Effects of Cadmium on the Rat Salivary Glands, During Lactation

Cadmium (Cd) in air, drinking water and food has the potential to affect the health of people, mainly those who live in highly industrialized regions. Cd affects placental function, can cross the placental barrier and directly modify fetal development. Once the organism is particularly susceptible to the exposition to the Cd during the perinatal period, and that this metal can be excreted in the milk, the aim of the present work was to study the effects of the constant exposition to drinkable water containing low levels of Cd during the lactation, on the salivary glands of the rat. Female rats received ad libitum drinking water containing 300mg/l of CdCl2 throughout the whole lactation. Control animals received a similar volume of water without Cd. Lactant rats (21 day old) were killed by lethal dose of anesthetic. The salivary glands were separated, fixed in ""alfac"" solution for 24 h, and serially sectioned. The 6 mu m thick sections were stained with hematoxylin and eosin. Nuclear glandular parameters were estimated, as well as cytoplasm and cell volume, nucleus/cytoplasm ratio, number and surface density, diameters and cell thickness. Mean body weight was 34.86 g for the control group and 18.56 g for the Cd-treated group. Histologically, the glandular acini were significantly smaller, the gland ducts were similar in both groups studied. The connective tissue was more abundant. In conclusion, the salivary glands (submandibular, parotid and sublingual) showed retarded growth after Cd intoxication.

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas` disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.

Effects of Cobalt Chloride in Junctional Epithelium and Reduced Enamel Epithelium of the Rat Maxillary First Molar

Cobalt is one of the main components of cast metal alloys broadly used in dentistry. It is the constituent of 45 to 70% of numerous prosthetic works. There are evidences that metal elements cause systemic and local toxicity. The purpose of the present study was to evaluate the effects of cobalt on the junctional epithelium and reduced enamel epithelium of the first superior molar in rats, during lactation. To do this, 1-day old rats were used, whose mothers received 300mg of cobalt chloride per liter of distilled water in the drinker, during lactation. After 21 days, the rat pups were killed with an anesthetic overdose. The heads were separated, fixed in ""alfac"", decalcified and embedded in paraffin. Frontal sections stained with hematoxylin and eosin were employed. Karyometric methods allowed to estimate the following parameters: biggest, smallest and mean diameters, D/d ratio, perimeter, area, volume, volume/area ratio, eccentricity, form coefficient and contour index. Stereologic methods allow to evaluate: cytoplasm/nucleus ratio, cell and cytoplasm volume, cell number density, external surface/basal membrane ratio, thickness of the epithelial layers and surface density. All the collected data were subjected to statistic analysis by the non-parametric Wilcoxon-Mann-Whitney test. The nuclei of the studied tissues showed smaller values after karyometry for: diameters; perimeter, area, volume and volume/area ratio. Stereologically, it was observed, in the junctional epithelium and in the reduced enamel epithelium, smaller cells with scarce cytoplasm, reflected in the greater number of cells per mm3 of tissue. In this study, cobalt caused epithelial atrophy, indicating a direct action on the junctional and enamel epithelium.

Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation. Reproduction (2011) 142 593-603

DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h: 12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Canga renatae, a new genus and species of Cyphophthalmi from Brazilian Amazon caves (Opiliones: Neogoveidae)

A new genus and species of Cyphophthalmi, Canga renatae gen. nov., sp. nov., is described in the family Neogoveidae from a system of caves in the Serra de Carajas, Para State, Brazil. Canga can be easily distinguished from other neogoveid genera by the presence of a dentate claw on leg I, a unique character among known cyphophthalmid species, and by the free coxa II, which is fused to coxae III and IV in all the other neogoveid species except for the North American Metasiro. The new genus also differs from other Neotropical neogoveids in the lack of a dorsal crest on the chelicerae and in the lack of opisthosomal glands. The finding of a neogoveid in the Para State greatly increases the known distribution of South American cyphophtalmids into the Eastern Brazilian Amazon forest.

Sexually Dimorphic Tegumental Gland Openings in Laniatores (Arachnida, Opiliones), With New Data on 23 Species

Sexually dimorphic glands often release sexual pheromones both in vertebrates and invertebrates. Species of Laniatores (Arachnida, Opiliones) seem to depend on chemical communication but few studies have addressed this topic. In this study, we review the literature for the Phalangida and present new data for 23 species of Laniatores. In 16 taxa, we found previously undescribed sexually dimorphic glandular openings on the femur, patella, metatarsus, and tarsus of legs I and metatarsus of legs III and IV For the other species, we provide scanning electron micrographs of previously undescribed sexually dimorphic setae and pegs located on swollen regions of the legs. We also list additional species in which males have swollen regions on the legs, including the tibia, metatarsus, and tarsus of legs I, trochanter and tibia of legs II, femur, metatarsus, and tarsus of legs III, and metatarsus and tarsus of legs IV. The function and biological role of the secretions released by these glands are discussed. J. Morphol. 271:641-653, 2010. (C) 2009 Wiley-Liss, Inc.

A new micro-whip scorpion species from Brazilian Amazonia (Arachnida, Schizomida, Hubbardiidae), with the description of a new synapomorphy for Uropygi

Surazomus uarini n. sp. is described and illustrated based on specimens collected by beating on understory vegetation of Amazonian ""terra firme"" upland rain forests. A new cuticular structure, possibly a gland opening, is described on the female tarsus I and terminal flagellum. A putatively homologous structure is reported from the same body parts in all Undescribed species of Rowlandius Reddell and Cokendolpher 1995; Stenochrus portoricensis Chamberlin 1922; Mastigoproctus maximus (Tarnani 1889), and Thelyphonellus amazonicus (Butler 1872); suggesting it new synapomorphy for the clade Uropygi (i.e., Schizomida + Thelyphonida).

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea - Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs. (C) 2008 Elsevier Inc. All rights reserved.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.