Basal Infoldings Research Articles

Expression of E-cadherin in pig kidney

E-cadherin is a cell adhesion molecule that plays an important role in maintaining renal epithelial polarity and integrity. The purpose of this study was to determine the exact cellular localization of E-cadherin in pig kidney. Kidney tissues from pigs were processed for light and electron microscopy immunocytochemistry, and immunoblot analysis. E-cadhedrin bands of the same size were detected by immunoblot of samples from rat and pig kidneys. In pig kidney, strong E-cadherin expression was observed in the basolateral plasma membrane of the tubular epithelial cells. E-cadherin immunolabeling was not detected in glomeruli or blood vessels of pig kidney. Double-labeling results demonstrated that E-cadherin was expressed in the calbindin D28k-positive distal convoluted tubule and H+-ATPase-positive collecting duct, but not in the aquaporin 1-positive, N-cadherin-positive proximal tubule. In contrast to rat, E-cadherin immunoreactivity was not expressed at detectable levels in the Tamm-Horsfall protein-positive thick ascending limb of pig kidney. Immunoelectron microscopy confirmed that E-cadherin was localized in both the lateral membranes and basal infoldings of the collecting duct. These results suggest that E-cadherin may be a critical adhesion molecule in the distal convoluted tubule and collecting duct cells of pig kidney.

Evaluation of Praziquantel effects on Echinostoma paraensei ultrastructure

Echinostomiasis is a food-borne, intestinal, zoonotic, snail-mediated helminthiasis caused by digenean trematodes of the family Echinostomatidae with seven species of the genus Echinostoma infecting humans or domestic and wildlife animals. Echinostoma paraensei is a peristomic 37-collar-spined echinostome belonging to the “revolutum group”.Praziquantel (PZQ) is the drug of choice for treatment and control of human schistosomiasis and food-borne trematodiasis. In the present study we used scanning and transmission electron microscopy to further elucidate the trematocidal effect of PZQ on adult E. paraensei and confirmed that this trematode is a suitable model to study anthelmintic drugs. Hamsters infected with E. paraensei were treated with a single dose of 30mgkg−1 of PZQ. The worms were recovered 15, 30, 90 and 180min after drug administration. There was a significant decrease in worm burden in the small intestine in the hamster-E. paraensei model at the intervals of 30, 90 and 180min after the treatment. The worms displayed damage of the peristomic collar with internalization of the spines and erosion of the tegument of the circumoral head-collar of spines. Ultrastructural analysis demonstrated an intense vacuolization of the tegument, appearance of autophagic vacuoles and swelling of the basal infolds of the tegumental syncytium. There was no change in the morphology of cells from the excretory system of adult E. paraensei, however, there was an apparent decrease of stores of glycogen particles in parenchymal cells in PZQ-treated worms. Our results demonstrated that PZQ promotes surface and ultrastructural damage of the tegument of adult E. paraensei supporting the idea that this trematode may constitute a good model to investigate drug effects mechanisms in vitro and in vivo.

The possible protective role of curcumin versus garlic in amiodarone-induced keratopathy in rats

Introduction Diabetes is a common serious metabolic disorder, and it may cause nephropathy. Garlic was experimentally documented to have an antidiabetic potential. Aim of the work Investigate the possible protective role of garlic in the structural changes of the renal cortex in diabetic male albino rats. Materials and methods Rats were divided into two main groups; group I included rats that were divided equally into a control group Ia (rats were administered distilled water) and a garlic group Ib (rats were administered 100▒mg/kg garlic powder). Group II (diabetic group) included rats in which diabetes was induced by a single intraperitoneal injection of 60▒mg/kg body weight streptozotocin; then, the rats were divided into two groups: an untreated diabetic group IIa (rats were administered distilled water) and a garlic-treated diabetic group IIb (rats were administered 100▒mg/kg garlic powder). Results At the end of the experiment, the rats in the untreated diabetic group showed a nonsignificant change in their body weight and a significant increase in the mean fasting blood glucose by 40.6%. Microscopic examination of the renal cortex showed structural changes in both the proximal convoluted tubules and distal convoluted tubules in the form of a luminal cast, less basal infolding, and fewer basal mitochondria with loss of its cristae. Loss of microvilli of proximal convoluted tubules, extravasation of red blood cells, and a significant increase in the thickness of the glomerular basement membrane were also observed. However, at the end of the experiment, the garlic-treated diabetic group showed a significant increase in body weight by 21.7%, a significant decrease in the mean fasting blood glucose level by 29.6%, and absence of the most of the structural changes of the renal cortex. Conclusion Garlic could protect against diabetes-induced structural changes in the renal cortex.

