Dilated Endoplasmic Reticulum Research Articles

The MMP-9/TIMP-1 System is Involved in Fluoride-Induced Reproductive Dysfunctions in Female Mice.

A total of 84 healthy female mice were kept with various concentrations of sodium fluoride (F) (0, 50, 100, 150mg F-/L in drinking water for 90days) and were then mated with healthy male mice for 1week to study the effect of excessive fluoride on female reproductive function, particularly in embryo implantation. The rate of pregnancy, litter size, and the birth weight of female mice were evaluated. Ultrastructural changes of uteri tissues were observed by transmission electron microscopy (TEM). The mRNA expression levels of MMP-9 and TIMP-1 were determined by quantitative real-time PCR. The protein expression levels of MMP-9 and TIMP-1 were analyzed by western blotting. Results showed a significant decrease of litter size in mice exposed to fluoride. TEM images of uteri tissue of mice that underwent a 150mg/L F- treatment for 90days showed a vague nucleus, reduced microvilli, increased lysosomes, a dilated endoplasmic reticulum, and a vacuolization mitochondrion when compared with the control group. Following the damage of the structure, the expression levels of MMP-9 and TIMP-1 in uteri tissues were significantly unregulated in the F 150 group. These results show that MMP-9/TIMP-1 system disturbance and changes of histological structure in uteri tissue are involved in fluoride-induced reproductive dysfunctions.

Histological and immunohistochemical study on the effect of gibberellic acid on the seminiferous tubules of testis of adult albino rat and the possible protective role of grape seeds proanthocyanidin extract

Background Gibberellic acid (GA) is widely used in Egypt and other countries to increase the growth of many fruits and vegetables. GA can induce disturbances in the testicular enzymes and hormones. Recent studies have revealed that grape seed proanthocyanidin extract (GSPE) has a potential protective effect in male reproductive disorders. Aim This work aimed to study the effect of GA on the seminiferous tubules of testis of adult albino rat and to evaluate the possible protective role of GSPE. Materials and methods Forty adult male albino rats were divided into four groups: control group, group II treated with GSPE (100 mg/kg once daily for 4 weeks), group III treated with GA (20 mg/kg once daily for 4 weeks), and group IV concomitantly treated with GSPE and GA with the same dose and duration of previous groups. Specimens from the testes were processed for light and electron microscopy. Immunohistochemical study was carried out using antibodies against Bcl-2. Results Specimens from GA-treated animals showed disturbance in the normal architecture with obvious structural changes. Some spermatogenic cells appeared disorganized with wide intercellular spaces, and others were exfoliated into the lumen of the tubules. Many spermatogonia showed vacuolated cytoplasm and deeply stained nuclei. Ultrastructurally, the cytoplasm of Sertoli cells showed markedly dilated smooth endoplasmic reticulum. Some primary spermatocytes appeared with vacuolated cytoplasm and dilated perinuclear cisternae. The early spermatids appeared with electron-dense bodies, concentric lamellar formations, and irregular outlined nuclei. Abnormally shaped spermatozoal heads were detected. The immunohistochemical study showed a highly significant decrease in Bcl-2 immunoreaction. In contrast, minimal changes were observed in rats treated concomitantly with both GSPE and GA, with a nonsignificant change in the immunoreaction. Conclusion GA induced structural changes of the seminiferous tubules of the rat testis that could be minimized by the coadministration of GSPE.

Withaferin A Induces ROS-Mediated Paraptosis in Human Breast Cancer Cell-Lines MCF-7 and MDA-MB-231

