Margination Of Nuclear Chromatin Research Articles

Phytochemical Profiling, Antioxidants, Antimicrobial and Anti‐Proliferative Effect of Senna hirsuta against PC‐3 Human Prostate Cancer Cells

AbstractThe phytochemical profile, antioxidant, antibacterial, and cytotoxicity of Senna hirsuta (M−Sh) leaf extracts were investigated in human prostate cancer PC‐3 cell lines. Antioxidant bioactive components like phenolics, tannins, and flavonoids were measured at 15.36±0.19, 2.97±0.11, and 19.77±0.51, respectively. An intense dose‐dependent radical scavenging activity was observed for M−Sh with 83 % in 500 μg/mL for DPPH. M−Sh boosted ABTS radical inhibition from 35 % at 100 μg/ml to 70 % at 500 μg/ml, whereas hydroxyl radical scavenging was 96 % at 500 μg/ml, significantly greater than butylated hydroxytoluene (91.91 %). The maximum NO scavenging activity of M−Sh was 83 %, and the lipid peroxidation inhibition was 87 % at 500 μg/ml. The M−Sh exhibited antibacterial efficacy against Escherichia coli (25 mm), Bacillus subtilis (24 mm), and Staphylococcus aureus (21 mm). The MTT assay was used to study anti‐proliferative activity, which inhibited PC‐3 cell growth with an IC50 of 257.13 μg/mL over 24 hours. These results demonstrate that M−Sh inhibits PC‐3 cell proliferation by shedding from the surface and assuming a spherical shape. Additionally, the morphology of PC‐3 cells showed shrinkage, blebbing membrane condensation, and nuclear chromatin margination, indicating that M−Sh was cytotoxic to PC‐3 Cells.

Capripoxviruses: Exploring the genetic relatedness between field and vaccine strains from Egypt.

Background and Aim:Lumpy skin disease (LSD) and sheep pox are economically important Capripoxvirus-induced diseases of cattle and sheep, respectively. Despite the extensive vaccination program adopted by Egyptian veterinary authorities, LSD and sheep pox are still prevalent and spread throughout the whole country. The current study was designed for molecular characterization and phylogenetic analysis of LSD virus (LSDV) and Sheep pox virus (SPPV) recovered from field cases in Egypt along with vaccinal strains to assess their genetic relatedness.Materials and Methods:Skin biopsies were collected from naturally infected cases of LSD in Ismailia (n=3 farms) and Beni-Suef (n=2 farms) Governorates and sheep pox in Beni-Suef (n=1 flock). Virus isolation was carried out on primary ovine fetal kidney and heart cell cultures. DNA was extracted from infected materials (skin lesions, infected cell cultures) as well as LSDV Neethling vaccine strain and Romanian SPPV vaccine strain. Polymerase chain reaction was performed using oligonucleotide primers targeting the entire open reading frame of G protein-coupled receptors (GPCR) gene and gene sequences were analyzed.Results:Virus isolation on primary ovine fetal kidney and heart cell culture revealed a cytopathic effect at the third passage characterized by rounding of infected cells and margination of nuclear chromatin. Comparative sequence analysis of GPCR gene revealed that Egyptian LSDV isolated from Ismailia and Beni-Suef shared 99:100% nucleotide and amino acid (AA) identities with each other. In comparison to the vaccinal strains, Egyptian LSDV isolates shared 98:99 nucleotide and AA identities with LSDV Neethling vaccine strain and 93:94% with SPPV Romanian vaccine strain. No differences at the nucleotide or AAs were observed between the SPPV vaccine and virulent strains (100% identity). Phylogenetic analyses revealed that LSDV Neethling vaccine strain is more related to field Egyptian LSDV and clustered within the LSDV group while Romanian SPPV vaccine strain clustered in a separate clade with SPPV field isolates.Conclusion:Comparative sequencing and phylogenetic analyses of the GPCR gene reveal a minimal genetic variation between LSDV field isolates from different locations and a close relationship between virulent field strains and homologous vaccines.

