Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

Summary The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.Research fellow of the Alexander von Humboldt Foundation     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged     

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone

Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.     

Adaptive mechanisms during food restriction in <Emphasis Type="Italic">Acomys russatus</Emphasis>: the use of torpor for desert survival

The golden spiny mouse (Acomys russatus) is an omnivorous desert rodent that does not store food, but can store large amounts of body fat. Thus, it provides a good animal model to study physiological and behavioural adaptations to changes in food availability. The aim of this study was to investigate the time course of metabolic and behavioural responses to prolonged food restriction. Spiny mice were kept at an ambient temperature of 27°C and for 3 weeks their food was reduced individually to 30% of their previous ad libitum food intake. When fed ad libitum, their average metabolic rate was 82.77±3.72 ml O₂ h^–1 during the photophase and 111.19±4.30 ml O₂ h^–1 during the scotophase. During food restriction they displayed episodes of daily torpor when the minimal metabolic rate gradually decreased to 16.07±1.07 ml O₂ h^–1, i.e. a metabolic rate depression of approximately 83%. During the hypometabolic bouts the minimum average body temperature T_b, decreased gradually from 32.6±0.1°C to 29.0±0.4°C, with increasing duration of consecutive bouts. In parallel, the animals increased their activity during the remaining daytime. Torpor as well as hyperactivity was suppressed immediately by refeeding. Thus golden spiny mice used two simultaneous strategies to adapt to shortened food supply, namely energysaving torpor during their resting period and an increase in locomotor activity pattern during their activity period.     

Voltage dependence of the basolateral membrane conductance in theAmphiuma collecting tubule

Summary The basolateral potassium conductance of cells of most epithelial cells plays an important role in the transcellular sodium transport inasmuch as the large negative equilibrium potential of potassium across this membrane contributes to the electrical driving force for Na⁺ across the apical membrane. In the present study, we have attempted to establish, theI-V curve of the basolateral membrane of theAmphiuma collecting tubule, a membrane shown to be K⁺ selective. TransepithelialI-V curves were obtained in short, isolated perfused collecting tubule segments. The shunt conductance was determined using amiloride to block the apical membrane Na⁺ conductance. In symmetrical solutions, the shuntI-V curve was linear (conductance: 2.2±0.3 mS·cm^–2). Transcellular current was calculated by subtracting the shunt current from the transepithelial current in the absence of amiloride. Using intracellular microelectrodes, it was then possible to measure the basolateral membrane potential simultaneously with the transcellular current. The basolateral conductance was found to be voltage dependent, being activated by hyperpolarization: conductance values at –30 and –80 mV were 3.6±1.0 and 6.6±1.0 mS·cm^–2, respectively. BasolateralI-V curves were thus clearly different from that predicted by the constant field model. These results indicate that the K⁺-selective basolateral conductance of an amphibian collecting tubule shows inward (anomalous) rectification. Considering the electrogenic nature basolateral Na–K-pump, this may account for coupling between pump-generated potential and basolateral K⁺ conductance.     

Ultrastructural and morphometric aspects of the juxtaglomerular apparatus in the monkey kidney

Summary The authors describe the ultrastructure of the juxtaglomerular apparatus in five adult male Cebus apella monkeys and communicate morphometric data of the macula densa.In comparison with several species of rodents examined before, the macula cells of the monkey contain many more mitochondria and possess a particularly thick basal membrane. The relative volume of the nuclei is slightly smaller than in rodents.The Goormaghtigh cells of the monkey resemble those of the other animals investigated.The epithelioid (or juxtaglomerular) cells do not contain secretory granules. This observation reminds one of the behavior of the epithelioid cells of guinea pigs.Dedicated to Professor Dr. med. G. Petry (Marburg/Lahn) on the occasion of his 65th birthdayThe authors wish to thank Professor Dr. G. Peters, Head of the Institute of Pharmacology, and Professor Dr. F. Roch-Ramel for kindly having provided the monkey kidneys     

Reactions to disturbances in the context of antipredatory behaviour in a solitary subterranean rodent

We investigated reactions of free-living silvery mole-rats (Heliophobius argenteocinereus) to anthropogenic disturbances. Mole-rats detected soil vibrations caused by man carefully walking at a distance of up to 6 m (proved by radio-telemetry). Occasionally, mole-rats encountered outside a nest retreated there after this type of disturbance. After having their burrows broken into, the mole-rats retreated into deep tunnels, separating themselves from the rest of the burrows by soil plugs. Trapping success of silvery mole-rats using live traps for subterranean rodents was low, which contrasts with data so far published on social bathyergids. Antipredatory function of examined behaviours is discussed.     

