Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Gap junction distribution in the Drosophila wing disc mutants vg,l(2)gd,l(3)c43hs1, and l(2)gl4

The density of gap junctions in four Drosophila melanogaster mutants with abnormal wing disc development has been determined using quantitative electron microscopy and compared with the gap junction density in wild-type wing discs. No appreciable differences relative to wild-type controls were found in the cell death mutant vestigial or in the mildly hyperplastic mutant lethal giant disc which could not be accounted for in terms of altered lateral plasma membrane surface density or as an extension of the gap junction growth which normally occurs during the third larval stage of development in wild-type wing discs. However, both the severely hyperplastic mutant l₍₃₎c43^hs1 and the neoplastic mutant lethal giant larva have significant reductions in the gap junction surface density, the number of gap junctions, and the gap junction areal fraction of the lateral plasma membrane compared with wild-type controls. These differences cannot be attributed to altered lateral plasma membrane surface densities which are not significantly different from wild-type control wing discs. The reduced gap junction density in severely hyperplastic and neoplastic wing discs suggests that alterations in the number or distribution of gap junctions may be as disruptive to normal growth and development as their complete absence.     

Changes in the distribution of gap junctions inDrosophila melanogaster wing discs during the third larval and early pupal stages of development

Summary Developmental changes in the distribution of gap junctions in early, mid and late third larval stage wing discs and in pupariation+6 h and pupariation+24 h stage wing discs fromDrosophila melanogaster were analyzed by quantitative electron microscopy. Gap junctions occur in all 12 intradisc regions examined in each of the five developmental stages. Their distribution is non-random and changes during development which suggests that they are developmentally regulated. The gap junctions are not static structures, rather they grow and regress during development. The changes tend to be gradual ones without sudden increases or decreases. Gap junctions continuously form and grow in size throughout the third larval stage and during the first 6 h following pupariation. Their surface density, number, percent of the lateral plasma membrane area, and absolute area as well as the lateral plasma membrane surface density all increase during this time. Between pupariation+ 6 h and pupariation+24 h all but one of these parameters decrease indicative of gap junctional breakdown. Gap junctions are most numerous and change least during development in the apical cell regions where intercellular contacts are close and stable. They change most in the basal cell regions where intercellular contacts tend to be looser and change during development. The most dramatic change is in the absolute area which increases by a factor of 23 between the early third larval stage and pupariation+24 h. At pupariation the rate of gap junction growth undergoes a transient increase before the phase of disassembly begins. Developmental changes in gap junction surface density are closely coupled with changes in the lateral plasma membrane surface density which suggests that these may be coregulated. Evidence from mutants suggests that when the number and density of gap junctions fail to increase in proportion to lateral plasma membrane growth, wing disc development will be abnormal. Our results support the idea that some minimum gap junction density is required for normal development and that this must increase as development proceeds. The results are consistent with the notion that gap junctions are involved in pattern formation and growth control and are discussed with respect to the acquisition of competence for metamorphosis, disc growth, disc morphogenesis and changes in the hormonal environment.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

The determination of aldehyde oxidase activity patterns in the wing discs of Drosophila melanogaster

Summary The pattern of aldehyde oxidase (AO) activity was determined in wing discs of Drosophila melanogaster larvae homozygous for the mutants apt ⁷³ⁿ, Beaded, and vestigial (vg) in order to determine if reduction in field size in the pouch could be related to alterations of the wild-type AO pattern, as suggested by the Kauffman (1978) hypothesis. The pattern in wild-type discs was resolved into six areas for comparison with mutant discs. vg discs developed at 25° C showed restriction of the pattern into a small area on the anterior side of the disc, and comparison of vg and wild-type prepupal wings allowed positive identification of the AO pattern elements which remained. AO patterns in vg wing discs grown at 27°, 29°, and 31° C were progressively more complete and similar to wild-type, reflecting the reduction in cell death in discs grown at higher temperatures. These results show that cell loss during the third instar in vg development at 25° C is responsible for the alteration of the AO pattern, rather than field size reduction, and that determination of the pattern must take place much earlier than the time of its first appearance during the third larval instar, and before cell death in vg discs begins. Thus mutants acting at earlier stages will be necessary for further tests of the Kauffman hypothesis.     