Advancement in cancer therapy requires a better understanding of the detailed mechanisms that induce death in cancer cells. Besides apoptosis, themode of other types of cell death has been increasingly recognized in response to therapy. Paraptosis is a non-apoptotic alternative form of programmed cell death, morphologically) distinct from apoptosis and autophagy. In the present study, Withaferin-A (WA) induced hyperpolarization of mitochondrial membrane potential and formation of many cytoplasmic vesicles. This was due to progressive swelling and fusion of mitochondria and dilation of endoplasmic reticulum (ER), forming large vacuolar structures that eventually filled the cytoplasm in human breast cancer cell-lines MCF-7 and MDA-MB-231. The level of indigenous paraptosis inhibitor, Alix/AIP-1 (Actin Interacting Protein-1) was down-regulated by WA treatment. Additionally, prevention of WA-induced cell death and vacuolation on co-treatment with protein-synthesis inhibitor indicated requirement of de-novo protein synthesis. Co-treatment with apoptosis inhibitor resulted in significant augmentation of WA-induced death in MCF-7 cells, while partial inhibition in MDA-MB-231 cells; implyingthat apoptosis was not solely responsible for the process.WA-mediated cytoplasmic vacuolationcould not be prevented by autophagy inhibitor wortmanninas well, claiming this process to be a non-autophagic one. Early induction of ROS (Reactive Oxygen Species)by WA in both the cell-lines was observed. ROS inhibitorabrogated the effect of WA on: cell-death, expression of proliferation-associated factor andER-stress related proteins,splicing of XBP-1 (X Box Binding Protein-1) mRNA and formation of paraptotic vacuoles.All these results conclusively indicate thatWA induces deathin bothMCF-7 and MDA-MB-231 cell lines byROS-mediated paraptosis.

Histological effect of nicotine on adrenal zona fasciculata and the effect of grape seed extract with or without withdrawal of nicotine

Cigarette smoking is harmful to the health of both smokers and nonsmokers. It is a major cause of death. This study aimed to investigate the structural changes in the zona fasciculata of albino rats caused by nicotine and the protective effect of grape seeds with or without the stoppage of nicotine administration. Thirty-five adult male rats were used and equally divided into five groups: negative and positive control groups (Groups I and II), nicotine-treated group (Group III), nicotine- and grape seed extract-treated group (Group IV), and nicotine withdrawal and grape seed extract-treated group (Group V). Adrenal glands were dissected and prepared for histological studies. The majority of zona fasciculata cells of Group III showed striking changes in terms of swelling of the cells with marked cytoplasmic vacuolation, many pyknotic nuclei, and increased immunoexpression to caspase 3 antibodies. By electron microscopy, a marked increase in lipid deposition with its appearance in the capillary between zona fasciculata cells was noticed. Heterochromatic nuclei and dilated smooth endoplasmic reticulum were noted. Degenerated mitochondria and some mitochondria that had cavitation with a progressive loss of their cristae were seen. The zona fasciculata cells of Group IV were partially improved, while in Group V, those cells showed complete improvement. We can conclude that nicotine causes severe histological changes in zona fasciculata cells. Grape seed extract can partially ameliorate these changes, and complete recovery is achieved with grape seed extract after the stoppage of nicotine administration.

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators.

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24-30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Histological alterations in Leydig cells and macrophages in azoospermic men.

The study aimed to compare the histological features of Leydig cells and macrophages in the testicular interstitium of obstructive versus nonobstructive azoospermia. Thirty-nine azoospermic men undergoing testicular sperm extraction during intracytoplasmic sperm injection were allocated into obstructive azoospermia group (GI) and nonobstructive azoospermia group (GII) which was subdivided into Sertoli cell-only syndrome (GIIA), germ cell arrest (GIIB) and hypospermatogenesis (GIIC) subgroups. Serum LH, FSH and testosterone levels were measured. Ultrastructural changes and the mean number of CD68-positive cells were estimated in the different groups. In GIIA, Leydig cells' processes came in contact with macrophages and showed smooth endoplasmic reticulum dilatation. In GIIB, Leydig cells showed apoptotic changes. Macrophages were commonly encountered in their vicinity demonstrating large number of lysosomes. In GIIC, Leydig cells showed euchromatic nuclei. Macrophages showed expulsion of their lysosomal contents in the interstitium surrounded by apoptotic bodies. The mean count of total CD68-positive macrophages was higher in cases of obstructive azoospermia with nonsignificant differences compared to nonobstructive azoospermia groups. Significant increase in FSH level was detected in GIIA compared to GI. It is concluded that structural interactions might take place between Leydig cells and macrophages in the interstitial tissue of azoospermic men.