Ultrastructural localization of the iodination centre in the endostyle of the adult amphioxus (Branchiostoma lanceolatum)

Summary The site of iodination in the endostyle of the adult amphioxus was examined by light-and electron-microscopic autoradiography. In accordance with previous studies, light-microscopic autoradiography showed a distinct accumulation of autoradiographic grains at the apical end of epithelial cells in the lateral part of the endostyle. In the electron microscope two distinct cellular zones were identified in an approximate position of the light-microscopic zone 5. Zone 5a, not previously recognized, was adjacent to zone 4 and consisted of six to nine rows of cells free of characteristic granules. Cells in zone 5b contained large mucous granules and had, in previous ultrastructural studies, been identified as belonging to the typical zone 5. Four or less incomplete rows of granule-containing cells, not observed in previous studies, marked the border between zones 5b and 6. After incubation in ¹²⁵I for 5 min, electron-microscopic autoradiography showed a selective concentration of label to zone 5a, which, thus, corresponds to the iodination centre seen in the light microscope. The grains were associated with cilia and microvilli in the lumen. After longer incubation times (30, 60, 90 min) grains were still concentrated at the surface of zone 5a but were also associated with the surface of zones 5b and 6. Grains were also located over the cytoplasm of all three zones. They were associated with vesicles and lysosome-like structures, suggesting secondary uptake of labelled products by endocytosis. Methimazole, an inhibitor of peroxidase, abolished the autoradiographic reaction. In conclusion, the site of iodination in the endostyle of amphioxus is located in zone 5a, which has not previously been ultrastructurally defined. Iodination in the endostyle is an extracellular process, but secondary uptake by endocytosis appears to occur.     

Nutritional responses to different diet quality in the subterranean rodent Ctenomys talarum (tuco-tucos)

Nutritional response to different diet quality was examined in the subterranean rodent Ctenomys talarum (tuco-tuco). Animals maintained in captive conditions were fed with three plant species that differed in their fibre content. Tuco-tucos showed the ability to perform adjusts in short time lapse in response to diet quality; food ingestion, egestion and feces ingestion changed in animals under different plant species diets. Time budget, mainly time devoted to feeding and activity accompanied such changes. Coprophagy was practiced along the day and night following the arrhythmic activity pattern found for this species. Feces reingestion was not associated to resting. Furthermore, it was observed during fresh food ingestion, being pellets chewed. Soft and hard feces differed in morphological and nutritional characteristics.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Ultrastructural changes in the fat body of adult female Aedes aegypti in relationship to vitellogenin synthesis

Summary The ultrastructure of the fat body of Aedes aegypti was followed from emergence through a blood meal. Changes in the volume of protein granules and lipid droplets were also examined. The relationship of these events to the known endocrinology of vitellogenin synthesis in mosquitoes is discussed.Massachusetts Experiment Station Paper No. 2130. Supported by the Massachusetts Agricultural Experiment Station, Project n° 356, and by NIH grant n° AI-11909     

Ultrastructural changes induced by precocene II and 3,4-dihydroprecocene II in the corpora allata of Blattella germanica

Summary Ultrastructural studies on corpora allata (CA) from different stages during the first gonadotropic cycle of the cockroach Blattella germanica have shown well defined changes which have a correspondence with oocyte length, CA volume and juvenile hormone (JH) biosynthesis. The most significant variations concern the mitochondria and the endoplasmic reticulum. Topically applied precocene II (P II) at a dose of 200 g induced a transient arrest of CA function, although cytotoxic effects were occasionally observed. When CA were maintained in vitro with 10^-3 M of P II, a relationship between the time of treatment (3, 6 or 9 h) and the intensity of the effects was apparent. The 9-h treatment led to an irreversible inhibition of JH production which parallels the severe damages observed in the CA (membrane lysis, nuclear pyknosis, vacuolization). Equivalent studies performed with the chroman derivative 3,4-dihydroprecocene II (DHP II) showed that it is less active than P II. Only treatments as severe as 12 h of incubation with a 10^-3 M concentration elicited cytotoxic effects which could be due to radical species involved in the in situ oxidative bioactivation of DHP II. Thus, this compound could be regarded as a new type of pro-allatocidin.     