Electron microscope immunolocation of gap junctions in Drosophila

Gap junction immunolocation was carried out in thin sections of Lowicryl K4M-embedded Drosophila imaginal wing discs using an affinity-purified polycolonal anti-18 kD gap junction protein anitbody and a colloidal gold-conjugated secondary antibody. Colloidal gold labelling was predominantly associated with obliquely-sectioned gap junctions, the ends of junctional profiles and other regions in which the adjacent junctional membranes were separated or distorted. The pattern of staining suggests that the determinant recognized by the antibody is relatively inaccessible, probably with a topological location in the transmembrane or extracellular domain of the membrane-spanning connexin protein.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Structural,immunocytochemical and initial biochemical characterization of NAOH-extracted gap junctions from an insect,Heliothis virescens

Subcellular fractions enriched in gap junctions with an ultrastructure similar to those in intact insect tissue have been obtained by extracting crude membranes from the tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) with 2.5 mM NaOH. n-Octyl--d-glucopyranoside (OG) was used to further purify integral membrane proteins in the NaOH-extracted fractions. A polyclonal antibody (R16) is described that specifically labels nonextracted and NaOH-extracted gap junctions in cell fractions by electron microscope immunocytochemistry. R16 immunostaining of sectioned Heliothis testis at the light-microscope level yields a pattern of immunoreactivity consistent with the distribution of gap junctions in the tissue. R16 identifies a 40-kDa protein as a candidate gap junction protein on immunoblots of crude membrane, NaOH-extracted and NaOH/OG-extracted fractions.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

幽门螺杆菌对胃上皮细胞间隙连接超微结构的影响

通过实验和临床观察幽门螺杆菌(Helicobacter pylori)对胃上皮细胞间隙连接超微结构的影响,从细胞间隙连接角度探讨H. pylori致癌机制．将不同H. pylori菌株与BGC-823细胞共培养24 h或 48 h,用原位固定与原位包埋法透射电镜观察细胞间隙连接超微结构变化．对70例胃癌患者,用快速尿素酶试验、碱性品红染色和¹⁴C尿素呼气实验检测H. pylori,PCR法检测H. pylori CagA基因,及透射电镜观察胃上皮细胞间隙连接超微结构变化．结果显示,未加H. pylori组BGC-823细胞可见较多细胞连接及连接复合体,加H. pylori各组细胞的连接数、单位周长连接数与单位周长连接长度均小于未加H. pylori组,而细胞间隙最小宽度大于未加H. pylori组(P < 0.001或P < 0.005),且CagA⁺ 的NCTC J99组、临床株GC 01组和NCTC 11639组细胞连接数、单位周长连接数均小于CagA^- 的NCTC 12908组(P < 0.001或P < 0.05),NCTC J99组与临床株GC 01组细胞单位周长连接长度短于NCTC 12908组(P < 0.001)．胃癌患者H. pylori感染组细胞连接数、单位周长连接数与单位周长连接长度均小于无H. pylori感染组,细胞间隙最小宽度大于无H. pylori感染组(P < 0.001),且CagA⁺ H. pylori感染者细胞连接数、单位周长连接数与单位周长连接长度均小于CagA^- H. pylori感染者,细胞间隙最小宽度大于CagA^- H. pylori感染者．上述结果表明,胃上皮细胞间隙连接改变与H. pylori感染,特别是CagA⁺ H. pylori感染有关．     

Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.     

Crustecdysone-induced modulation of electrical coupling and gap junction structure in crayfish hepatopancreatocytes

Crustecdysone, the hormone responsible for onset and regulation of the molt cycle in Crustacea, causes an increase in ionic coupling of cells of the hepatopancreas concomitant with the events of the molt. Hepatopancreatic tissue incubated for up to 4 hr in modified Eagle Basal Medium containing crustecdysone, exhibited an approximate 29% decrease in intercellular resistance as compared with tissue incubated in control medium. This represents a 29% increase in ionic coupling between hepatopancreatocytes following treatment with crustecdysone. Examination of platinum replicas of freeze-fractured, crustecdysone-treated hepatocyte plasma membrane revealed that most of the gap junction plaques were round with tightly packed intramembrane particles; a condition indicative of highly coupled cells. Similar preparations of control plasmalemmae demonstrated many gap junction plaques which were round or irregular in shape with very loosely packed particles and were indicative of uncoupled junctions. Results of this study are identical to those from a previous investigation of the electrophysiology and freeze-fracture morphology of hepatopancreatocytes during the molt cycle (McVicar and Shivers, 1984), and are thus presumed to reflect a crustecdysone-controlled increase in cell communications in vivo.     