Age-associated histological changes of gastric fundal mucosa and ultrastructure of gastric chief cells in human and Beagle dogs

Objective To investigate the effect of aging on gastric pepsinogen secretion through observing histological changes of gastric fundal mucosa and ultrastructure of gastric chief cells in human and Beagle dogs at different ages. Methods Fifty middle-aged and elderly indigestion patients with gastroscopy were selected as study objects and divided into young and middle age group (age 20-59 years, n=19), junior elderly group (age 60-69 years, n=11), middle elderly group (age 70-79 years, n=10), and senior elderly group (no less than 80 years, n=10). In addition, nineteen healthy Beagle dogs were also selected as study objects and divided into young and middle age group (age between 1 to 5 years, n=8), junior elderly group (age six to eight years, n=5), and senior elderly group (no less than 9 years, n=6). The histology and morphology of gastric fundal mucosa of human and Beagle dogs were observed under light microscope. Then the thickness of lamina propria was measured and the number of gastric chief cells was counted. The ultrastructure of gastric chief cells was observed under electron microscope, and the area percentages of secretory granule(also called mucous granule)area in cytoplasm of chief cell was calculated.The data was analyzed by one-way analysis of variance. Results No significant histological and morphological changes of gastric fundal mucosa were observed in human and Beagle dogs at different ages.There was no significant difference in the thickness of lamina propria among different ages in human and Beagle dogs (all P>0.05). The number of human chief cells of young and middle age group, junior elderly group, middle elderly group and senior elderly group was 71.79±16.85, 52.73±16.60, 57.10±20.21 and 43.70±16.89/high power field (HPF), respectively, which decreased as age increased (F=6.431, P=0.001). The numbers of chief cells of young and middle age group, junior elderly group, and senior elderly group of Beagle dogs were 328.38±32.36, 341.20±42.49 and 225.67±52.19/HPF, respectively, which decreased as age increased (F=13.647, P<0.01). Aging-related degeneration was founded in ultrastructure of gastric chief cells in human and Beagle dogs including rough endoplasmic reticulum dilation, ribosomes missing, secretory granule decreasing and so on. The area percentages of secretory granule area in cytoplasm of human chief cells of young and middle age group, junior elderly group, middle elderly group and senior elderly group were (67.28±3.79)%, (66.88±4.84)%, (65.63±7.10)% and (56.25±8.47)%, respectively, which decreased as age increased (F=6.069, P=0.002). The area percentages of secretory granule area in cytoplasm of Beagle dogs′ chief cells of young and middle age group, junior elderly group, and senior elderly group were (58.83±3.07)%, (52.34±4.73)% and (39.89±4.46)%, respectively, which decreased as age increased (F=38.837, P<0.01). Conclusion As age increased, the ability of pepsinogen secretion of fundal gland decreased, which may be one of the reasons of functional dyspepsia in the eldly. Key words: Chief cells, gastric; Ultrastructure; Aging; Pepsinogens; Beagle dog

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators.

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24–30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Deoxyrhapontigenin, a Natural Stilbene Derivative Isolated From Rheum undulatum L. Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Human Breast Cancer Cells.

Although current chemotherapeutic agents are active at the beginning of therapy, the most common risk is the development of resistance during later stages in almost all cancer types including breast cancer. Hence, investigation of novel drugs is still a priority goal for cancer treatment. The objective of the present study is to investigate the anticancer effect of a derivative of stilbene, deoxyrhapontigenin (DR) isolated from Rheum undulatum L. root extracts against the chemoresistant MCF-7/adr and its parental MCF-7 human breast cancer cells. The morphological images indicate that DR induces an extensive cytoplasmic vacuolation in breast cancer cells. Mechanistic investigations revealed that DR treatment causes endoplasmic reticulum (ER) dilation and upregulated the expression of ER stress markers GRP78, IRE1α, eIF2α, CHOP, JNK, and p38. Subsequently, we also identified that DR increases the levels of apoptotic fragment of PARP (89 kDa) in breast cancer cells. Blocking the expression of one of the components of the ER stress–mediated apoptosis pathway, CHOP using siRNA significantly decreased DR-induced apoptotic cleavage of PARP. In summary, the present study suggests that the induction of ER stress–mediated apoptosis by DR may account for its cytotoxic effects in human breast cancer cells.