The synaptic organization of visual interneurons in the lobula complex of flies

Summary The synaptic organization of three classes of cobalt-filled and silver-intensified visual interneurons in the lobula complex of the blowfly Calliphora (Col A cells, horizontal cells and vertical cells) was studied electron microscopically. The Col A cells are regularly spaced, columnar, small field neurons of the lobula, which constitute a plexus of arborizations at the posterior surface of the neuropil and the axons of which terminate in the ventrolateral protocerebrum. They show postsynaptic specializations in the distal layer of their lobula-arborizations and additional presynaptic sites in a more proximal layer; their axon terminals are presynaptic to large descending neurons projecting into the thoracic ganglion. The horizontal and vertical cells are giant tangential neurons, the arborizations of which cover the anterior and posterior surface of the lobula plate, respectively, and which terminate in the perioesophageal region of the protocerebrum. Both classes of these giant neurons were found to be postsynaptic in the lobula plate and pre- and postsynaptic at their axon terminals and axon collaterals. The significance of these findings with respect to the functional properties of the neurons investigated is discussed.     

Responses of Etiella zinckenella to habitat fragmentation of Caraganas in desert steppe in Ningxia, China

Habitat fragmentation is a common cause for which species becomes threatened or endangered. Existence of viable habitat is critical to the survival of any species, so habitat fragmentation is the main reason for the changes in distribution and abundance of organisms, and is usually considered to have negative effect on the abundance, species richness and population of organisms in a specific landscape. But this effect may also depend on whether some species could use one or more types of habitat in a specific landscape. Because of its well resistance to stress, Caragana is one of predominant shrub in desert region for forest planting and desert preventing, which plays a critical role in desert control and ecosystem stabilization. Baijitan National Nature Reserve, located in Lingwu County, Ningxia Hui Autonomous Region, China, is typical of desert nature reserve in which the Caragana spp., Oxytropisaciphylla and other desert plants are protected. The Caragana woodlands in this region show a pattern of dots, patches and strips separated by natural and cultivated forest, thereby leading to a typical fragmented landscape. Etiellazinckenella (Lepidoptera, Pyralidae) is one of seed pests of Caragana. In order to illuminate the responses of E. zinckenella to the habitat fragmentation of Caraganas woodlands, the present study focused on the effects of habitat area, habitat fragmentation, as well as matrix composition on the population density and damage ratio of E. zinckenella in desert steppe. From May 2008 to June 2009, by using parallel jump sampling method, 13 Caragana woodland patches representing four landscapes from Baijitan National Nature Reserve were investigated and totally 15,117 pods were inspected. Then, the landscape fragmentation indices, population density and damage ratio of E. zinckenella in Caragana woodlands were calculated. The statistic analysis of the data indicated that the four landscapes have a significant difference in the population density and damage ratio of E. zinckenella; and in the same landscape, the neighbor patches also have a significant difference in the damage ratios of E. zinckenella. E. zinckenella seems to prefer some species of Caragana, for instance, the damage ratio of E. zinckenella to Caragana microphylla is found the highest, followed by the damage ratio to Caraganadavazamcidamage, while the damage ratio to Caraganakorshinskii is found the lowest. The coverage of Caragana is found positively related to the damage ratio of E. zinckenella with hinge damage ratio in high coverage of Caragana forest. The regression analysis shows that the latitude (R_s = 0.5724), longitude (R_s = 0.5577), altitude (R_s = 0.4614) and patch area (R = 0.3012) were not significantly associated with population density and damage ratio of E. zinckenella. However, the population density and damage ratio of E. zinckenella decreased with the increasing in patch area. The landscape patch fragmentation index (R = 0.91129) and the patch density index (R = 0.89864) show a positive correlation with damage ratio. The fragmentation shape index (R = ?0.89675) and inside habitat area fragmentation index (R = ?0.77646) show a negative correlation with the damage ratio. As a result, the population of E. zinckenella was suppressed by the landscape fragmentation, but the patch isolation and complementary resources in the landscape matrix may also have a positive impact on the population density of E. zinckenella.     

Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems

The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO₂ fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per m²), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing k_mt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.Abbreviations DPIP 2,6-dichloroindophenol - EF and PF exoplasmic and protoplasmic fracture faces, respectively - PS I and PS II photosystems I and II, respectively     

Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria,with special reference to vitelline membrane formation

Summary Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed.Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.This work was supported by the Volkswagenstiftung, HannoverI wish to thank Prof. Dr. H. Emmerich, Techn. Hochschule Darmstadt, for valuable discussions     

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1^−/− renal cells displayed aberrant cell–cell contacts and tubulomorphogenesis. The Pkhd1^−/− cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1^−/− mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1^−/− kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.     

Alteration in N-acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N-acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E₄-PHA. The increased GnT-V activity led to the elevation of the β1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.