Gross morphology and ultrastructure of salivary glands of the mute cicada Karenia caelatata Distant (Hemiptera: Cicadoidea)

Salivary glands of the cicada Karenia caelatata Distant were investigated using light microscopy and transmission electron microscopy. The salivary glands are paired structures and consist of principal glands and accessory glands. The principal gland is subdivided into anterior lobe and posterior lobe; the former contains about 34–39 long digitate lobules, while the latter contains approximately 30–33 long digitate lobules and 13–22 short digitate lobules. These short digitate lobules, about one fifth or sixth as long as the long digitate lobules, locate at the base of the long digitate lobules of posterior lobe. All of these digitate lobules vary in size, disposition, length and shape. The anterior lobe and the posterior lobe are connected by an anterior-posterior duct. Two efferent salivary ducts, which connect with the posterior lobe, fuse to form a common duct. The accessory gland is composed of three parts: a greatly tortuous and folded accessory salivary tube, a circlet of gular gland constituting of several acini of the same size, and a non-collapsible accessory salivary duct. The digitate lobules and gular glands possess secretory cells containing abundant secretory granules vary in size, shape, and electron density, as might indicate different materials are synthesized in different secretory regions. The anterior-posterior duct lines with a player of cuticular lining, and cells beneath the cuticular lining lack of basal infoldings, as suggests the duct serves just to transport secretions. The accessory salivary duct is lined with cuticular lining; cells of the duct have well developed basal infoldings associated with abundant mitochondria, as probably suggests the duct is a reabsorptive region of ions. The cells of the accessory salivary tube possess deep basal infoldings and well developed apical dense microvilli, indicating the cells of the tube are secretory in function. Concentric lamellar structures and a peculiar structure with abundant membrane-bound vesicles and secretory granules are observed for the first time, but their derivation and function remain unclear. The morphology and ultrastructure differences observed in the principal glands and accessory gland of the salivary glands of K. caelatata indicate that the sheath saliva was secreted by the principal glands, and the watery saliva was secreted by the accessory salivary glands. Rod-shaped microorganisms are found in the salivary glands (i.e., accessory salivary duct, gular gland, and long digitate lobule of salivary glands) for the first time, and their identity, function, and relationship to microorganisms residing in the salivary glands and/or other parts of alimentary canal of other cicadas need to be investigated further.

Distribution, fate and histopathological effects of ethion insecticide on selected organs of the crayfish, Procambarus clarkii

This study aims to investigate the fate and histopathological effects of ethion on selected organs of the crayfish, Procamabrus clarkii. Crayfish were exposed to 1mgl−114C-ethion and the concentrations of ethion and its possible degradation products were measured in water and different organs of the crayfish over both the exposure and recovery periods. Chromatographic analysis revealed that ethion was degraded into ethion monooxon, ethion dioxon, O,O-diethyl phosphorothioate, O-ethyl phosphorothioate and one unknown compound. At the end of exposure period, ethion was accumulated in different organs of the crayfish especially in the hepatopancreas and gills. Following the transfer of crayfish to clean water for seven days, the concentration of insecticide residues were decreased in both the hepatopancreas and gills suggesting that these organs play an important role in elimination of ethion. On the other hand, the exposure of the crayfish to ¼ 96h-LC50 (0.36mgl−1) of ethion caused extensive ultrastructural alterations to both hepatopancreas and gill epithelial cells. In the hepatopancreas, the most notable pathological features included vacuolation, degradation and distinct cell lysis. In the gill epithelium, the histopathological alterations included infiltration of hemocytes, cytoplasmic vacuolation and a decrease in the number of basal plasma membrane infoldings.

Ultrastructural features of the larval Malpighian tubules of the flesh fly Sarcophaga ruficornis (Diptera: Sarcophagidae)

The larval Malpighian tubules of Sarcophaga ruficornis Fab. consist of two types of cells, namely principal and stellate cells. The principal cells are characterized by a large euchromatic nucleus, well-developed basal plasma membrane infoldings associated with mitochondria and prominent luminal microvilli containing mitochondria with well-developed cristae. The central cytoplasm of the cells contains clear vacuoles, lysosomes, mineral concretions or spherocrystals, lipid droplets and peroxisomes. The stellate cells are thin with extremely short basal plasma membrane infoldings and luminal microvilli devoid of mitochondria. The ultrastructural features of the principal cells show the characteristic of transporting cells engaged in water and ion transport. The cells are separated by septate junctions. The tubules are richly supplied by the tracheae.

X-Box Binding Protein 1 Is Essential for the Anti-Oxidant Defense and Cell Survival in the Retinal Pigment Epithelium

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

Fasciola gigantica: The in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80μg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.

Natural History of Age-Related Retinal Lesions That Precede AMD in Mice Fed High or Low Glycemic Index Diets

Epidemiologic data indicate that people who consume low glycemic index (GI) diets are at reduced risk for the onset and progression of age-related macular degeneration (AMD). The authors sought corroboration of this observation in an animal model. Five- and 16-month-old C57BL/6 mice were fed high or low GI diets until they were 17 and 23.5 months of age, respectively. Retinal lesions were evaluated by transmission electron microscopy, and advanced glycation end products (AGEs) were evaluated by immunohistochemistry. Retinal lesions including basal laminar deposits, loss of basal infoldings, and vacuoles in the retinal pigment epithelium were more prevalent in the 23.5- than in the 17-month-old mice. Within each age group, consumption of a high GI diet increased the risk for lesions and the risk for photoreceptor abnormalities and accumulation of AGEs. Consuming high GI diets accelerates the appearance of age-related retinal lesions that precede AMD in mice, perhaps by increasing the deposition of toxic AGEs in the retina. The data support the hypothesis that consuming lower GI diets, or simulation of their effects with nutraceuticals or drugs, may protect against AMD. The high GI-fed C57BL/6 mouse is a new model of age-related retinal lesions that precede AMD and mimic the early stages of disease and may be useful for drug discovery.

