A therapeutic intervention for Alzheimer's disease using ginsenoside Rg3: its role in M2 microglial activation and non-amyloidogenesis.

Previously, we have reported that ginsenoside Rg3 has typical activities for neuroprotection and Aß42 clearance by modulating microglia. In this study, we determined the pivotal role of ginsenoside Rg3 in microglia and neuronal cells. In human microglia, Rg3 and its stereoisomers significantly restored inflammatory M1 to normal M0 state and promoted M2 activation by up-regulating acute cytokines such as interleukin-10 and Arginase 1. Moreover, scavenger receptor type A (SRA) was significantly elevated in the presence of ginsenoside Rg3 and 20(S)-Rg3. This indicated that ginsenoside Rg3 could play a crucial role in Aß uptake and clearance under activated M2 state. We also observed that soluble amyloid precursor protein-alpha (sAPPα) and ADAM10 levels were increased in APP swe-transfected Nuro-2a neuronal cells, whereas sAPPß was not processed, suggesting that ginsenoside Rg3 was involved in non-amyloidogenic processing. In immunocytochemistry, SRA and a disintegrin and metalloproteinase 10 (desintegrin and metalloproteinase-containing protein 10, ADAM10) were coincidently upregulated in the presence of ginsenoside Rg3 and its stereoisomers compared to those in normal control. Taken together, these results suggested that ginsenoside Rg3 could boost acute activation of microglia, promote Aß uptake, and elevate the sAPPα processing under activated M2 state. Although in vivo studies need to be performed, it is certain that ginsenoside Rg3 is highly involved in ameliorating the pathogenesis of neurodegeneration and can be a promising candidate for treating Alzheimer's disease as a new therapeutic intervention.

The correlation between amylin and insulin by treatment with 2-deoxy-D-glucose and/or mannose in rat insulinoma ins-1E cells.

Amylin or islet amyloid polypeptide (IAPP) is a peptide synthesized and secreted with insulin by the pancreatic ß-cells. A role for amylin in the pathogenesis of type 2 diabetes (T2D) by causing insulin resistance or inhibiting insulin synthesis and secretion has been suggested by in vitro and in vivo studies. These studies are consistent with the effect of endogenous amylin on pancreatic ßcells to modulate and/or restrain insulin secretion. Here, we reported the correlation between amylin and insulin in rat insulinoma INS-1E cells by treating 2-deoxy-D-glucose (2-DG) and/or mannose. Cell viability was not affected by 24 h treatment with 2-DG and/or mannose, but it was significantly decreased by 48 h treatment with 5 and 10 mM 2-DG. in the 24 h treatment, the synthesis of insulin in the cells and the secretion of insulin into the media showed a significant inverse association. in the 48-h treatment, amylin synthesis vs. the secretion and insulin synthesis vs. the secretion showed a significant inverse relation. The synthesis of amylin vs. insulin and the secretion of amylin vs. insulin showed a significant inverse relationship. The p-ERK, antioxidant enzymes (Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase), and endoplasmic reticulum (ER) stress markers (cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78) were significantly increased or decreased by the 24 h and 48 h treatments. These data suggest the relative correlation to the synthesis of amylin by cells vs. the secretion into the media, the synthesis of amylin vs. insulin, and the secretion of amylin vs. insulin under 2-DG and/or mannose in rat insulinoma INS-1E cells. Therefore, these results can provide primary data for the hypothesis that the amylin-insulin relationships may be involved with the human amylin toxicity in pancreatic beta cells.

Potassium-dependent sodium/calcium exchanger 3 (Nckx3) depletion leads to abnormal motor function and social behavior in mice.

Transcellular calcium transport is an essential activity in mineralized tissue formation, including that in nervous systems. Dysregulation of Ca2+ homeostasis can induce excitotoxicity and neurodegeneration in the central nervous system. Nckx3, a potassium-dependent Na+/Ca2+ exchanger, is most abundant in the brain and has a critical role in the transport of intracellular calcium across the cell membrane. However, the roles of Nckx3 in neuron development and function remain unreported. Herein, we examined the behaviors of Nckx3-knock-out mice at the age of six weeks. Detailed behavioral analyses showed Nckx3-/- mice exhibited an increase in moving distances in the open field test. Additionally, the rotarod test revealed motor learning defects in Nckx3-/- mice. Both Nckx3+/- and Nckx-/- mice also exhibited deficits in sociability and social novelty preference. Furthermore, Nckx-/- mice displayed increased depression-related behavior. However, there was no significant change in cognition function detected in Nckx-/- mice. This study demonstrates that NCKX3 is involved in behavior and neuronal function.

