Guadalupian algae-sponge reefs in siliciclastic environments - the reefs at Lengwu (South China) compared with the reef at Iwaizaki (Japan)

Guadalupian reefs occur locally in Guangxi, Guizhou, Yunnan and Western Zhejiang, South China. Two types of Guadalupian reefs can be recognized, one is developed in carbonate platforms, e.g. those in the juncture areas of Guangxi, Yunnan and Guizhou; the other occurs in a littoral clastic shelf. The Lengwu reef in Western Zhejiang is a representative of the latter type, which is a major topic of this paper. Lengwu algae-sponge reef, more than one hundred meters in thickness, are composed mainly of sponges, hydrozoans, algae, bryozoans, microbes and lime mud. Reef limestones sit on the mudstone interbedded with fine sandstone of the proximal prodelta facies and are overlain by coarse clasts of the delta front sediments. Lengwu reef displays a lens-shaped relief, dipping and thinning from the reef core, which is remarkably different from the surrounding sediments, showing a protruding relief. Sponges and microbe/algae form bafflestone, bindstone and framestone of the reef core facies. Fore-reef facies is characterized by lithoclastic rudstone and bioclastic packstone. Reef limestone sequence is composed of three cycles and controlled by sea level changes and sediment influx. Such reef is unique among the Guadalupian reefs in South China, but seems similar in some aspects to lwaizaki reef limestones of south Kitakami in Japan. Algae and microbes growing around sponges to form rigid structure in Lengwu reef are a typical feature, which is distinctly different to Guadalupian reefs in a stable platform facies of Guizhou, Yunnan and Guangxi, South China.

Bond strength of Epiphany (TM) Sealer combined with different adhesive systems photo-activated with LED and QTH

The Epiphany (TM) Sealer is a new dual-curing resin-based sealer and has been introduced as an alternative to gutta-percha and traditional root canal sealers. The canal filling is claimed to create a seal with the dentinal tubules within the root canal system producing a `monoblock` effect between the sealer and dentinal tubules. Therefore, considering the possibility to incorporate the others adhesive systems, it is important to study the bond strength of the resulting cement. Forty-eight root mandibular canines were sectioned 8-mm below CEJ. The dentine discs were prepared using a tapered diamond bur and irrigated with 1% NaOCl and 17% EDTA. Previous the application Epiphany (TM) Sealer, the Epiphany (TM) Primer, AdheSE, and One Up Bond F were applied to the root canal walls. The LED and QTH (Quartz Tungsten Halogen) were used to photo-activation during 45 s with power density of 400 and 720 mW/cm(2), respectively. The specimens were performed on a universal testing machine at a cross-head speed of 1 mm/min until bond failure occurred. The force was recorded and the debonding values were used to calculate Push-out bond strength. The analysis of variance (ANOVA) and Tukey`s post-hoc tests showed significant statistical differences (P < 0.05) to Epiphany (TM) Sealer/Epiphany (TM) Primer/QTH and EpiphanyTM Sealer/AdheSE/QTH, which had the highest mean values of bond strength. The efficiency of resin-based filling materials are dependent the type of light curing unit used including the power density, the polymerization characteristics of these resin-based filling materials, depending on the primer/adhesive used.

The anatomy of two-rooted mandibular canines determined using micro-computed tomography

P>Aim To investigate the internal and external anatomy of extracted human mandibular canines with two roots and two distinct canals using micro-computed tomography (mu CT). Methodology Fourteen two-rooted human mandibular canines were scanned using a high-resolution mu CT system (SkyScan 1174v2; SkyScan N.V., Kontich, Belgium). The images were processed to evaluate the size of the roots, the furcation regions, the presence of accessory canals, the mean distances between several anatomical landmarks, the position of the apical foramina, the direction of root curvatures, the cross-sectional appearances (SMI index), the volume and surface areas of the root canals. Results Root bifurcation was located in both apical (44%, n = 6) and middle (58%, n = 8) thirds of the root. The size of the buccal and lingual roots was similar in 29% of the sample. From a buccal view, no curvature towards the lingual or buccal direction occurred in either roots. From a proximal view, no straight lingual root occurred. In both views, S-shaped roots were found in 21% of the specimens. Location of the apical foramen varied considerably, tending to the mesio-buccal aspect of both roots. Lateral and furcation canals were observed mostly in the cervical third in 29% and 65% of the sample, respectively. The structure model index (SMI) index ranged from 1.87 to 3.86, with a mean value of 2.93 +/- 0.46. Mean volume and area of the root canals were 11.52 +/- 3.44 mm3 and 71.16 +/- 11.83 mm2, respectively. Conclusions The evaluation of two-rooted mandibular canines revealed that bifurcations occurred in the apical and middle third. S-shaped roots were found in 21% of the specimens. Mean volume, surface area and SMI index of the root canals were 11.52 mm3, 71.16 mm2 and 2.93, respectively.