Di-(2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in quail (Coturnix japonica) via suppression of the heat shock response

Di-(2-ethylhexyl) phthalate (DEHP) is a widespread environmental toxicant that severely impacts agricultural production and animal and human health. Nevertheless, DEHP-induced hepatotoxicity at the molecular level in quail remains unexplored. The heat shock response (HSR), involving heat shock proteins (HSPs) and heat shock transcription factors (HSFs), is a highly conserved molecular response that is triggered by stressors, especially exposure to toxicants. To explore the DEHP-induced hepatotoxicity that occurs via regulation of HSR in birds, female quail were dosed with DEHP by oral gavage (0, 250, 500 and 1000 mg/kg) for 45 days. Based on histopathological analysis, the livers of the DEHP-treated groups exhibited structural alterations of hepatocytes, including mitochondrial swelling, derangement of hepatic plates, inflammatory cell infiltration and adipose degeneration. Ultrastructural evaluation of the livers of DEHP-treated quail revealed swollen mitochondria, partial disappearance of mitochondrial membranes and cristae, nuclear chromatin margination and nuclear condensation. The expression of HSF1 and HSF3 significantly decreased after DEHP exposure. The levels of HSPs (HSP10, HSP25, HSP27, HSP40, HSP47, HSP60, HSP70 and HSP90) were significantly downregulated in the livers of DEHP-treated quail. In this study, we concluded that DEHP exposure resulted in liver function damage and hepatotoxicity by reducing the expression of HSFs and HSPs in quail liver, which inhibited the protective effect of the HSR signaling pathway.

Role of the Nrf2-ARE pathway in perfluorooctanoic acid (PFOA)-induced hepatotoxicity in Rana nigromaculata

Perfluorooctanoic acid (PFOA) is widely distributed in various environmental media and is toxic to organisms. This study demonstrated that PFOA induces hepatotoxicity in the frog and evaluated the role of CYP3A and the Nrf2-ARE signaling pathway in regulating responses to PFOA-induced hepatotoxicity. Rana nigromaculata were exposed to 0, 0.01, 0.1, 0.5, or 1 mg/L PFOA solutions in a static-renewal system for 14 days. Liver tissue samples were collected 24 h after the last treatment. Hepatic histology was observed by HE staining and transmission electron microscopy. The oxidative stress levels in the liver were measured. The expression levels of CYP3A, Nrf2, NQO1, and HO-1 mRNA were measured by quantitative reverse transcription–polymerase chain reaction. PFOA-treated frog liver tissue exhibited diffuse cell borders, cytoplasmic vacuolization, broken nuclei, nuclear chromatin margination, and swollen mitochondria. In addition, the livers of PFOA-treated frogs showed a significantly elevated content of reactive oxygen species, malondialdehyde, glutathione and glutathione S-transferase activity compared to the livers of control frogs. However, the glutathione peroxidase activities concomitantly decreased in PFOA-treated frogs compared to those in the control group. Furthermore, compared with control frogs, the expression levels of CYP3A, Nrf2, and NQO1 mRNA significantly increased in PFOA-treated frogs. HO-1 mRNA expression remarkably increased only in groups treated with 0.5 or 1 mg/L PFOA. Our results indicate that PFOA induces hepatotoxicity in a dose-dependent manner. Furthermore, the results of the comparison analysis between different gender groups illustrated that PFOA is more toxic to female frogs than male frogs. Our results demonstrated that PFOA causes liver damage and that CYP3A enhances PFOA-induced female frogs hepatotoxicity are more virulent than male through biotransformation, and the activation of the Nrf2-ARE pathway is induced to protect against hepatotoxicity in Rana nigromaculata, all of which provide the scientific basis for the protection of amphibians against environmental contaminants.

