.

Areca quid chewing is a major risk factor for oral submucous fibrosis and oral cancer. Clinical evidence suggests that the pathophysiology of the oral diseases is closely associated with immune deterioration. The objective of the present studies was to investigate the pro-apoptotic effect of areca nut extract (ANE) in lymphocytes. Exposure of naïve splenic lymphocytes to ANE significantly enhanced apoptosis in a time- and concentration-dependent manner. Results from Hoechst staining confirmed the morphological features characteristic of apoptosis in ANE-treated cells. ANE treatment induced the depolarization of mitochondrial membrane potential (Deltapsi(m)), which preceded the occurrence of apoptosis. In parallel with the disruption of Deltapsi(m), ANE induced the release of cytochrome c, and the activation of caspase-9, indicating the activation of the mitochondrion-dependent pathway. Moreover, an increased level in the intracellular reactive oxygen species was detected in ANE-treated lymphocytes undergoing apoptosis. ANE-mediated apoptosis, caspase-9 activation and ROS production, but not Deltapsi(m) depolarization, were partially but significantly attenuated in the presence of the antioxidant N-acetyl-L-cysteine (NAC). Collectively, these results demonstrated the pro-apoptotic effect of ANE in primary lymphocytes, which was mediated, at least in part, by the activation of the mitochondrion-dependent pathway and oxidative stress.

Morphologic and cytochemical characteristics of Chinese striped-necked [corrected] turtle (Ocadia sinensis) blood cells and their hematologic and plasma biochemical reference values.

Hematologic analyses are useful for the monitoring of animal health and diseases and for the differentiation of physiologic processes for clinicians and conservationists. In order to establish hematology reference values for the Chinese striped-necked [corrected] turtle (Ocadia sinensis) and to produce an accurate baseline of clinical laboratory data for O. sinensis with regard to sex and season, 50 (24 males and 26 females) adult captive individuals of O. sinensis were studied. Blood samples from the jugular veins of the turtles were collected in January, April, June, and November. Data were analyzed using repeated measures analysis of variance for significant (P < 0.05) variation by sex, season, and the interaction between sex and season. Significant sex differences were observed for the parameters of packed cell volume, eosinophil count, heterophils and monocytes ratio, total protein, albumin, uric acid, aspartate aminotransferase, alanine aminotransferase, triglycerides, cholesterol, and alkaline phosphatase. Marked seasonal variation was noted in all parameters except mean cell hemoglobin, monocytes and heterophils ratio, and creatinine. Differences between sexes and seasons were primarily associated with the reproductive cycle. Heterophils had a strong positive reaction and eosinophils had a moderate positive reaction to benzidine peroxidase stain. Thrombocytes had a positive reaction to periodic acid-Schiff stain. Surface morphologic study using scanning electron microscopy of blood cells showed that white blood cells of O. sinensis had no distinctive surface characteristics.

CD5-low expression lymphocytes in canine peripheral blood show characteristics of natural killer cells.

NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5-low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 +/- 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8alpha (77.7%), CD8beta (53%), alpha/beta TCR (83%), and CD11/18 (80%), but the expression of gamma/delta TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5lo cells were larger than CD5-high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL-2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL-2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5hi cells. In addition, IL-2-stimulated, CD5lo-depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.