Morphological Alterations of Radicular Dentine Pretreated With Different Irrigating Solutions and Irradiated With 980-nm Diode Laser

Background: The topographical features of intraradicular dentine pretreated with sodium hypochlorite (NaOCl) or ethylenediamine tetraacetic acid (EDTA) followed by diode laser irradiation have not yet been determined. Purpose: To evaluate the alterations of dentine irradiated with 980-nm diode laser at different parameters after the surface treatment with NaOCl and EDTA. Study design: Roots of 60 canines were biomechanically prepared and irrigated with NaOCl or EDTA. Groups were divided according to the laser parameters: 1.5 W/CW; 1.5 W/100 Hz; 3.0 W/CW; 3.0 W/100 Hz and no irradiation (control). The roots were splited longitudinally and analyzed by scanning electron microscopy (SEM) in a quali-quatitative way. The scores were submitted to two-way Kruskal-Wallis and Dunn`s tests. Results: The statistical analysis demonstrated that the specimens treated only with NaOCl or EDTA (control groups) were statistically different (P < 0.05) from the laser-irradiated specimens, regardless of the parameter setting. The specimens treated with NaOCl showed a laser-modified surface with smear layer, fissures, and no visible tubules. Those treated with EDTA and irradiated by laser presented absence of smear layer, tubules partially exposed and melting areas. Conclusions: The tested parameters of 980-nm diode laser promoted similar alterations on dentine morphology, dependent to the type of surface pretreatment. Microsc. Res. Tech. 72:22-27, 2009. (C) 2008 Wiley-Liss, Inc.

Temperature variation at the external root surface during 980-nm diode laser irradiation in the root canal

Objective: To assess the temperature variation in the cervical, middle and apical thirds of root external wall, caused by 980-nm diode laser irradiation with different parameters. Methods: The roots of 90 canines, had their canals instrumented and were randomly distributed into 3 groups (n = 30) according to the laser potency (1.5 W, 3.0 W and 5.0 W). Each group was subdivided into 3 (n = 10) according to the frequency (CM, 100 Hz and 1000 Hz), and each subgroup divided into 2 (n = S): dried canal or filled with distilled water. The maximum temperature values were collected by 3 thermocouples located at each third of the root external wall and recorded by digital thermometers. Results: The groups irradiated in the continuous mode (CM) presented the highest values (11.82 +/- 5.78), regardless of the canals were dry or not, which were statistically different (p < 0.01) from those obtained with 100 Hz (6.22 +/- 3.64) and 1000 Hz (6.00 +/- 3.36), which presented no statistical difference between them (p > 0.01). The groups irradiated with 5.0 W presented the greatest temperature variation (12.15 +/- 5.14), followed by 3.0 W (7.88 +/- 3.92) and 1.5 W (4.02 +/- 2.16), differing between them (p < 0.01). The cervical third of the root presented the highest temperature rises (9.68 +/- 5.80), followed by the middle (7.66 +/- 4.87) and apical (6.70 +/- 4.23), with statistical difference among them (p < 0.01). After 30 s from the end of irradiation, all the specimens presented temperature variation lower than 10 degrees C. Conclusions: Application of 980-nm diode laser in the root, at 1.5 W in all operating modes, and 3.0 W, in the pulsed mode, for 20 s, can safely be used in endodontic treatment, irrespective of the presence of humidity. (C) 2008 Elsevier Ltd. All rights reserved.