Evaluation of the effect of Gallic acid against carbon tetrachloride-induced liver injury in albino rats: histological, immunohistochemical and biochemical study

Introduction: Carbon tetrachloride (CCL4) is used for induction of liver fibrosis. Liver fibrosis has no standard treatment. Gallic acid (GA) is a naturally occurring phenolic compound having anti-inflammatory and anti-cancer activities.Aim of the work: This work aimed to evaluate the preventive role of GA against CCL4 induced liver fibrosis.Materials and Methods: Forty adult male albino rats were divided into 4 groups; Group I: without treatment .GroupII: received olive oil. Group III: received 1.5 mL/kg of CCL4 twice a week. Group IV: received CCL4 with GA 100mg/kg. Animals were sacrificed after 8 weeks. Liver specimens were processed for light and electron microscopic (EM) study. Area % of collagen and α-SMA expression, levels of AST, ALT, ALP, reduced GSH, MDA, SOD and hydroxyproline were measured and evaluated statistically.Results: Group III revealed foci of altered hepatocytes with dense nuclei and vacuolated cytoplasm. Dilatation and congestion of central and portal veins with mononuclear cell infiltration were observed. There was increased collagen deposition around the central vein. Intense stained α-SMA-positive cells were observed. CCL4 increased collagen and α-SMA expression area %. EM showed electronlucent areas in the cytoplasm of the hepatocytes, vacuolation and margination of nuclear chromatin. CCL4 increased AST, ALT, ALP, hepatic MDA and hydroxyproline levels. Moreover, it decreased the activities of SOD and reduced GSH. The co-administration of GA prevented most of these histological and biochemical changes.Conclusions: The use of natural antioxidants as GA can be promising in ameliorating liver fibrosis better than the drugs and their side effects.

Molecular detection and pathology of spontaneous cases of Chronic Respiratory Disease in chicken

Chronic Respiratory Disease (CRD) is a major problem causing morbidity and mortality which leads to economic loss to the poultry industry. The present research was carried out to study the gross, microscopic and ultrastructural changes in target tissues of natural cases of CRD and to detect the presence of causative pathogenic M. gallisepticum and M. synoviae. Routine post mortem examination of birds submitted to Avian Health Laboratory, DPR, Hyderabad were carried out and from selected CRD cases, the presence of M. gallisepticum and M. synoviae was confirmed by PCR. Results showed a higher prevalence of M. synoviae (25.98%) than M. gallisepticum (9.45%) in samples collected from CRD affected birds. Gross lesions included cloudiness of air sacs, presence of caseous mass in the air sacs, congested tracheal mucosa with catarrhal exudates and caseous materials and congestion and oedema of lungs. Occasionally, sinusitis, conjunctivitis, pericarditis, perihepatitis and salpingitis were also observed. Microscopic changes were edema, congestion and infiltration of mononuclear cells. Ultrastructural studies showed nucleoplasmolysis, margination of nuclear chromatin and swelling of perinuclear cisternae, dilatation of RER and swollen and damaged mitochondria in the air sac and trachea of birds suffering from CRD and positive for M. synoviae.

Identification and characterization of ostreid herpesvirus 1 associated with massive mortalities of Scapharca broughtonii broodstocks in China.

In the early summer of 2012 and 2013, mass mortalities of blood ark shell (Scapharca [Anadara] broughtonii), broodstocks were reported in several hatcheries on the coast of northern China. Clinical signs including slow response, gaping valves and pale visceral mass were observed in diseased individuals. In response to these reported mortalities, 238 samples were collected from hatcheries at 6 sites. Microscopic changes including lysed connective tissue, dilation of the digestive tubules, eosinophilic inclusion bodies, nuclear chromatin margination and pyknosis were found in affected animals. Transmission electron microscopy (TEM) revealed herpes-like viral particles within the connective tissue of the mantle. Quantative PCR (qPCR) and nested PCR (nPCR) analysis using primers specific for ostreid herpesvirus 1 (OsHV-1) indicated significant higher prevalence of OsHV-1 DNA in cases associated with mass mortalities than those without mass mortalities (p = 0.0012 for qPCR, p < 0.0001 for nPCR). qPCR also indicated that samples associated with mass mortalities carried high viral DNA loads, while the loads in apparently healthy samples were significantly lower (t = 3.15, df = 92, p = 0.002). Sequence analysis of the C2/C6 region of nPCR products revealed 5 newly described variants, which were closely related to each other. Phylogenetic analysis of the 5 virus variants and 48 virus variants reported in previous studies identified 2 main phylogenetic groups, and the 5 virus variants identified here were allocated to a separate subclade. To our knowledge, this is the first report of mass mortalities of bivalve broodstocks associated with OsHV-1 infection.