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) digestive tract: Histology, ultrastructure and its physiological significance

The Sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae), is a key pest of wheat in the Middle East and some other areas which causes severe qualitative and quantitative damage. The objective of the current work is to describe the morphology of the midgut of E. integriceps adult. Microscopic studies revealed that foregut consists of oral cavity, pharynx and oesophagus likely other phytophagous Hemiptera. In the Midgut, four anatomical regions could be identified: the first ventriculus (V1), the second ventriculus (V2), the third ventriculs (V3), and the fourth ventriculus (V4). The microvilli and perimicrovillar membrane (PMM) were found in V1–V3 regions with columnar cells characterized by presence of mitochondria, rough endoplasmic reticulum and basal infoldings in the basal portion. However, V2 and V3 showed less developed basal plasma membrane infoldings. Three cell types: columnar, endocrine and regenerative cells were found in V1–V3. The V4 region showed different histological features from the other three midgut regions by showing a vacuolated epithelium with crypts storing symbiotic bacteria. The hindgut had a short ileum followed by a well-developed rectum with an epithelial cell layer and a thin cuticular intima. The current results suggest V1–V3 midgut regions play a role in enzyme and absorption, whereas V4 seems to have no function in digestion.

Erratum to: Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10mg/kg live weight and ketoconazole at a dosage of 10mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96h p.t., and sub-tegumental flooding was seen from the 72h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ+inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.

Additional Ultrastructural Observations of the First Segments of Malpighian Tubules in Cenocorixa bifida (Hemiptera: Corixidae) in Relation to Reabsorption of Solutes

Abstract The ultrastructure of the first segment of the four-segmented Malpighian tubules of Cenocorixa bifida Hungerford (Hemiptera: Corixidae) is described. The first segment of each of the four tubules empties proximally into the midgut—hindgut junction. Its squamous epithelium is composed of a single layer of binucleated cells containing two types of mitochondria. The cells rest on a prominent basal lamina and possess short, shallow, widely spaced basal plasma membrane infoldings closely associated with large mitochondria and a few short blunt microvilli devoid of mitochondria projecting into the lumen. In sharp contrast, the fourth segment cells that are secretory in function, display numerous, long basal membrane infoldings that penetrate deeply into the cytoplasm and numerous long microvilli at the luminal border. The cytoplasm of the first segment cells contains dense bodies, small mitochondria, microtubules, vesicles, and vacuoles and is rich in smooth endoplasmic reticulum, giving rise to clear vacuoles, but rough endoplasmic reticula and Golgi apparati are sparse. The apical plasma membrane is covered with glycocalyx and displays coated pits pinching off coated vesicles. The apical cytoplasm displays numerous vesicles containing fine granular material, smooth vesicles, dense tubules, large vacuoles, primary lysosomes, and multivesicular bodies. The lateral plasma membranes are linked by extensive junctional complexes each composed of an apical smooth septate junction, intercalated gap junctions, and a basal heterocellular scalariform junction. The contrast in ultrastructural features between of the first segment and fourth segment cells suggests that the first segment is involved in reabsorption than secretion of inorganic and organic solutes.

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.

Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration

Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated. The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy. The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age. The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ.

Time-dependent changes to the tegumental system and gastrodermis of adult Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48h, 72h and 96h post-treatment (pt). Two control sheep were euthanized alongside the 96h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps liver and, if present, from the gall bladder and they were processed for transmission electron microscopy (TEM). Disruption to the ultrastructure of the tegument became increasingly severe over time pt. Flukes recovered at 48h pt showed widespread blebbing of the apical plasma membrane and swelling of the mucopolysaccharide masses surrounding the basal infolds. There was evidence of reduced secretory activity in the tegumental cells and spacing between the cells. Sloughing of the tegumental syncytium was observed at 72h pt. The subtegumental musculature, parenchyma and tegumental cells were severely disrupted. At 96h pt, all of the flukes were totally devoid of tegument. Disruption to the subtegumental tissue and somatic musculature was severe, and was so extreme in some specimens that the tegumental cells were barely discernible. Disruption to the gastrodermis was also progressive, though not as severe as disruption to the tegument. There was a general decline of secretory activity with time pt. Autophagic activity was apparent from 48h pt and became more widespread with increasing time, culminating in breakdown of the gastrodermal cell cytoplasm. The mitochondria were swollen and electron-lucent and the cisternae of the granular endoplasmic reticulum were dilated and fragmented from 72h pt.

KCNJ10 gene mutations causing EAST syndrome (epilepsy, ataxia, sensorineural deafness, and tubulopathy) disrupt channel function

Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.

Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment

Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocarboxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.