Shear Bond Strength and Ultrastructural Interface Analysis of Different Adhesive Systems to Bleached Dentin

Background: It remains unclear as to whether or not dental bleaching affects the bond strength of dentin/resin restoration. Purpose: To evaluated the bond strength of adhesive systems to dentin submitted to bleaching with 38% hydrogen peroxide (HP) activated by LED-laser and to assess the adhesive/dentin interfaces by means of SEM. Study design: Sixty fragments of dentin (25 mm(2)) were included and divided into two groups: bleached and unbleached. HP was applied for 20 s and photoactivated for 45 s. Groups were subdivided according to the adhesive systems (n = 10): (1) two-steps conventional system (Adper Single Bond), (2) two-steps self-etching system (Clearfil standard error (SE) Bond), and (3) one-step self-etching system (Prompt L-Pop). The specimens received the Z250 resin and, after 24 h, were submitted to the bond strength test. Additional 30 dentin fragments (n = 5) received the same surface treatments and were prepared for SEM. Data were analyzed by ANOVA and Tukey`s test (alpha = 0.05). Results: There was significant strength reduction in bleached group when compared to unbleached group (P < 0.05). Higher bond strength was observed for Prompt. Single Bond and Clearfil presented the smallest values when used in bleached dentin. SEM analysis of the unbleached specimens revealed long tags and uniform hybrid layer for all adhesives. In bleached dentin, Single Bond provided open tubules and with few tags, Clearfil determined the absence of tags and hybrid layer, and Prompt promoted a regular hybrid layer with some tags. Conclusions: Prompt promoted higher shear bond strength, regardless of the bleaching treatment and allowed the formation of a regular and fine hybrid layer with less deep tags, when compared to Single Bond and Clearfil. Microsc. Res. Tech. 74:244-250, 2011. (C) 2010 Wiley-Liss, Inc.

Effect of Chelating Solutions on the Microhardness of Root Canal Lumen Dentin

Introduction: The greatest reduction in microhardness of the most superficial layer of dentin of the root canal lumen is desired. The use of chelating agents during biomechanical preparation of root canals removes smear layer, increasing the access of the irrigant into the dentin tubules to allow adequate disinfection, and also reduces dentin microhardness, facilitating the action of endodontic instruments. This study evaluated the effect of different chelating solutions on the microhardness of the most superficial dentin layer from the root canal lumen. Methods: Thirty-five recently extracted single-rooted maxillary central incisors were instrumented, and the roots were longitudinally sectioned in a mesiodistal direction to expose the entire canal extension. The specimens were distributed in seven groups according to the final irrigation: 15% EDTA, 10% citric acid, 5% malic acid, 5% acetic acid, apple vinegar, 10% sodium citrate, and control (no irrigation). A standardized volume of 50 mu L of each chelating solution was used for 5 minutes. Dentin microhardness was measured with a Knoop indenter under a 10-g load and a 15-second dwell time. Data were analyzed statistically by one-way analysis of variance and Tukey-Kramer multiple-comparison test at 5% significance level. Results: EDTA and citric acid had the greatest overall effect, causing a sharp decrease in dentin microhardness without a significant difference (p > .05) from each other. However, both chelators differed significantly from the other solutions (p < .001). Sodium citrate and deionized water were similar to each other (p > .05) and did not affect dentin microhardness. Apple vinegar, acetic acid, and malic acid were similar to each other (p > .05) and presented intermediate results. Conclusion: Except for sodium citrate, all tested chelating solutions reduced microhardness of the most superficial root canal dentin layer. EDTA and citric acid were the most efficient. (J Endod 2011;37:358-362)

Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days. which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V-max of brush-border membrane (BBM) Na/Pi cotransport activity (1943 95 in control vs. 1183 +/- 99 pmol/5 sec/mg BBM protein in K deficiency. P < 0.02). Surprisingly. the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1). and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin. glucosylceramide. and ganglioside GM, content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficit nt rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 11.52 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias

KCNQ1 (K(V)LQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study. the properties and regulation of the cloned sK(V)LQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (

Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Depositional environments for strata cored in CRP-3 (Cape Roberts Project), Victoria Land Basin, Antarctica: palaeoglaciological and palaeoclimatological inferences