First ultrastructural observations on gastritis caused by Physaloptera clausa (Spirurida: Physalopteridae) in hedgehogs (Erinaceus europeaus).

Ultrastructural changes of gastritis due to infection with Physaloptera clausa in 12 fresh carcasses of euthanized European hedgehogs (Erinaceus europaeus) collected from different part of Urmia, Iran, in which they were highly populated with this animal, six females and six males were subjected to detail necropsy with special reference to the stomach. Macroscopic changes of stomach were recorded and some of the worms collected. Based on number of parasites present in the stomach, they were divided into light infection, mild infection, and severe infection. Parasites were collected, and worms identification of the species was confirmed on the basis of light microscope examination with reference to keys. Tissues fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide and processed and plastic embedded; ultrathin sections of 60-70 nm were cut and stained with uranyl acetate and lead citrate; electron microscopic observations showed that, in light infection some changes were observed in gastric cells such as dilatation and vesiculation of the endoplasmic reticulum, large numbers of lipid granules, mitochondrial swelling, nuclear chromatin margination, and some nucleus showed washed out appearance. Other cells showed some alterations in mitochondria, dilatation of smooth endoplasmic reticulum, loss of both free and bound ribosomes, vesiculation in cytoplasm, and increase Golgi apparatus and secretory vesicles. The inflammatory cells including lymphocytes, macrophages, mast cells, and predominantly eosinophils were identified. In moderate infection, the cellular pattern of gastric mucosa replaced with inflammatory cells. The marked increase of macrophages and other inflammatory cell was observed. A particular finding in our study was the presence of globule leukocyte in the moderate stage. Moreover, scant formation and distribution of collagen fibers as well as fibroblasts were also noted. In severe infection, the most obvious observation was marked distribution of collagen fibers around the mucosal cells. The fibroblastic cells with elongated nucleus and extensive indentation were noticed. In conclusion, the result of our study revealed P. clausa could be a cause of gastritis and according to cellular pattern of inflammatory reaction, with the increase of worm burden and development of infection, chronic gastritis was stabilized. Present investigation documented the ultrastructural changes during verminous gastritis in hedgehogs.

Effect of Atorvastatin (ATOR) on the Cardiac Muscle Fibres in Hyperlipidemic Adult Male Albino Rats (Structural and Biochemical Study)