Electron microscope immunolocation of gap junctions in Drosophila

Gap junction immunolocation was carried out in thin sections of Lowicryl K4M-embedded Drosophila imaginal wing discs using an affinity-purified polycolonal anti-18kD gap junction protein antibody and a colloidal gold-conjugated secondary antibody. Colloidal gold labelling was predominantly associated with obliquely-sectioned gap junctions, the ends of junctional profiles and other regions in which the adjacent junctional membranes were separated or distorted. The pattern of staining suggests that the determinant recognized by the antibody is relatively inaccessible, probably with a topological location in the transmembrane or extracellular domain of the membrane-spanning connexin protein.     

Extra DNA replication and cell proliferation in wing imaginal discs of aDrosophila species hybrid

Summary Species hybrids obtained from corsses betweenD. azteca andD. athabasca display characteristic growth anomalies. These are restricted to hybrid males while hybrid females resemble the pure species. The ratio of the wing length to body length is 1 in pure species, 1.3 in hybrid males from the crossD. azteca x D. athabasca and 0.65 in hybrid males from the reciprocal cross. This indicates a disproportionate enlargement and diminuation, respectively, restricted to some tissues. Whereas the number of ommatidia in the compound eye is normal, the number of wing cells is approximately twice as high in hybrid giant males as in hybrid females and pure species. Microdeterminations of the DNA content of wing discs show that an extra DNA replication step occurs during the early pupal stage of hybrid giant males; the pupal stage of these males lasts about 58 h longer than that of the pure species. In eye-antenna discs no extra DNA synthesis has been found. Incorporation experiments reveal a delayed uptake of radioactively labeled thymidine into the DNA of wing discs in late third instar larvae and early pupae of hybrid giant males. The results of this study are interpreted to indicate that a disturbed balance between sex-linked and autosomal genes in hybrid males is responsible for the modified rhythm of DNA synthesis and of cell proliferation in certain cell types.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart

Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofiuorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution. © 1994 Wiley-Liss, Inc.     

Intercalated discs of mammalian heart: a review of structure and function

Intercalated discs are exceptionally complex entities, and possess considerable functional significance in terms of the workings of the myocardium. Examination of different species and heart regions indicates that the original histological term has become out-moded; it is likely, however, that all such complexes will continue to fall under the generic heading of 'intercalated discs'. The membranes of the intercalated discs establish specific associations with a variety of intracellular and extracellular structures, as well as with numerous types of proteins and glycoproteins. Characterization of discs and their components has already brought together a large number of research disciplines, including microscopy, cytochemistry, morphometry, cell isolation and culture, cell fractionation, cryogenics, immunology, biochemistry, and electrophysiology. The continued dissection of substance and function of intercalated discs will depend on such interdisciplinary approaches. The intercalated disc component which continues to attract the greatest amount of interest is the so-called gap junction. All indications thus far point to a great deal of inherent lability in the architecture of the gap junction. There is thus considerable potential for the creation of artefact while preserving and observing gap junctions, and this problem will doubtless continue to hamper the understanding of their functions. A question of special interest concerns whether the gap junctions of intercalated discs are required for transfer of electrical excitation between cells, or maintain cell-to-cell adhesion, or in fact subserve both electrical and structural phenomena. Two schools of thought exist with respect to cell-to-cell coupling in the heart. One proposes that low-resistance junctions in the discs mediate electrical coupling, whereas the other supports the possibility of coupling across ordinary high-resistance membranes. Thus the intercalated discs continue to be a source of controversy, just as they have been since they were originally discovered in heart muscle over a century ago.     

Relationship of Cytoskeletal Filaments to Annular Gap Junction Expression in Human Adrenal Cortical Tumor Cells in Culture

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed α₁gap junctions at areas of cell to cell contact. In addition, α₁gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 μg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.