Cape Roberts Project drill core 3 (CRP-3) was obtained from Roberts ridge, a sea-floor high located at 77°S, 12 km offshore from Cape Roberts in western McMurdo Sound, Antarctica. The recovered core is about 939 m long and comprises strata dated as being early Oligocene (possibly latest Eocene) in age, resting unconformably on ∼ 116 m of basement rocks consisting of Palaeozoic Beacon Supergroup sediments. The core includes ten facies commonly occuring in five major associations that are repeated in particular sequences throughout the core and which are interpreted as representing different depositional environments through time. Depositional systems inferred to be represented in the succession include: outer shelf, inner shelf, nearshore to shoreface each under iceberg influence, deltaic and/or grounding-line fan, and ice proximal-ice marginal-subglacial (mass flow/rainout diamictite/subglacial till) singly or in combination. The record is taken to represent the initial talus/alluvial fan setting of a glaciated rift margin adjacent to the block-uplifted Transantarctic Mountains. Development of a deltaic succession upcore was probably associated with the formation of palaeo-Mackay valley with temperate glaciers in its headwaters. At that stage glaciation was intense enough to support glaciers ending in the sea elsewhere along the coast, but a local glacier was fluctuating down to the sea by the time the youngest part of CRP-3 was being deposited. Changes in palaeoenvironmental interpretations in this youngest part of the core are used to estimate relative glacial proximity to the drillsite through time. These inferred glacial fluctuations are compared with the global δ18O and Mg/Ca curves to evaluate the potential of glacial fluctuations on Antarctica for influencing these records of global change. Although the comparisons are tentative at present, the records do have similarities, but there are also some differences that require further evaluation.

A revision of Benedenia Diesing, 1858 including a redescription of B-sciaenae (van Beneden, 1856) Odhner, 1905 and recognition of Menziesia Gibson, 1976 (Monogenea : Capsalidae)

Benedenia Diesing, 1858, a genus of capsalid (benedeniine) monogeneans, is redefined. The generic diagnosis is amended to include: the path of tendons in the haptor from extrinsic muscles in the body; presence and form of the marginal valve; a penis occupying a penis canal with weakly muscular wall; a weakly muscular accessory gland reservoir proximal to the penis and enclosed by a proximal extension of the wall of the penis canal; male and female genital apertures usually common, rarely separate; vagina with pore usually close to the common genital pore but may open in mid body between the germarium and the common genital pore, or anterior to the common genital pore. A conservative approach is adopted and the generic diagnosis is clarified and broadened to accommodate species that display some variation in reproductive anatomy, especially of the female system. We argue against potential alternative actions such as defining Benedenia strictly to contain species with separate male and female genital apertures and against recognition of a separate genus, Tareenia Hussey, 1986, for species with a vaginal pore anterior to the common genital pore. Under our conception, Benedenia comprises 21 species: B. sciaenae (van Beneden, 1856) Odhner, 1905 (type species); B. acanthopagri (Hussey, 1986) comb. nov.; B. anticavaginata Byrnes, 1986; B. bodiani Yamaguti, 1968; B. elongata (Yamaguti, 1968) Egorova, 1997; B. epinepheli (Yamaguti, 1937) Meserve, 1938; B. hawaiiensis Yamaguti, 1968; B. hendorffi(von Linstow, 1889) Odhner, 1905; B. hoshinai Ogawa, 1984; B. innobilitata Burhnheim Gomes and Varela, 1973: B. jaliscana Bravo-Hollis, 1952; B. lolo Yamaguti, 1968; B. lutjani Whittington and Kearn, 1993: B. monticellii (Parona and Perugia, 1895) Johnston, 1929; B. ovata (Goto, 1894) Johnston. 1929: B. pompatica Burhnheim, Gomes and Varela, 1973; B. rohdei Whittington, Kearn and Beverley-Burton, 1994; B. scari Yamaguti, 1968; B. sekii (Yamaguti, 1937) Meserve, 1938; B, seriolae (Yamaguti, 1934) Meserve, 1938; and B. synagris Yamaguti, 1953. The type species, B. sciaenae, is redescribed based on new material from Australia. No types for this taxon were designated and we have assigned a series of voucher specimens. Tareenia acanthopagri Hussey, 1986 becomes B. acanthopagri (Hussey, 1986) comb. nov. and T. anticavaginata (Byrnes, 1986) Egorova, 1997 and T. lutjani (Whittington and Kearn, 1993) Egorova, 1997 are returned to Benedenia as B. anticavaginata and B. lutjani Benedenia akaisaki Iwata, 1990 is considered a synonym of B. ovata and B. kintoki Iwata, 1990 is considered a synonym of B. elongata. Two species, B, madai Ishii and Sawada, 1938 and B. pagrosomi Ishii and Sawada, 1938, are considered species inquirendae. Based on the redefinition of Benedenia, the diagnosis for the Benedeniinae is amended. Tareenia is synonymized with Benedenia but Menziesia Gibson, 1976 is recognized and its generic diagnosis amended to include: anterior attachment organs tending to form a 'hooded' appearance; prominent anterior gland cells between the pharynx and the anterior margin of the body: long penis, tapering proximally, occupying a penis canal with weakly muscular wall: penis canal and penis describe a sigmoid; accessory gland reservoir dorsal and alongside, or posterior and lateral to, proximal end of the penis and enclosed by a proximal extension of the wall of the penis canal. Under this conception. Menziesia comprises: M. noblei (Menzies. 1946) Gibson, 1976 (type species); M. malaboni (Velasquez. 1982) comb. nov.: M. merinthe (Yamaguti, 1968) Gibson. 1976: M. ovalis (Yamaguti, 1968) Gibson, 1976: and M. sebastodis (Yamaguti, 1934) comb, nov. A key to valid species of Benedenia and Menziesia is provided and a list is presented of published records of undescribed or unattributed species of Benedenia. Some protocols are suggested for preparation of benedeniine material to enhance future taxonomic studies and comparisons. The host-specificity and geographic distribution of species in these revised genera are discussed. The composition of the Capsalidae is discussed and some difficulties in defining and distinguishing between its different subfamilies are considered.