Background:Atorvastatin (ATOR) used as a cholesterol-lowering drug. Ator is one of statins widely used, commercially available and increasingly used day by day.Few studies and limited data were existed on atorvastatin induced histological and ultrastructural changes in the cardiac muscle fibres. The present study was conducted to evaluate the potential toxicity of the human equivalent therapeutic doses of Atorvastatin on cardiac muscle fibres in induced hyperlipidemic adult male albino rats. Material and Methods: thirty adult male albino rats were used in this study, the rats used weighing (150±10) gm. Rats were divided into groups,Group one (5 rats), served as the control group (C) .Other twenty five rats were subjected to high fat diet (25% fat and 2% cholesterol) for 3 weeksand thenthey subdivided into three groups.Group two (five rats) was considered as a hyperlipidemic group (H).Group three, treated group one (T1) (ten rats) received atorvastatin(Ator 20mg) (1.5 mg / day / rat)for three weeks , by the end the third week five rats weresacrificed and other five rats were considered as the recovery group one (R1) , they received normal diet only for another three weeks. Group four , treated group two (T2) (ten rats) received Atorvastatin (Ator 20mg) (1.5 mg / day / rat)for other six weeks , by the end of the 6th week five rats weresacrificed and other five rats were considered as the recovery group two (R2) , they received only normal diet for another six weeks. At the end of each step of the experiment, rats were sacrificed and specimens of cardiac muscle fibres prepared by different methods for light and electron microscopic studies. Also blood sampling was obtained forthebiochemical study.Results: the histological data obtained from the different groups showed many changes, in hyperlipidemic group (H) indistinct and distorted striation and separation of cardiac muscle fibres , ,vesicular nuclei , presence of myofibroblasts with congested and dilated blood vessels, loss of some nuclei and condensation of nuclear chromatin with normal mitochondria were detected. Treated group one (T1) showed that cardiac muscle fibres restored their striation with separated and splitted myofibres and pyknotic nuclei, increased myofibrobasts ,lymphocytic infiltration ,congested blood vessels and mitochondrial vacuolation with disorganization of their cristae. In treated group two (T2) patchy loss of striation was observed with splitted cardiac muscle fibres ,pyknotic nuclei of cardiomyocytes, margination of nuclear chromatin ,congested blood vessels, lymphocytic infiltration, swelling of mitochondria with disorganization of their cristae. In recovery group one (R1) cardiac muscle fibres restored their striation. In recovery group two (R2) cardiac muscle fibres restored their normal architecture. The statistically evaluated data showed a significant difference in parentage of collagen and optical density of PAS + ve materials in the different groups when compared to the control group specially in T2.The laboratory evaluated data showedsignificant increase inlevels of cholesterol (C) , triglycerides (TG), low density lipoprotein- cholesterol (LDL-C), Very low density lipoprotein (vLDL), Atherogenic index(AI) and decrease in high density lipoprotein- cholesterol (HDL-C) between the control and the hyperlipidemic group. Administration of ATOR to the treated groups induce significant decrease inlevels of C, TG, LDL-C , (vLDL), (AI) and increase of HDL-C when compared with the hyperlipidemic group .Also there were significant decreasein levels of C, TG, LDL-C and increase of HDL-Cin recovery groups when compared with the hyperlipidemic group. Conclusion: administration of ATOR for long time led to adverse degenerative effects on structure of the cardiac muscle fibres, although it has a potent lowering effect on lipid profile in hyperlipidemia.

Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs.

The microcystins-induced DNA damage in the liver and the heart of zebrafish, Danio rerio

Microcystins (MCYST) are the freshwater cyanobacterial toxins, known to induce hepatocellular carcinoma, necrosis, intrahepatic bleeding, as well as human and livestock mortality. Within hepatocytes, MCYST selectively bind to protein phosphatases 1 and 2A, resulting in severe liver damage. The toxicology of MCYST in mice and rats has been well studied, but little is known regarding genotoxicity in aquatic animals. In this study, the zebrafish, Danio rerio was exposed to crude extract of Microcystis aeruginosa bloom. Liver and heart were examined for MCYST-induced toxicity. Light microscopy at 36 h revealed severe, widespread apoptotic necrosis of the majority of hepatocytes, and cytoskeletal deformation in myocardiocytes. Hepatocytes were dissociated with cell shrinkage and margination of nuclear chromatin. Laddering of genomic DNA from the liver and heart of the exposed fish in an increment of 180–200 bp was consistent with apoptosis. Fluorimetric analysis of DNA unwinding was carried out to determine the DNA strand breakage. After 36 h exposure, the % double-stranded DNA was significantly reduced in hepatocytes and myocardiocytes. In conclusion, the results obtained in this study indicate that, the extract of M. aeruginosa bloom is genotoxic to fish. The DNA damage observed in this study may be attributed to the activation of DNA endonucleases. This model of DNA damage may contribute for identifying novel molecular mechanisms of interest for therapeutic application.