Abstract 2187: Garcinone inhibits nasopharyngeal carcinoma cell growth and arrests cell cycle via modulating the expression of ATR/Stat3/4E-BP1

Abstract Nasopharyngeal carcinoma (NPC) is one of the common head and neck malignancies. Radiotherapy and chemotherapy are the standard treatments for NPC. Garcinone, a natural compound isolated from Garcinia oblongifolia Champ., is a xanthone derivative and has been shown to have potential cytotoxic effects on certain cancer types. However, there have been limited studies performed on its effects on NPC cells and the mechanisms of these effects remain unknown. In this study, we firstly found that garcinone significantly inhibited cell viability of human NPC cell lines CNE1, CNE2, HK1, and HONE1. This inhibition was in a time- and dose-dependent manner. Then, flow cytometry analysis was performed on cell cycle analysis. We observed that garcinone arrested cell cycle at S phase. With 10μM of high-dose garcinone treatment, the cells exhibited necrotic morphology changes including cell swelling, rough endoplasmic reticulum (ER) degranulation, ER dilatation, mitochondria swelling, and vacuolar degeneration. Finally, we found that garcinone stimulated the expression levels of ATR and 4E-BP1, while efficiently inhibited the expression levels of Cyclin B1, Cyclin D1, Cyclin E2, cdc2, CDK7, and Stat3. Collectively, the ability of garcinone to inhibit NPC in vitro suggested that garcinone might be a novel agent for the management of NPC. Supported by NSFC Key Project 81130046; NSFC Project 81171993 and NSFC81272415; Guangxi Key Projects 2013GXNSFEA053004 and 2012GXNSFCB053004; Guangxi Ministry of Education 201202ZD022 and 201201ZD004. Citation Format: Yudui Xia, Xia Liu, Chunllin Zou, Hongwei Guo, Yeguo Yang, Yong Lei, Jian Zhang, Yi Lu. Garcinone inhibits nasopharyngeal carcinoma cell growth and arrests cell cycle via modulating the expression of ATR/Stat3/4E-BP1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2187.

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway.

Endoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here, we investigated the effects and underlying mechanism of ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) on cardiac ER stress. Cell death, apoptosis and ER stress-related signalling pathways were measured in cultured neonatal rat cardiomyocytes, treated with the ER stress inducers tunicamycin, hydrogen peroxide and thapsigargin. Acute myocardial infarction was established using left coronary artery occlusion in mice, and infarct size was measured by triphenyltetrazolium chloride staining. Echocardiography was used to assess heart function and transmission electron microscopy for evaluating ER expansion. Ginkgolide K (GK) significantly decreased ER stress-induced cell death in both in vitro and in vivo models. In ischaemic injured mice, GK treatment reduced infarct size, rescued heart dysfunction and ameliorated ER dilation. Mechanistic studies revealed that the beneficial effects of GK occurred through enhancement of inositol-requiring enzyme 1α (IRE1α)/X box-binding protein-1 (XBP1) activity, which in turn led to increased ER-associated degradation-mediated clearance of misfolded proteins and autophagy. In addition, GK was also able to partly repress the pro-apoptotic action of regulated IRE1-dependent decay and JNK pathway. In conclusion, GK acts through selective activation of the IRE1α/XBP1 pathway to limit ER stress injury. GK is revealed as a promising therapeutic agent to ameliorate ER stress for treating cardiovascular diseases.

Electron microscopic ultrastructural study on the toxicological effects of AgNPs on the liver, kidney and spleen tissues of albino mice.

