The lectin KM+ induces corneal epithelial wound healing in rabbits

Neutrophil influx is essential for corneal regeneration (Gan et al. 1999). KM+, a lectin from Artocarpus integrifolia, induces neutrophil migration (Santos-de-Oliveira et al. 1994). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6,0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 mu g/ml) every 2 h, The control eyes received buffer, The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured, The animals were killed 12 h (group 1, n = 5), 24 h (group 2, n = 10) and 48 h (group 3, n = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p&3, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls, Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 mu g/quarter per d with or without 10 mu g of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered similar to 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Insulin Affects Tissue Organization and the Kinetics of Epithelial Cell Death in the Rat Ventral Prostate After Castration

Prostate growth and physiology are regulated by steroid hormones and modulated by multiple endocrine factors We investigated the action of insulin on the tissue organization and kinetics of epithelial cells in the rat ventral prostate (VP) in response to castration up to 120 hours after surgery by using an acute protocol of alloxan induced diabetes Diabetes caused a reduction in volume density (Vv(o)/) and volume of the epithelium The effects of castration on the epithelium were accelerated in the diabetic animals as determined by changes in V(o)/, and volume The smooth muscle cells became atrophic and apparently relaxed in response to castration in contrast to the spinous aspect observed in nondiabetic castrated rats Counting of apoptotic nuclei in the epithelium showed the classical apoptosis peak at 72 hours in nondiabetic rats and an advance of the apoptosis peak to 48 hours after castration in diabetic rats Insulin restored the time of the peak to 72 hours These results were confirmed after immunostaining for cleaved caspase 3 and suggest a survival and antiapoptotic effect on VP epithelial cells in both the presence and absence of androgen stimulation This idea is supported by the observation that insulin also reduced the overall rate of apoptosis at all experimental points analyzed before and after castration

The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis

Background Prolonged exposure of the lip to sunlight may cause actinic cheilitis (AC) and squamous cell carcinoma (SCC). Maspin is a serpin with tumor suppressor functions. This work analyzed the presence and distribution of maspin in AC and lip SCC. Methods Sections from 36 cases diagnosed as AC (18 cases with mild epithelial dysplasia, 11 with moderate and 7 with severe), 18 cases diagnosed as lip SCC and 7 specimens containing normal lip vermillion epithelium were submitted for immunohistochemical analysis to detect maspin. Results All AC cases with mild and two cases with moderate dysplasia were scored 3. The remaining nine cases with moderate dysplasia were identified as score 2, whereas all cases with severe dysplasia were scored 1. Positive staining for maspin decreased from the basal layer to the surface. Among the 18 lip SCCs studied, 15 cases showed abundant staining for maspin. Epithelium adjacent to the SCCs also showed intense positive staining in all cells. Conclusions Our results suggest that the loss of maspin expression occurs from the basal layer to the surface. Lip SCCs related to solar radiation show an intense presence of maspin protein in almost all tumor cells as well as the neighboring epithelium. Fontes A, Sousa SM, Santos E, Martins MT. The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis.

Recurrent bilateral gingival peripheral calcifying epithelial odontogenic tumor (Pindborg tumor): A case report

Calcifying epithelial odontogenic tumor (CEOT) is an extremely rare, benign neoplasm, accounting for approximately 1% of all odontogenic tumors. Peripheral CEOTs commonly resemble oral hyperplastic or reactive lesions and are histologically similar to their intraosseous counterparts. We report an unusual case of multifocal peripheral CEOT. A 40-year-old female presented with bilateral soft, painful, erythematous, gingival swellings localized in premolar areas of the mandibular gingiva. The presumptive diagnosis was bilateral pyogenic granuloma. The masses were surgically excised under local anesthesia without bone curettage and both recurred 12 months later. Morphologic features, and histochemical and immunohistochemical tests revealed bilateral peripheral calcifying odontogenic epithelial tumor. There is no clinical or radiographic evidence of recurrence 3.5 years after excision. This multifocal phenomenon has been reported previously only for intraosseous CEOT. Gingival masses must be carefully evaluated for clinical and histologic evidence of neoplasia. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e66-e71)