The pathology of ‘scale drop syndrome’ in Asian seabass, Lates calcarifer Bloch, a first description

This is the first pathological description of 'scale drop syndrome' (SDS) in Asian seabass, Lates calcarifer Bloch. Cumulative mortality was estimated at 40-50%. The vasculitis in all major organs including the skin and associated tissue necrosis was distinctive. The dermis overlying scale beds was often necrotic and associated with scale loss. Necrosis of splenic ellipsoids, renal glomeruli and choroid rete glands of eye were further hallmarks of a disease with systemic vascular involvement. The brain was not spared vascular damage, and the resulting multifocal encephalomalacia probably accounts for the spiral swimming behaviour in some affected fish. Other lesions included accentuated hepatic lobulation and gastric gland necrosis. Nuclear chromatin margination and karyolysis in hepatocytes, renal tubular epithelium and gastric and intestinal epithelium suggest specific targeting of cells. Basophilic cytoplasmic inclusions were present in spleen, kidney, liver, heart and choroid rete, but they were not prominent. Using transmission electron microscopy, two morphological forms of virions were observed: single- and double-enveloped hexagonal virions. Based on size and morphology, these virions resemble iridovirus or herpesvirus. The cause of SDS is unknown, but the pathological changes, especially the vasculitis, suggest an infectious aetiology, possibly viral.

Cutaneous Viral Papilloma with Local Extension and Subungual Cyst Formation in a Dog

A viral-induced digital cutaneous exophytic papilloma was diagnosed in a 2-year-old, spayed, female Siberian husky dog with lameness. Digital pain and lameness persisted after removal of the initial papilloma, and the fifth lateral digit was subsequently amputated. Upon histologic examination of the digit, a de novo digital, cutaneous, inverted, viral papilloma and subungual cyst were diagnosed. The inverted cutaneous papilloma, located at the junction of the digital paw pad and ventral nail, extended focally through the nail into the subungual space, where an expansile cyst was formed. Cellular changes suggestive of papillomavirus infection were present in the epithelium of the original exophytic papilloma, as well as the endophytic mass and subungual cyst. Cytopathic effects included ballooning degeneration of keratinocytes, koilocytosis, irregularity of keratohyalin granules, and margination of nuclear chromatin. Numerous faintly basophilic to eosinophilic intranuclear inclusions measuring 10-15 microm in diameter were present within keratinocytes of the exophytic, endophytic, and subungual cystic lesions. Electron microscopy was performed on tissues from all lesions and revealed numerous 40-45 nm diameter hexagonal virions characteristic of papillomavirus that were arranged in crystalline arrays and dense clusters within affected nuclei.

Histological and haematotoxic effects of Azadirachta indica (Neem) leaf extract in albino rats

The crude methanolic extract of Azadirachta indica leaves was administered intraperitoneally once daily for ten days at varied doses of (100, 200 and 500mg/kg) in 24 albino rats. The acute histopathological changes induced in the spleen, liver, kidney, heart and lungs of the albino rats were studied. Changes in serum biochemical and haematological parameters were also studied [1]. The histopathological results showed congestion and haemosiderosis at a dose level of 200mg/kg, while sections of the lungs showed evidence of interstitial pneumonia and interlobular oedema, the hepatocytes showed necrosis, margination of nuclear chromatin and hyperplastic bile duct epithelial cells. The control group (0mg/kg) and 100mg/kg treatment groups had no lesion. It was then concluded that the administration of the extracts to the rats at doses of 200mg/kg i.p and above, induces histological changes in the spleen, liver and lung which are dose dependent. On the haematotoxic effects, administration of the extract at a dose level of 100mg/kg did not produce any toxic symptoms but at 200 and 500mg/kg, the animals showed high incidence of mortality, even though no significant difference in weight gain, food consumption and fluid intake was observed. There was a fall in packed cell volume (PCV) and a significant elevation of total leucocyte (WBC) count serum trasaminases (ALT and AST) showed enhanced activity and there was an elevation in the levels of urea and bilirubin. Hence, the extracts suggest haematotoxicity in rats at the doses of 200 and 500mg/kg bwt.

Cigarette smoke extract inhibits the proliferation of alveolar epithelial cells and augments the expression of P21WAF1

Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type II and its relationship with P21WAF1, the alveolar epithelial type II cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type II cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke extract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose-and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic microscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type II and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.