The present study deals with the intraperitoneal administration of 500, 1000, 3000, and 5000mg/kg of AgNPs in albino mice for 28 days to evaluate the potential toxicological effects of AgNPs on blood biochemical parameters and to investigate the light and electron microscopic histopathological alterations on three major targets organs i.e., liver, kidney and spleen. The AgNPs was well tolerated and no mortality was observed even at the highest dose i.e., 5000mg/kg. Mice treated with 500 and 1000mg/kg AgNPs did not show significant behavioral, biochemical and ultrastructural pathological changes. Mice treated with 1000mg/kg AgNPs produces little ultrastructural alteration in liver, kidney and spleen. However, mice treated with 3000 and 5000mg/kg AgNPs revealed significant changes in biochemical parameters. Electron microscopic ultrastructural investigation of liver and kidney shows that the administration of 3000 and 5000mg/kg AgNPs revealed irregularity in the nuclear membrane, nuclear chromatin condensations, degenerated hepatocytes, swollen and pleomorphic mitochondria with distorted cristae, extensive dilation of rough endoplasmic reticulum, destructed cytoplasm, hypertrophied and fused podocytes and thickened basement membrane in the endothelial cells of the proximal tubules. The spleen sections at 3000 and 5000mg/kg AgNPs revealed megakaryocytes hyperplasia, lobulations, invaginations and folding of nuclei and nuclear membrane. The present research indicates that AgNPs were well tolerated at the lower doses, but significant alterations in liver, kidney and spleen were observed at the higher doses tested. It is, therefore, suggested that further studies are needed for the minimization of the observed side effects, especially at higher doses before AgNPs being applied in pharmaceutical application.

Immunoreactions for P53 isoforms are associated with ultrastructural proliferative profiles in benign thyroid nodules.

P53 isoforms originate from the alternative initiation of P53 gene translation through usage of an internal promoter located in intron 4. All P53 isoforms are spliced in intron 9 and may modulate cell proliferation and cell fate outcome in response to DNA damage. To examine immunoexpression of P53 isoforms in benign proliferative lesions occurring in multinodular thyroids and to assess the ultrastructural phenotype of P53 distribution in the thyrocytes of those lesions by electron microscopy. By immunohistochemistry and transmission electron microscopy (TEM), we evaluated 38 multinodular thyroids containing a total of 102 benign lesions: 38 nodular goiters (NG; colloid=20, parenchymatous=18), 52 follicular adenomas (FA) and 12 Hashimoto's thyroditis (HT). FA were classified into 10 normo-follicular, 9 macro-follicular, 28 micro-follicular and 5 solid variants. Immunoreaction for P53 isoforms was observed in approximately 50% of all lesions, except macrofollicular variant FA (33%). At TEM analysis, immunoreactive NG, FA and TH lesions showed signs of proliferation by simultaneous appearance of dispersed chromatin, increased amounts of cytoplasmic organelles and dilation of the rough endoplasmic reticulum. TEM signs of apoptosis and proliferation were also detected in FA, but with different rates compared to NG. The immunohistochemical expression of P53 isoforms in NG, FA and HT suggests their role in the development of these lesions. Ultrastructural findings support the hypothesis that P53 immunoexpression correlates with reactive proliferative changes in thyrocytes.

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment.

Phthalimido-thiazoles as building blocks and their effects on the growth and morphology of Trypanosoma cruzi

Chagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affects approximately 6–7 million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases; however, its efficacy during the symptomatic chronic phase is controversial. The present work reports the synthesis and anti-T. cruzi activities of a novel series of phthalimido-thiazoles. Some of these compounds showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in spleen cells, and the resulting structure-activity relationships are discussed. We also showed that phthalimido-thiazoles induced ultrastructural alterations on morphology, flagellum shortening, chromatin condensation, mitochondria swelling, reservosomes alterations and endoplasmic reticulum dilation. Together, these data revealed, for the first time, a novel series of phthalimido-thiazoles-structure-based compounds with potential effects against T. cruzi and lead-like characteristics against Chagas disease.

Gasoline induced pulmo- oxidative damage in mice model

The present work aims to evaluatethe cytotoxic effects on lung cells exerted by gasoline. 60 male albino mice were used in the present experiment, divided equally into 3 groups: first group (control) was left access to fresh air, second group (experimental) was subjected to gasoline 80- vapor/1h./day and third group (experimental) was subjected to gasoline 90-vapor/1h./day along eight consecutive weeks. The study comprises determination of body weight, bioaccumulation of some heavy metals ,histological and ultra-changes . Histological changes increased according to duration of exposure as, infiltration of inflammatory cells, detachment and necrosis of the epithelial cells .The electron micrographs revealed dilatation of the smooth endoplasmic reticulum, loss of the secretory granules in the Clara cells and loss of cilia in the ciliated cells that exhibited bleb formation. Necrotic type II pneumocytes, exhibited vacuolation, fragmentation of the rough endoplasmic reticulum,mitochondrial degeneration , nuclear alterations, degeneration of lamellar bodies and microvillar atrophy. In conclusion, gasoline vapour inhalation induced lung tissue injury and cellular damage concomitant with impairment of the lung antioxidant defense system. These effects were more pronounced with the unleaded than with the leaded gasoline.