Second-phase validation study of an alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies.

The embryoid body test (EBT) is a developmental toxicity test method that measures the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The previous pre-validation study confirmed the high accuracy (above 80%) of EBT using 26 coded test chemicals. This second-phase validation study assessed the inter-laboratory reproducibility (5 chemicals in common) and predictive capacity (10 chemicals in each laboratory) test using the coded test chemicals at three laboratories. For the prediction model, the accuracy is increased when more data is accumulated. Therefore, we updated the prediction model and analyzed the results of the second year with the newly created-prediction model. Statistical analysis of the inter-laboratory reproducibility test results indicated that accuracy, sensitivity, and specificity were 87%, 78%, and 100%, respectively. The results of the statistical analysis of the predictive capacity test showed an accuracy of 80%, sensitivity of 78%, and specificity of 81%. In conclusion, the EBT can accurately classify various embryotoxicants within a short period and with relatively little effort. Therefore, EBT can be used as a good way to test developmental toxicity.

[Melatonin Induces Apoptotic Cell Death in 3T3-L1 Preadipocytes].

Obesity is a major disease that causes significant complications. Inhibition of preadipocyte proliferation has the potential to prevent obesity and metabolic diseases. Melatonin is a pineal gland hormone that has various effects on cells and tissues. In this research, we investigated whether melatonin induces apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured until confluence and then treated with 0, 10, 100, and 1000 µM melatonin for 1, 3, and 5 days. A cell viability assay kit was used for determining cell viability. Cell death marker proteins were assessed by Western blot analysis using GAPDH for control. Apoptotic morphological changes with nuclei fragmentation were observed using DAPI staining. Melatonin treatment decreased the phosphorylated extracellular signal-regulated kinases (p-ERK) activation while increasing the activation of caspase-3, 8, and 9. Furthermore, melatonin not only increased Bcl-2-associated X protein (Bax) but decreased B-cell lymphoma 2 (Bcl-2) expression as dose increases from 0 to 1000 µM. The melatonin treatment also suppressed the growth of preadipocytes with increasing concentration. These effects were attenuated by luzindole, a melatonin receptor antagonist and U0126, an inhibitor of p-ERK activation. In conclusion, melatonin can induce apoptosis of 3T3-L1 preadipocytes via p-ERK decrease.

Nanomolar melatonin influences insulin synthesis and secretion in rat insulinoma INS-1E cells.

Pancreatic beta-cell dysfunction results in reductions of insulin synthesis/secretion, cell survival, and insulin sensitivity thereby inducing diabetes mellitus. In this study, how nanomolar melatonin regulates insulin synthesis and secretion in rat insulinoma INS-1E cells was investigated. At melatonin concentrations of 10 - 100 nM for 48 hours, melatonin significantly increased the insulin protein level in INS-1E cells above the level in control cells without melatonin or glucose treatments and decreased the insulin level in media with glucose: increases in insulin synthesis and decreases in insulin secretion occurred in dose-dependent manners. Luzindole or 4-phenyl-2-propionamidotetralin (4P-PDOT), melatonin receptor antagonists, inhibited the melatonin-induced insulin level in cells and media. Levels of membrane vesicle trafficking-related proteins including Rab5, GOPC, phospho-caveolin-1, EEA1, and clathrin proteins significantly increased with melatonin treatment above that in control cells without melatonin or glucose treatments, whereas expressions of APPL1 and syntaxin-6 proteins significantly decreased with melatonin treatment. The increases in the phosphorylation of mammalian target of rapamycin (p-mTOR), raptor protein, and mTOR complex 1 (mTORC1) levels were consistent with the increments in the expressions of p-Akt (Ser473, Thr308) and stress-induced IRE1α/p-eIF2α proteins in the endoplasmic reticulum following melatonin treatment. also, expression levels of Bcl-2 and Bcl-xl proteins were significantly increased compared to those in control cells without melatonin or glucose treatments, whereas the Bax protein level decreased. These results indicate that nanomolar melatonin regulates insulin synthesis and secretion associated with membrane vesicle trafficking-related proteins, including Rab5, GOPC, p-Caveolin-1, EEA1, and clathrin, through the Akt/mTOR pathway.

