Role of MMP9 on Invadopodia Formation in Cells From Adenoid Cystic Carcinoma. Study by Laser Scanning Confocal Microscopy

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type I matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMR Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After I h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMR Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech. 73:99-108, 2010. (C) 2009 Wiley-Liss, Inc.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxa, Brycon cephalus Gunther 1869 (Teleostei-Characidae)

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus, the head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column, the parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located, Such cells were interpreted as noradrenaline cells, Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

Sebaceous metaplasia in a canine mammary gland non-infiltrative carcinoma with myoepithelial component

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Absence of DNA damage in multiple organs (blood, liver, kidney, thyroid gland and urinary bladder) after acute fluoride exposure in rats

Fluoride has been widely used in dentistry as a caries prophylactic agent. However, there has been some speculation that excess fluoride could cause an impact on genome integrity. In the current study, the potential DNA damage associated with exposure to fluoride was assessed in cells of blood, liver, kidney, thyroid gland and urinary bladder by the single cell gel (comet) assay. Male Wistar rats aging 75 days were distributed into seven groups: Groups 1 (control), 2, 3, 4, 5, 6 and 7 received 0 (deionized water), 10, 20, 40, 60, 80 and 100 mgF/Kg body weight from sodium fluoride (NaF), respectively, by gastrogavage. These groups were killed at 2 h after the administration of the fluoride doses. The level of DNA strand breaks did not increase in all organs evaluated and at all doses of NaF tested, as depicted by the mean tail moment. Taken together, our results suggest that oral exposure to NaF did not result in systemic genotoxic effect in multiple organs related to fluoride toxicity. Since DNA damage is an important step in events leading to carcinogenesis, this study represents a relevant contribution to the correct evaluation of the potential health risk associated with chemical exposure.

Changes in the morphology and ultrastructure of the Dufour's gland during the life cycle of the bumble bee queen, Bombus terrestris L-(Hymenoptera : Bombini)

The Dufour's gland is found closely associated with the sting apparatus of all female hymenopterans, playing multiple roles among bees. In some species of Bombus the gland may be involved in production of nestmate recognition pheromones, but in B. terrestris its function is not certain yet. The morphology of the :Dufour's gland of B. terrestris queens and the ultrastructural features of its cells were studied in different ages and behavioural stages using routine transmission electron microscopy. Measurements of the length and the diameter of the gland in the same conditions were also made. The Dufour's gland of the queen increases significantly in size (both in length and in diameter) with age and reproductive activity the ultrastructural features of the gland show electrondense material that comes from the haemolymph. This material is also present in the intercellular spaces, and is conducted to the subcuticular space, to be released directly into the glandular lumen. Hence at least part of the secretion is probably taken up directly from the haemolymph. The ultrastructural features indicate a more active phase of the gland corresponding to the period of egg-laying of the queen, and a decrease in activity when the queen is in hibernation as well as after the competition point. In conclusion, the gland is probably involved in reproduction, more specifically, in the marking off eggs.

Venom gland of Pachycondyla striata worker ants (Hymenoptera : Ponerinae). Ultrastructural characterization

Morphological data concerning the venom gland of worker ants of Pachycondyla striata revealed that this gland consists of three distinct regions: an external secretory portion, composed by a secretory filament that bifurcates in order to give rise to other two filaments; an internal secretory portion, represented by the convoluted gland; and a storage portion, represented by a sac-shaped reservoir. The ultrastructural analysis showed that the reservoir is enveloped by a simple pavementous epithelium, coated internally with a cuticle. The external secretory portion is composed by cells forming a simple cubic epithelium, in which the apical portion presents numerous microvilli while the basal portion of the cells shows infoldings of the plasma membrane containing numerous mitochondria. The convoluted gland possesses cells of irregular morphology with nuclei containing condensed chromatin, suggesting inactivity. However, these cells are in fact undergoing secretory activity, which is probably added to the final secretion produced by the gland. The cytoplasm of these cells contains several elements distributed therein, such as ribosomes and polyribosomes, lipid droplets, and protein inclusions in the form of crystals, thus Suggestive of protein storage, which would be used by the insect when metabolically required. (c) 2005 Elsevier Ltd. All rights reserved.

Morphometric analysis of the venom gland in worker bees of Apis mellifera (Hymenoptera : Apidae) from the pantanal region of Mato Grosso do Sul

In Apis mellifera the acid or venom gland is composed of secretory cells that surround a channel that opens into a reservoir devoid of musculature. This gland can at times present apical branching. In this study we recorded the frequency of branched venom glands in workers of Africanized bees (Apis mellifera Linnaeus) from six localities in the Pantanal region of Mato Grosso do Sul, and analyzed the relation among the length of the main duct, the length of the duct from the reservoir to the beginning of branching, the length of the branched segment (when present) and the total length of the gland. We sought to determine the probable genotypes of the bees from each population by using the model proposed by Alves-Junior. The frequency of branched glands varied from 50% to 83% in the worker bees coming from those places, indicating that this characteristic is primitive in these bees. The results of the Analysis of Discriminant Functions indicated significant differences in the morphometrical segments of the venom gland (Wilk's Lambda = 0.065; F-(27,F-30) = 4.507; P < 0.001), and permitted a differentiation of the populations studied. The genotypes inferred for the bees of each locality agree with the results obtained in the Analysis of Discriminant Functions and form three distinct groups, with some overlapping areas among them. In all of the populations considered the phenotype largevenom gland was predominant. It is inferred that bees with this phenotype (venom gland larger than S. 15 mm) have Gm(1) Gm(1) genotype, being therefore homozygotes for the major alleles and also for the modifier genes that codify this morphological trait. The high frequency of worker bees with large venom gland in all the places considered makes viable the development of a selection program in order to obtain bees with longer venom glands, aimed at the commercial production of venom by the beekeepers of the Pantanal region of Mato Grosso do Sul.