Peroxisome proliferator activated receptor gamma coactivator-1 induces apoptosis in human epithelial ovarian cancer cells

To study the apoptosis-inducing effects of peroxisome proliferator activated receptor gamma (PPARgamma) coactivator-1 (PGC-1alpha) on the human epithelial ovarian cancer cell. Human epithelial ovarian cancer cell of the line HO8910 were cultured and divided into 3 groups Ad- PGC-1alpha group (infected with adenovirus. containing PGC-1alpha), Ad- GFP group (infected with adenovirus containing green fluorescent protein, and blank control group. Forty-eight hours later, light microscopy was used to observe the morphological changes of the cells, and apoptosis of the cells was observed by Hoechst staining assay. RT-PCR was used to measure the expression of intracellular Bax and Bcl-2 mRNA. Chinese hamster ovary cells of the line CHO were cultured, infected by adenovirus containing PGC-1alpha and GFP and negative adenovirus respectively, and then underwent Hoechst staining assay and flow cytometry. Light microscopy and Hoechst staining assay showed that there were many morphological characteristics of apoptosis including compaction and margination of nuclear chromatin, nuclear fragments, and apoptotic bodies in the Ad- PGC-1alpha. RT-PCR showed the mRNA expression of intracellular Bax was up-regulated by 1.5 times and the mRNA expression of Bcl-2 was down-regulated by 69% in the Ad- PGC-1alpha group. However, PGC-1alpha treatment did not induce apoptosis in the CHO cells. PGC-1alpha induces apoptosis in epithelial ovarian cancer cells in vitro, which may be a result of up-regulation of the pro-apoptotic gene Bax and down-regulation of the anti-apoptotic gene Bcl-2. And over-expression of PGC-1alpha results in no apoptosis in CHO.

Mortality and herpesvirus infections of the Pacific oyster Crassostrea gigas in Tomales Bay, California, USA

Seed losses of Pacific oysters Crassostrea gigas have been associated with an ostreid herpesvirus-1 (OsHV-1) in Europe, and in 2002, a similar OsHV was detected in Tomales Bay, California, USA. In May of 2003, 5 stocks of seed Pacific oysters were planted at 2 sites (Inner Bay and Outer Bay) in Tomales Bay and monitored for mortality, presence/prevalence of OsHV (using polymerase chain reaction [PCR] and histology), and growth. Temperature (degrees C) and salinity data were collected every half an hour at each site. OsHV was detected at both the Inner and Outer Bay sites on the same sample date and mean temperature predicted OsHV presence (p < 0.005). High levels of mortality occurred 2 wk (Inner Bay site) and 4 wk (Outer Bay site) after OsHV detection. OsHV presence predicted mortality (p = 0.01). Temperature maximums and overall temperature exposure were greater at the Inner Bay site and may explain why mortality affected these oysters sooner than oysters planted at the Outer Bay site. Differences in cumulative mortality were significant among stocks (p < 0.0001), but not between sites (p > 0.05). OsHV prevalence was similar among stocks (p > 0.05) and between sites (p > 0.05). No evidence of herpesvirus-induced Cowdry type A nuclear inclusions or other pathogens were observed. Changes in tissue and cellular architecture including dilation of the digestive tubules and nuclear chromatin margination and pycnosis were observed in OsHV-infected oysters, consistent with previously observed OsHV infections. Stocks with smaller oysters had higher mortality rates than those with larger oysters; growth rate did not correlate with mortalities (p > 0.05). Taken together, these data suggest that the OsHV may cause or act in synergy with temperature to kill Pacific oyster seed in Tomales Bay, but further investigation of OsHV etiology in seed oysters is needed.