Cigarette smoke extraxt influences intracellular calcium concentration in A549 cells.

Cigarette smoking is a major risk factor for pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and cancer. Cigarette smoke is reported to contain over 4,000 chemical compounds. Therefore, it needs to study the effects of cigarette smoke extract (CSE) administration on intracellular calcium concentration. In this study, we investigated how CSE influences intracellular calcium concentration in human lung adenocarcinoma A549 cells. The CSE concentrations used (0.4, 2, 3%) did not influence cell viability. However, at these CSE concentrations, calcium influx transient receptor potential vanilloid 4 (TRPV4) and transient receptor potential vanilloid 6 (TRPV6) proteins significantly increased, whereas calcium efflux sodium-calcium exchanger (NCX1) and plasma membrane Ca2+ ATPase (PMCA1) proteins significantly decreased from those of the control cells. The 3% CSE treatment produced an intracellular calcium concentration higher than that of the control treatment through methods of co-transfection of pGP-CMV-GCaMP6f/CMV-R-GECO1.2 and Rhod-4 Assay. CSE induced concentration-dependent increments in hypoxia-inducible factor (HIF)-1α and HIF-2α protein levels. Moreover, phosphorylation of ERK and Akt was induced by CSE treatment. Also, mitochondrial marker B-cell lymphoma 2 (Bcl-2) protein level decreased and Bcl-2-associated X (Bax) protein level increased following CSE treatment. Also, endoplasmic reticulum (ER) stress markers BiP, CHOP, p-SAPK, and p-eIF2α levels were increased by CSE treatment. These results suggest that CSE may increase the concentration of intracellular calcium, thus increasing mitochondrial and ER stress.

Inhibitory effect of octyl-phenol and bisphenol A on calcium signaling in cardiomyocyte differentiation of mouse embryonic stem cells.

Endocrine-disrupting chemicals (EDCs) have structures similar to steroid hormones and can interfere with hormone synthesis and normal physiological functions of reproductive organs. For example, sex steroid hormones influence calcium signaling of the cardiac muscle in early embryo development. To confirm the effect of progesterone (P4), octyl-phenol (OP), and bisphenol A (BPA) on early differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes, mESCs were treated with P4, OP, and BPA two days after attachment and media were replaced every two days. In addition, cells were treated with mifepristone (RU486), a synthetic steroid that has an affinity for progesterone receptor (Pgr), for one day starting on day 11. Beating ratio was decreased with P4, OP, and BPA treatment. The Pgr mRNA level was significantly increased in the P4-, OP- and BPA-treated groups. However, the mRNA level of the calcium channel gene (Trpv2), contraction-related genes (Ryr2, Cam2, and Mylk3) and cardiac development and morphogenesis genes (Rbp4, Ly6e, and Gata4) were significantly decreased in the P4-, OP-, and BPA-treated groups. Interestingly, treatment with RU486 rescued the altered calcium channel gene, contraction-related genes, and cardiac development and morphogenesis genes. P4, OP, and BPA treatments reduced the intracellular calcium level. Taken together, these results indicate that EDCs (OP and BPA) has a structure similar to that of endogenous steroid hormones such as progesterone and estrogen, and OP and BPA act like progesterone to inhibit and disrupt cardiomyocyte differentiation of mESCs.

Regulatory effect of dexamethasone on tracheal calcium processing proteins and mucosal secretion.

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.

Melatonin influences the expression and oligomerization of amylin in rat INS-1E cells.