SEC23A Functionally Compensates for SEC23B Deficiency in Mice

Congenital Dyserythropoietic Anemia type II (CDAII) is a disease of ineffective erythropoiesis characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroid precursors. CDAII is an autosomal recessive disease resulting from mutations in SEC23B. SEC23 is a core component of COPII vesicles, which transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi. Despite identification of the underlying genetic defect, the molecular mechanism by which SEC23B deficiency produces the unique CDAII phenotype remains unknown. We previously reported that mice homozygous for a Sec23b null allele die perinatally, exhibiting massive pancreatic degeneration, precluding evaluation of the adult erythroid compartment. To examine the impact of SEC23B deficiency on adult murine hematopoiesis, we generated mice with erythroid specific and pan-hematopoietic deficiency for SEC23B by crossing a second, conditional Sec23b allele (Sec23bfl), in which exons 5 and 6 are flanked by loxP sites, to an EpoR-Cre and Vav1-Cre, respectively. These mice did not exhibit anemia or any other CDAII characteristic. Similarly, mice transplanted with SEC23B-deficient fetal liver cells harvested from E17.5 embryos also failed to recapitulate any features of the CDAII phenotype. We next generated mice deficient in SEC23B exclusively in the pancreas by crossing the Sec23bfl allele to either p48-Cre or Pdx1-Cre. These mice exhibit a phenotype indistinguishable from mice with germline deletion of Sec23b, indicating that loss of pancreatic SEC23B is sufficient to explain the perinatal-lethality of global SEC23B deficiency in mice. The mammalian genome contains two paralogs for SEC23, SEC23A and SEC23B. These paralogs are highly identical at the amino acid level (~85%). We examined the relative expression of SEC23B/SEC23A in WT tissues from both humans and mice. This ratio is higher in human BM compared to pancreas, while it was higher in mouse pancreas compared to BM. In order to determine if SEC23A can rescue the phenotype of SEC23B deficient mice when expressed under the regulatory control of Sec23b, we genetically engineered the Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23a-b) in mouse embryonic stem cells via recombinase mediated cassette exchange. A heterozygous Sec23a-b intercross yielded the expected number of mice homozygous for the Sec23a-b allele. These mice exhibited normal survival, development, and fertility. Pancreas tissues dissected from Sec23a-b/a-b mice had normal weights, were histologically indistinguishable from WT controls, and did not exhibit dilated ER by transmission electron microscopy. Western blot analysis confirmed the absence of SEC23B in pancreata of Sec23a-b/a-b mice, with high levels of SEC23A expression. These data demonstrate that the SEC23A and SEC23B proteins overlap significantly (or completely) at the level of protein function, and suggest that the distinct phenotypes of human and mouse SEC23 deficiency are the result of an evolutionary shift in the tissue-specific gene expression programs of SEC23A and SEC23B. These findings also suggest that therapies that increase the expression of either SEC23 paralog in erythroid cells might be effective in CDAII. DisclosuresNo relevant conflicts of interest to declare.

Hydrogen sulfide suppresses endoplasmic reticulum stress-induced endothelial-to-mesenchymal transition through Src pathway.