Autophagic Degeneration of Motor Neurons in a Model of Slow Glutamate Excitotoxicity in Vitro

There is increasing evidence that so-called “autophagic cell death” participates in cell degeneration in certain pathological conditions. Autophagy might be involved in some neurodegenerative processes, including lateral amyotrophic sclerosis (SLA). The exact mechanism leading to progressive motor neuron (MN) loss remains unclear, but glutamate-mediated mechanism is thought to be responsible. Previous ultrastructural studies by the authors performed on a model of SLA in vitro, based on chronic glutamate excitotoxicity, revealed a subset of morphological features characteristic to different modes of neuronal death, including autophagic degeneration. The contribution of this pathway of MNs death is evaluated in organotypic cultures of rat lumbar spinal cord chronically exposed to specific glutamate uptake blockers: DL-threo-β-hydroxyaspartate (THA) and L-transpyrrolidine-2,4-dicarboxylate (PDC). The study documents the various steps of authophagy in slowly evolving process of MN neurodegeneration. The cells undergoing autophagy usually exhibited sequestration of some parts of cytoplasm with normal and/or degenerated organelles, whereas other parts of cytoplasm as well as neuronal nucleus remained unchanged. The advanced autophagic changes were often associated with other modes of MN death, especially with apoptosis. Numerous MNs revealed apoptotic nuclear features with typical peripheral margination of nuclear chromatin, accompanied by severe autophagic or autophagic-necrotic degeneration of the cytoplasm. These results support the opinion of unclear distinction between different modes of cell death and indicate the involvement of autophagey in MNs neurodegeneration in vitro.

Fluorescence and ultrastructural localization of actin distribution patterns in the nucleus of HL-60 and K-562 cell lines treated with cytostatic drugs

Actin has been studied extensively for decades, but so far its well-defined role has been found localized to the cytoplasm. The present study characterizes the distribution pattern of actin in the nucleus of HL-60 and K-562 cell lines treated with etoposide and doxorubicin. F-actin labeled with TRITC-phalloidin was found in the nucleus in both cell lines independently of the cytostatic drugs used. Using confocal microscopy F-actin is seen in the center of the nucleus showed labeling of the nucleoli by phalloidin. There are also distinct tracts of F-actin seen from nucleoli to the nuclear envelope in some sections. HL-60 cells treated with 200 micro M etoposide and 5, 10 micro M doxorubicin showed cells with characteristic features of apoptosis at the ultrastructural level. Intense immunogold labeling for actin was seen in nucleus of HL-60 cells with compaction and margination of chromatin. In K-562 cells more intense labeling was often found in the cytoplasm of the cells. Positive labeling for actin was not found after control incubations. F-actin is present in the nucleus and there is labeling of nucleoli by phalloidin. The observation at the ultrastructural level suggests that actin can be involved in chromatin reorganization during the process of apoptosis. Increase of labeling at the ultrastructural level in the nucleus of HL-60 cells in relation with compaction and margination of nuclear chromatin might also suggest translocation of actin from cytoplasm to the nucleus in connection with chromatin reorganization.

Ultrastructural changes during in situ early postmortem autolysis in kidney, pancreas, liver, heart and skeletal muscle of rats

Many morphological studies of the postmortem interval were carried out under conditions in which the tissue was incubated in vitro after extirpation. However, the extirpation affects cell viability. We examined the ultrastructural changes in the kidney, pancreas, liver, heart and skeletal muscle of male Wistar rats occurring postmortem in situ. In each organ, cell edema (cell swelling), appearance of amorphous dense deposits in the mitochondria, loss of glycogen granules, dilation of the endoplasmic reticulum, clumping and margination of nuclear chromatin, and/or condensation of nuclear chromatin were observed, but the duration of the period of ultrastructural change was organ specific. Most of the ultrastructural changes occurred earlier in kidney. In hepatocytes, the morphological degeneration occurred later than in the renal tubule epithelium and earlier than that in the myocardium. Of the five organs we examined, skeletal muscle showed the greatest delay in postmortem change. In the distal tubule epithelium and pancreatic acinar cells, two forms of nuclear change were seen: one resembled necrotic change and the other resembled apoptotic change. The effect of lysosomes and hydrolytic enzymes was not as great as previous findings.