The present study investigated whether melatonin influences the expression/oligomerization of amylin with endoplasmic reticulum (ER) stress in rat insulinoma INS-1E cells. No change in cell survival after exposure to thapsigargin- and tunicamycin-combined melatonin treatment or melatonin-only treatment was observed when compared with the normal control cells. With thapsigargin-only or combined tunicamycin-melatonin treatments, phosphorylation of extracellular signal-regulated kinase (ERK) was significantly increased compared with control and melatonin-only treatments. A significant increase was observed in the levels of ER stress markers, namely, phosphorylated inositol-requiring protein 1α (p-IRE1α), CCAAT enhancer binding proteins (C/EBP)-homologous protein, p-eukaryotic translation initiation factor 2α and cleaved caspase-12, in the thapsigargin-combined melatonin-treated cells as compared with the tunicamycin-combined or only melatonin treatment. The melatonin-only treatment resulted in increased levels of amylin expression/oligomerization in 15-25 kDa and insulin proteins, compared with the thapsigargin- and tunicamycin-combined melatonin treatments. Treatment with ER stress inhibitor 4-phenylbutyric acid (4-PBA) did not suppress amylin expression/oligomerization or insulin production with thapsigargin or tunicamycin treatment. Levels of cleaved caspase-12 were significantly decreased in the thapsigargin- or tunicamycin-4-PBA combination treatments. Therefore, whether melatonin regulates the amylin expression/oligomerization in thapsigargin- or tunicamycin-combined with Bafilomycin A1 (autophagy inhibitor) or MG132 (proteasome inhibitor) treatments were investigated. Amylin expression/oligomerization with melatonin treatment was significantly decreased in the thapsigargin- or tunicamycin-combined Bafilomycin A1 or MG132 treatments. Since these outcomes are involved in cell viability, they indicate that increased cell death leads to decreased amylin expression/oligomerization, however, the effects of melatonin treatment on amylin expression/oligomerization induce proliferation of pancreatic ß cells and improve the cellular functions of pancreatic ß cells.

Anti-asthmatic effects of volatile organic compounds from Chamaecyparis obtusa, Pinus densiflora, Pinus koraiensis, or Larix kaempferi wood panels.

Asthma is a common chronic inflammatory disease in which lung airways narrow and produce extra mucus. Numerous medications, such as steroids, are used to prevent or control asthmatic symptoms, but side effects are associated with those medications. There are reports of anti-inflammatory, antibacterial, and antiparasitic effects of terpene, a volatile organic compound (VOC) in conifers. VOCs easily enter a gaseous form, and wood products are good sources of VOCs. However, only a few studies have been conducted on the effect on asthma of VOCs emitted by wood. In this study, we examined the effects of VOCs diffused from wood panels on ovoalbumin (OVA)-induced asthma in a mouse model. The mice were intraperitoneally sensitized with 10 µg of OVA with aluminum hydroxide on days 0, 7, and 14. From day 21 to day 26, the mice were challenged with 2% OVA intranasally for 30 min. For VOC treatment, asthma model mice were placed in polyacrylamide chambers containing wood panels of Chamaecyparis obtusa, Pinus densiflora, Pinus koraiensis, or Larix kaempferi. On day 27, serum, lung tissue, and bronchoalveolar lavage fluids were prepared for H&E staining, qRT-PCR, ELISA, and Diff-Quik staining, as appropriate. OVA treatment induced hypertrophy of the bronchiolar wall. The budesonide group and all four of the wood panel-exposed groups showed less thickening of the bronchiolar wall and downregulated transcriptional expressions of cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13). The serum tumor necrosis factor-α (TNF-α) mRNA expression level was significantly decreased only in the C. obtusa group, but the serum IL-4 levels were decreased in all wood panel treatment groups. Diff-Quik staining of bronchoalveolar lavage fluids revealed a decrease in the number of granulocytes in all wood panel treatment groups. The results suggest that VOCs from C. obtusa, P. densiflora, P. koraiensis and L. kaempferi produce antiasthmatic effects by regulating the production of IL-4, IL-9, IL-13, TNF-α.

Articular cartilage repair with autografting under the influence of insulin-like growth factor-1 in rabbits.