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain

Eag1 (K(v)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K+ channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Relationship Between Expression of Voltage-Dependent Anion Channel (VDAC) Isoforms and Type of Hexokinase Binding Sites on Brain Mitochondria

Voltage-dependent anion channels (VDAC) are pore-forming proteins found in the outer mitochondrial membrane of eukaryotes. VDACs are known to play an essential role in cellular metabolism and in early stages of apoptosis. In mammals, three VDAC isoforms have been identified. A proteomic approach was exploited to study the expression of VDAC isoforms in rat, bovine, and chicken brain mitochondria. Given the importance of mitochondrially bound hexokinase in regulation of aerobic glycolysis in brain, we studied the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A/type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was significantly higher in bovine than in rat brain. Chicken brain mitochondria showed the highest VDAC1 expression and the lowest of VDAC2. Bovine brain mitochondria had the highest VDAC2 levels. We concluded that the nature of hexokinase binding site is not determined by the expression of a single VDAC isoform.

Rosiglitazone prevents sirolimus-induced hypomagnesemia, hypokalemia, and downregulation of NKCC2 protein expression

Alexandre CS, Braganca AC, Shimizu MH, Sanches TR, Fortes MA, Giorgi RR, Andrade L, Seguro AC. Rosiglitazone prevents sirolimus-induced hypomagnesemia, hypokalemia, and downregulation of NKCC2 protein expression. Am J Physiol Renal Physiol 297: F916-F922, 2009. First published August 5, 2009; doi:10.1152/ajprenal.90256.2008.-Sirolimus, an antiproliferative immunosuppressant, induces hypomagnesemia and hypokalemia. Rosiglitazone activates renal sodiumand water-reabsorptive pathways. We evaluated whether sirolimus induces renal wasting of magnesium and potassium, attempting to identify the tubule segments in which this occurs. We tested the hypothesis that reduced expression of the cotransporter NKCC2 forms the molecular basis of this effect and evaluated the possible association between increased urinary excretion of magnesium and renal expression of the epithelial Mg(2+) channel TRPM6. We then analyzed whether rosiglitazone attenuates these sirolimus-induced tubular effects. Wistar rats were treated for 14 days with sirolimus (3 mg/kg body wt in drinking water), with or without rosiglitazone (92 mg/kg body wt in food). Protein abundance of NKCC2, aquaporin2 (AQP2), and TRPM6 was assessed using immunoblotting. Sirolimus-treated animals presented no change in glomerular filtration rate, although there were marked decreases in plasma potassium and magnesium. Sirolimus treatment reduced expression of NKCC2, and this was accompanied by greater urinary excretion of sodium, potassium, and magnesium. In sirolimus-treated animals, AQP2 expression was reduced. Expression of TRPM6 was increased, which might represent a direct stimulatory effect of sirolimus or a compensatory response. The finding that rosiglitazone prevented or attenuated all sirolimus-induced renal tubular defects has potential clinical implications.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca(2+) Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 mu M) concentrations of forskolin, respectively. The expression of GLP-1 receptors in a cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on a cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates a cell electrical activity, increases [Ca(2+)] enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP]). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP],.

Role of a Novel Tetrodotoxin-Resistant Sodium Channel in the Nitrergic Relaxation of Corpus Cavernosum from the South American Rattlesnake Crotalus Durissus Terrificus

Introduction. Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown. Aim. To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity. Methods. Hemipenes were removed from anesthetized South American rattlesnakes (Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths. Main Outcome Measures. Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of N omega nitro-L-arginine methyl ester (L-NAME; 100 mu M), 1H-[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mu M) and tetrodotoxin (TTX; 1 mu M). Results. The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by alpha-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC(50)) values of 5.84 +/- 0.17 and 5.10 +/- 0.08 (N = 3-4), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX. Conclusions. Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus. Capel RO, Monica FZ, Porto M, Barillas S, Muscara MN, Teixeira SA, Arruda AMM, Pissinatti, L, Pissinatti A, Schenka AA, Antunes E, Nahoum C, Cogo JC, de Oliveira MA, and De Nucci G. Role of a novel tetrodotoxin-resistant sodium channel in the nitrergic relaxation of corpus cavernosum from the South American rattlesnake Crotalus durissus terrificus. J Sex Med 2011;8:1616-1625.

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.