Spermatozoal ultrastructure of spiny oysters (Spondylidae, Bivalvia) including a comparison with other bivalves

Sperm ultrastructure in three representative species of the marine bivalve family Spondylidae (spiny or thorny oysters) is examined and compared with available data on other bivalves, especially other families of the subclass Pteriomorphia. Spondylid spermatozoa are of the externally fertilizing aquasperm. type (ect-aquasperm). The acrosomal vesicle is conical with a deep basal invagination extending almost the full length of the vesicle. Vesicle contents are divisible into an inner, highly electron-dense anterior layer and a less dense posterior layer. The anterior layer is folded back on itself posteriorly and exhibits radiating plates (best developed peripherally). The vesicle rests on, and is partially embedded in, an extensive granular deposit of subacrosomal. material at the nuclear apex. This deposit extends partly into acrosomal vesicle invagination and also fills a broad depression in the anterior of the nucleus. No pre-formed axial rod (perforatorium) is present. The nucleus is round-pyriform and its contents coarsely fibrogranular. At the base of the nucleus, four broad depressions partially accommodate the midpiece mitochondria. The midpiece consists the four spherical mitochondria and the proximal and distal centrioles. The centrioles are arranged at approximately 90degrees to each other, and each consists of nine, angularly-oriented, microtubular triplets embedded in a granular matrix. A short, periodically banded rootlet connects the proximal centriole to the nuclear fossa, whereas the distal centriole, which forms the basal body to the flagellar axoneme, is anchored to the plasma membrane by nine terminally forked satellite fibres. Extensive deposits of putative glycogen rosettes surround the centrioles and mitochondria. The flagellum consists of a 9+2 axoneme sheathed by the plasma membrane. Spondylid spermatozoa strongly resemble those of the Pectinidae, further confirming the traditional view (based on comparative anatomy and shell morphology) of a close relationship between the Spondylidae and the Pectinidae. Differences in acrosomal shape and dimensions were noted between the three species examined, indicating potential taxonomic utility for comparative sperm ultrastructure within the Spondylidae.

Gill pathology caused by infestations of adult and preadult Dissonus manteri Kabata (Copepoda : Dissonidae) on coral trout, Plectropomus leopardus (Lacepede), (Serranidae)

Adult and preadult Dissonus manteri attached to the gills of Plectropomus leopardus cause significant pathology in the form of large hyperplastic nodules on the afferent (leading), edges of gill filaments. Nodules result from the dual actions of parasite attachment and feeding. The host response is characterized by severe epithelial hyperplasia, supplemented by fibroplasia and inflammation. Parasites attach close to the gill arch near the base of filaments. They have little effect on gill vasculature as the maxillipeds penetrate the filament superficial to the efferent filament artery and do not interfere with the blood vessels of the secondary lamellae. Tissue proliferation is limited to the wide portion of filament 'edge' epithelium in the proximal third and also does not extend to the secondary lamellae. Nodules are most numerous towards the ends of hemibranchs and are generally absent from the central regions. Leading hemibranchs bear significantly more nodules than their trailing counterparts. Of the total number of nodules, 20.5% are located on the pseudobranchs. Distribution patterns are considered to be primarily the result of D. manteri avoiding strong water currents, although this cannot explain the difference between numbers on leading and trailing hemibranchs.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.