The intramandibular gland of leaf-cutting ants (Atta sexdens rubropilosa Forel 1908)

The eusociality developed in Hymenoptera and Isoptera is driven by an efficient interaction between exocrine glands and jointed appendages, both in close interaction with the environment. In this context, the mandible of ants plays an important role, since, in addition to being the main jointed appendage, it possess glandular functions. As an example we might name the two glands associated with the mandible: the mandibular and the intramandibular glands. The intramandibular gland is found inside the mandible and consists of a hypertrophied secretory epithelium and secretory cells in the mandible's lumen. The secretion of the secretory epithelium is liberated through intracuticular ducts that open at the base of hairs at the mandible's surface. The secretion of the intramandibular gland (epithelium and secretory cells) reacted positively to tests for the detection of polysaccharides and proteins, thus suggesting that it consists of glycoproteins. The ultrastructure of the secretor epithelium presents variations related to the developmental stage of the individual, showing a large number of ribosomes and microvilli close to the cuticle in young individuals, while in the older specimens it was possible to note the formation of ail intracellular reservoir. These variations of secretory epithelium, as also the interaction between the cellular groups inside the mandible, are important information about this gland in leaf-cutting ants. (c) 2005 Elsevier Ltd. All rights reserved.

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L)

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed. (C) 2000 Academic Press.

A new type of sternal gland present in Oxaea flavescens (Hymenoptera, Oxaeinae): location and histology

The sternal glands of the abdomen of Oxaea flavescens consist of class ill glandular cells, with the excretory canaliculus linking to the lateral intersegmental membrane of segments ill, TV and V. The intersegmental membrane is augmented and folded into several lobes forming a reservoir covered by secretory cells. The intersegmental membrane is then transformed into an intima that lines a reservoir space containing secretions of a type of mucus which is periodic acid-Schiff positive. The storage of a great amount of secretion suggests that it is not used continuously. These glands are absent from males, indicating that their products must have a specific function linked to the female sex.

Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae)

The sternal gland is considered the only source of trail pheromones in termites. The morphology of the sternal gland was investigated in workers of Coptotermes gestroi using transmission and scanning electron microscopy. The results showed a small bilobed gland at the anterior part of the fifth abdominal sternite. The cuticular surface of the sternal gland showed a V-shaped structure with two peg sensilla in elevated socket and various campaniform sensilla. Pores and cuticular scale-like protuberances also occur in the glandular area. The ultrastructure showed a gland composed of class I cells and two different types of class 3 cells distinguished by location, different size and electron-density of secretory vesicles. Small class 3 cells (type 1) of the anterior lobe are inserted among class I cells and have weakly electron-dense vesicles associated with mitochondria, glycogen and smooth endoplasmic reticulum. The class 3 cells (type 2) of posterior lobe showed many round electron-lucent vesicles of secretion, abundant free ribosomes and a well-developed Golgi apparatus. Each class 3 cell is connected to the cuticle by a cuticular duct constituted by the receiving canal and the conducting canal. The secretion of class I cells is stored in an inner subcuticular reservoir that is delimited by the microvilli of these cells. This inner reservoir is large and crossed by the campaniform sensilla and ducts of two types of class 3 cells that open outside of the insect body. An exterior reservoir also is present between the fourth and fifth sternite. The complex structure of the sternal gland suggests multicomponents for the trail pheromone in the worker of C gestroi. (c) 2005 Elsevier Ltd. All rights reserved.

Secretory cycle of the Dufour's gland in workers of the bumble bee Bombus terrestris L-(Hymenoptera : Apidae, Bombini)

The Dufour's gland of Bombus terrestris workers, of different ages and with varying degrees of ovary development, was studied with the aim to verify its involvement in reproduction. Measurements of the diameter and the length of the gland were made using an ocular micrometer adapted to a microscope. Transmission electron microscopy was used to study the morphology of the secretory cells and to analyze the secretory cycle. The glandular cells were considered to be near type II cells of NOIROT and QUENNEDEY (1974), a type that has not been described before in Hymenopterans. The results show that there is a correlation between the degree of ovary development and Dufour's gland activity of workers. The diameter of the gland and the secretory cell activity increased with increasing oocyte size in the ovary. Regressive conditions of the gland were observed, which are probably related to increasing worker age. To elucidate the production and releasing process of the secretion and to establish its precise function, a comparative analysis of the secretion process of the Dufour's gland of queens and workers is needed.

The functional morphology of the metapleural gland of the leaf-cutting ant Atta laevigata (Formicidae: Attini)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MRT Letter: A Novel Tegumental Gland in Female Imagoes of the Neotropical Termite Cornitermes cumulans (Isoptera, Termitidae, Syntermitinae)

In general, the exocrine glands of social insects are structures involved in the chemical communication associated with social life. Here, we report the discovery of an unknown tegumental gland that is present in the female imagoes of Cornitermes cumulans and occurs next to the well-developed tergal glands that have previously been described. The tegumental glands release their secretion in the intersegmental membrane and are composed of bicellular units, a secretory cell and a canal cell, that are closely located to the epidermal cells in the inferior part of the eighth and ninth tergites. The ultrastructure of the glandular cells showed abundant smooth endoplasmic reticulum, suggesting that the secretion may be pheromonal, although its function is still unknown. These exocrine structures are facing the tergal glands, and we hypothesized that they act synergistically with the tergal glands to generate short-range attraction during tandem behavior. Microsc. Res. Tech. 73: 1005-1008, 2010. (C) 2010 Wiley-Liss, Inc.

Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)