Insulin-like growth factor (IGF)-1 has been successfully demonstrated to stimulate proteoglycan synthesis, slow down its catabolism and promote cartilage formation through well defined in vitro studies. It was therefore, assumed that IGF-1 would eventually serve to augment current cartilage repair techniques in vivo. Study was therefore, designed to determine the influence of IGF-1 in cartilage repair with or without autografting. For this purpose articular cartilage repair model was created in the left knee of 48 New Zealand white rabbits of either sex, 6-7 months old, weighing 1-2 kg. The articular cartilage defect was created in the femoral groove of femoro-patellar joint using hand held trephine under xylazine and ketamine anaesthesia in all the animals. The defect created was 3 mm in diameter and 2 mm in depth. For autografting, osteochondral tissues harvested from the proximal patellar groove of the femur were placed in the distal defect and vice versa. The experimental animals were divided mainly into four groups, i.e. Group A (control), Group B (autografting), Group C (control + IGF-1) and Group D (autografting + IGF-1). Animals of group A and B were provided only with collagen scaffolds at 10 mug/cm(2) whereas animals of treatment group C and D were provided with collagen scaffolds holding 30 ng/30 mul of IGF-1 into the defect. Evaluation of cartilage repair was done on days 15, 30 and 45 after ethically killing the animals. Initially IGF-1 had shown the tendency for either in the maintenance of autografted cartilage or helped in proliferation of chondroblast for the repair process. However, later in the process, cartilage formation apparently declined and appeared to converge to osseous tissue. Collectively, non-responsiveness of osteoarthritic chondrocytes to IGF-1 could be partially attributed to either increased IGF-binding proteins in the joint space, micromovement of the graft, lack of nutrition, dose of IGF-1 or its half life in the current study.

Transdifferentiation of porcine satellite cells to adipoblasts with ciglitizone.

Ciglitizone, a class of thiazolidinediones, acts as a potent activator of the adipose differentiation program in established preadipose cell lines. Thiazolidinediones have also been investigated in diabetic patients and have been reported to act as peroxisome proliferator-activated receptor-gamma ligands. Intramuscular adipogenesis or marbling through transdifferentiation of satellite cells in cattle was successfully conducted earlier. In this report, the effects of ciglitizone on the differentiation pathway of porcine myogenic satellite cells was investigated. Semitendinosus muscle was aseptically taken from 10-d-old piglets under general anesthesia, and porcine satellite cells were obtained and grown to near confluence. Postconfluent cells (d 0) were further cultured in differentiation medium containing an adipogenic mixture plus ciglitizone (10 microM) for 48 h. From d 2 onward, the cells were cultured only in the presence of ciglitizone until d 10. Controls were cultured in differentiation medium only. Exposure of porcine satellite cells to the adipogenic mixture plus ciglitizone generated lipid droplets on d 2, and subsequently, exposure of cells to ciglitizone alone helped in cytoplasmic lipid filling, providing them with the acquisition of adipocyte morphology. An increase (P < 0.05) in the fusion (structures containing 2 to 3 nuclei) of satellite cells was observed, and myosin heavy chain appeared with greater intensity (immunohistochemistry) in the control group from d 2 onward. Adipocyte-specific transcriptional factors (i.e., CCAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma) were predominant during transdifferentiation and were observed with immunohistochemistry, Western blot (approximately 47.2 and approximately 60.4 kDa, respectively), and real-time PCR. Ciglitizone appeared to convert the differentiation pathway of satellite cells into that of adipoblasts.

A research note on predicting the carcass yield of Korean native cattle (Hanwoo).

This study developed a new equation to predict the carcass yield of Hanwoo cattle, as well as re-evaluating a previous prediction equation. Experimental animals comprised 79 cows, 79 steers, and 79 bulls. A stepwise model selection was performed to determine the most practical carcass traits for predicting the yield. Cold carcass weight (CW), backfat thickness (BFT), and ribeye area (REA) accounted for 52% variation in yield, and these were determined as the final parameters. When the kidney, pelvic, and heart fat were included in the model, this increased the percentage variation explained (R(2)) by 4%. To compare the newly developed equation (Yield=64.65-0.0198×CW-5.2264×BFT+0.1339×REA) and the previously used one, carcass yields of 377,048 industrial animals (145,695 cows, 225,926 bulls, and 5427 steers) were assessed. For the newly developed equation, the mean difference between experimental animals industrial animals differed only by 1.99-2.68% for all sex groups. In the case of the previous prediction equation, the mean difference ranged from 6.06% for bulls to 11.05% for steers. The results demonstrated that the prediction equation should use a mixture of sex and market weight groups. The results also support our decision to use the newly developed model in predicting the yield of Hanwoo.

Hemoglobin toxicity in experimental bacterial peritonitis is due to production of reactive oxygen species.