Hydrogen sulfide (H2S) ameliorates cardiac fibrosis in several models by suppressing endoplasmic reticulum (ER) stress. Endothelial-to-mesenchymal transition (EndMT) is implicated in the development of cardiac fibrosis. Therefore, we investigated whether H2S could attenuate EndMT by suppressing ER stress. ER stress was induced by tunicamycin (TM) and thapsigargin (TG) and inhibited by 4-phenylbutyrate (4-PBA) in human umbilical vein endothelial cells (HUVECs). ER stress and EndMT were measured by Western blot, Real-Time PCR and immunofluorescence staining. Inhibition Smad2 and Src pathway were performed by specific inhibitors and siRNA. Ultrastructural examination was detected by transmission electron microscope. The functions of HUVECs were investigated by cell migration assay and tube formation in vitro. Under ER stress, the expression of endothelial marker CD31 significantly decreased while mesenchymal markers α-SMA, vimentin and collagen 1 increased which could be inhibited by 4-PBA. Moreover, HUVECs changed into a fibroblast-like appearance with the activation of Smad2 and Src kinase pathway. After inhibiting Src pathway, EndMT would be significantly inhibited. TM reduced H2S levels in cell lysate and H2S pretreatment could preserve endothelial cell appearance with decreased ER stress and ameliorated dilation of ER. H2S could also downregulate the mesenchymal marker expression, and upregulate the endothelial markers expression, accompanied with the suppression of Src pathway. Moreover, H2S partially restored the capacity of migration and tube formation in HUVECs. These results revealed that H2S could protect against ER stress-induced EndMT through Src pathway, which may be a novel role for the cardioprotection of H2S.

Endoplasmic reticulum pathology and stress response in neurons precede programmed necrosis after neonatal hypoxia-ischemia

The endoplasmic reticulum (ER) is tasked, among many other functions, with preventing excitotoxicity from killing neurons following neonatal hypoxia-ischemia (HI). With the search for delayed therapies to treat neonatal HI, the study of delayed ER responses becomes relevant. We hypothesized that ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal HI. Since necrostatin-1 (Nec-1), an inhibitor of programmed necrosis, provides delayed neuroprotection against neonatal HI in male mice, Nec-1 is an ideal tool to study delayed ER responses. C57B6 male mice were exposed to right carotid ligation followed by exposure to FiO2=0.08 for 45min at p7. Mice were treated with vehicle or Nec-1 (0.1μl of 8μmol) intracerebroventricularly with age-matched littermates as controls. Biochemistry assays at 3 and 24h and electron microscopy (EM) and immunohistochemistry at 96h after HI were performed. EM showed ER dilation and mitochondrial swelling as apparent early changes in neurons. With advanced neurodegeneration, large cytoplasmic fragments containing dilated ER “shed” into the surrounding neuropil and calreticulin immunoreactivity was lost concurrent with nuclear features suggestive of programmed necrosis. Nec-1 attenuated biochemical markers of ER stress after neonatal HI, including PERK and eIF2α phosphorylation, and unconventional XBP-1 splicing, consistent with the mitigation of later ER pathology. ER pathology may be an indicator of severity of neuronal injury and potential for recovery characterized by cytoplasmic shedding, distinct from apoptotic blebbing, that we term neuronal macrozeiosis. Therapies to attenuate ER stress applied at delayed stages may rescue stressed neurons after neonatal HI.

Ultrastructural characterization of Gonadotrophs in the Wild caught female bat Taphozous nudiventris kachhensis (Dobson)

Present study has been design to observe the ultramicroscopic structure of Gonadotrophs in the female bat Taphozous nudiventris kachhensis during the various phases of reproductive cycle. During the present study specimen were collected during Estrous and pregnancy to know the probable role of Gonadotrophs (FSH, LH) in respective stage of reproductive cycle. After collecting female specimen they were weight and dissect out for the pituitary gland and then those were fixed in ice cold gluteraldehyde. Gonadotrophs (FSH and LH) are mostly frequently observed cell types after Somatotrophs cells in the pars distalis of the female bat Taphozous nudiventris kachhensis. FSH cell during estrus is large, ovoid to polyhedral in shape with irregular shaped nucleus. Cell cytoplasm shows well developed Golgi apparatus, rough endoplasmic reticulum and mitochondria this indicating active state. During early pregnancy cytoplasm of FSH cell appears vacuolated because of dilation of rough endoplasmic reticulum. Mitochondria are spherical to rod shaped with lamellar cristae. During late pregnancy rough endoplasmic reticulum shows dilation. Golgi complex is juxtanuclear in position and mitochondria are numerous. LH cells during early part of gestation are large with bilobed nucleus. Cell cytoplasm shows well developed rough endoplasmic reticulum heavily dotted with ribosomes and contains large number of secretory granules. Hypertrophied LH cell in pars distalis of bat during late pregnancy shows well developed rough endoplasmic reticulum occupies major part of cytoplasm. Golgi zone is well developed with dilated saccules. Secretory granules are very few.