Hemoglobin (Hb) is a toxic molecule responsible for the extreme lethality associated with experimental Escherichia coli peritonitis, but the mechanism has yet to be elucidated. Hb, but not globin, showed toxic effects in a live E. coli model but not in a model using killed E. coli. Methemoglobin, hematin, and the well-known Fenton reagents iron and iron-EDTA demonstrated the same lethal effect in E. coli peritonitis as Hb, while the addition of the Fenton inhibitors desferrioxamine (DF) and diethylenetriamine pentaacetate removed most of the cytotoxic activity of iron. Administration of a combined dose of superoxide dismutase and catalase minimized the action of Hb and iron-EDTA, suggesting that both O(2)(.-) and H(2)O(2) are involved in the toxic action of Hb in this rat model. The combination of the antioxidative enzymes and DF further suppressed iron-mediated lethality. An electron spin resonance technique with the spin-trapping reagent 5, 5-dimethyl-1-pyroline-N-oxide (DMPO) showed O(2)(.-) generation in the peritoneal fluid of rats injected with E. coli alone or E. coli plus iron-DF, and (.)OH generation was detected in the peritoneal fluid of the rats injected with iron-EDTA. Hb did not show any spin adduct of oxygen radicals, suggesting that Hb produces non-spin-trapping radical ferryl ion, which decayed the spin adduct of DMPO. In the presence of Hb or iron-EDTA, O(2)(-)-generating activity and viability of phagocytes decreased, whereas lipid peroxidation of peritoneal phagocytes increased. Generation of oxygen radicals and lipid peroxidation did not differ in the live and dead bacterial models. Bacterial numbers in the peritoneal cavity and blood were markedly increased in the live bacterial model with Hb and iron-EDTA. The Fenton inhibitor iron-DF prevented the loss of phagocyte function, lipid peroxidation, and bacterial proliferation. These results led us to conclude that the lethal toxicity of Hb in bacterial peritonitis is associated with a Fenton-type reaction, the products of which decrease phagocyte viability, through the induction of lipid peroxidation, allowing bacterial proliferation and resulting in mortality.

Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling.

Although nitric oxide (NO) induces neuronal cell death under some conditions, it also can prevent apoptosis resulting from growth factor withdrawal. We investigated the molecular mechanism by which NO protects undifferentiated and differentiated PC12 cells from trophic factor deprivation-induced apoptosis. PC12 cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and cytochrome c release after 24 hr of serum withdrawal. The apoptosis of PC12 cells was inhibited by the addition of NO-generating donor S-nitroso-N-acetylpenicillamine (SNAP) (5-100 microM) and the specific caspase-3-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) but not the YVADase (or caspase-1-like protease) inhibitor N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevented the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppression of DEVDase activity was only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO did not S-nitrosylate caspase-3-like proteases in PC12 cells. Western blot analysis showed that NO inhibited the proteolytic activation of caspase-3. The cGMP analog 8-bromo-cGMP (8-Br-cGMP) blocked apoptotic cell death, caspase-3 activity and activation, and cytochrome c release. The soluble guanylyl cyclase inhibitor 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (CODQ) significantly attenuated NO-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death, PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, the protein kinase G inhibitor KT5823 reversed both SNAP- and 8-Br-cGMP-mediated anti-apoptotic events. All these apoptotic phenomena were also suppressed by NO production through neuronal NO synthase gene transfer into PC12 cells. Furthermore, similar findings were observed in differentiated PC12 cells stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting the activation of caspase proteases through cGMP production and activation of protein kinase G.

Inaccuracy and inefficiency of urinary sediment analysis.

When the efficiency of urine sediment analysis was studied: we found that: (1) In tests of repeatability, counts of red blood cells, white blood cells, and epithelial cells, but not of casts, showed high precision, even when they were conducted by relatively inexperienced technologists; (2) Correlation of counts of red blood cells, white blood cells, and epithelial cells was good for results obtained by an expert technologist and those obtained by relatively inexperienced technologists, but the correlation was low in the case of casts, perhaps because of their low incidence in urine sediment; (3) Hematuria and leukocyturia were often detected in specimens positive for occult blood, albumin, or both; (4) Most casts were hyaline, which are physiologic in nature. Pathologic casts such as granular casts, were found in specimens containing red and white blood cells; (5) Although urine sediment analysis was frequently requested by clinicians, most analyses showed negative results; (6) Microscopic sediment analysis requires considerable time, labor, and experience, and is more sensitive than tape tests. The results of our study indicate that there are advantages to the present method for analyzing urine sediment, but that its efficiency is doubtful.