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Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/β-catenin dependent (canonical) and Armadillo/β-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Salim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Virginia opossum (Didelphis virginiana) is widespread in the USA, ranging south through Latin America. The ecology of opossums is such that they are in frequent contact with soils, suggesting that they may function as a valuable bioindicator for chemical contamination in terrestrial environments. Surprisingly, there have been virtually no toxicology studies on opossums. Here, we provide the first analysis of metal contaminants in opossum liver tissues. Liver samples were obtained from 471 opossums, collected from 2003 to 2006, at four sites in North Florida and South Georgia, USA, and concentrations of copper, lead, nickel, selenium, and zinc were measured. We found little evidence of age differences in the concentration of any of the metals. However, there were at least some significant differences between years, males and females, and between sites for each metal, although the pattern of these differences was not always consistent across metals. Concentrations of metals in liver tissue were positively correlated with one another, primarily of each metal (except Pb) with zinc. Reference levels of metal contaminants are not available for opossums, but concentrations of Cu, Ni, Pb, and Zn in our samples were for the most part significantly higher than those reported from liver tissues of nine-banded armadillos (Dasypus novemcinctus) collected at the same sites and in the same years. Data from other small mammals studied elsewhere further indicate that metal concentrations in opossums were high, but at this time, it is not possible to determine if these elevated levels generated toxicity. The substantial temporal and spatial variation we found in metal concentrations suggests that determination of baseline levels for opossums may not be straightforward. Nonetheless, this is the first study quantifying metal accumulation in the livers of Didelphis virginiana and, as such, provides an important starting point for future research.

Understanding the basic reproductive biology and limitations to successful breeding of the southern three-banded armadillo (Tolypeutes matacus) is necessary to maintain viable zoo populations. Our objectives were to: 1) describe the reproductive biology using non-invasive, fecal hormone analysis; 2) assess influence of season on gonadal hormonal patterns in both the sexes; 3) characterize reproductive cyclicity and pregnancy in the female; and 4) characterize the onset of sexual maturity in males. Nineteen armadillos were monitored including: 13 (7 males, 6 females) from Lincoln Park Zoo and six (3 males, 3 females) from San Antonio Zoological Garden. Fecal samples (n=5220; 275/animal/yr) were collected 5 to 7 times a week for 1 year. Hormones were extracted from feces and analyzed for progestagen (females) and androgen (males) metabolite concentrations using enzyme immunoassays. Mean estrous cycle length (26.4±1.3 days) did not vary (P<0.05) among individuals (n=9). Mean gestation length (n=3) was 114.0±0.6 days long with mean fecal progestagen metabolites increasing 10-fold during pregnancy. Seasons did not influence (P<0.05) fecal androgen or progestagen metabolites. These data can assist with management decisions, which will directly affect the success of this species in zoos., (Published by Elsevier B.V.)

The prevailing view of canonical Wnt signaling emphasizes the role of beta-catenin acting downstream of Wnt activation to regulate transcriptional activity. However, emerging evidence indicates that other armadillo catenins in vertebrates, such as members of the p120 subfamily, convey parallel signals to the nucleus downstream of canonical Wnt pathway activation. Their study is thus needed to appreciate the networked mechanisms of canonical Wnt pathway transduction, especially as they may assist in generating the diversity of Wnt effects observed in development and disease. In this chapter, we outline evidence of direct canonical Wnt effects on p120 subfamily members in vertebrates and speculate upon these catenins' roles in conjunction with or aside from beta-catenin., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Improving the husbandry in the southern three-banded armadillo (Tolypeutes matacus) through gaining knowledge of its stress physiology is imperative to maintaining a healthy, zoo-housed population. Our objectives were to: 1) validate the use of fecal hormone analysis for monitoring adrenocortical activity using both an adrenocorticotropic hormone (ACTH) challenge and biological events; and 2) characterize longitudinal adrenocortical activity in male and female southern three-banded armadillos. An ACTH injection was given intra-muscularly to one male (4IU/kg; 5.6IU total) and one female (5.5IU/kg; 8IU total) southern three-banded armadillo. Fecal samples were collected 1 day pre- and continued 5 days post-ACTH to capture the physiological response measured by elevated fecal glucocorticoid metabolites (FGM) to validate these techniques. Additionally, natural and routine events, including pairing individuals for breeding and veterinary procedures/handling, were used to biologically validate these techniques. To characterize adrenocortical activity, fecal samples (∼3025 total; n=275/animal/yr) were collected from 11 (5 males; 6 females) southern three-banded armadillos 5-7 times a week for 1 year at Lincoln Park Zoo (Chicago, IL). A cortisol enzyme immunoassay was used for FGM analysis. The ACTH challenge in the male resulted in a twofold increase of FGM (1123.2±36.2 ng/g dry feces) above baseline (675.7±10.0 ng/g dry feces) at approximately 54-94h post- injection. The female exhibited a twofold increase (1635.4 ng/g dry feces) over baseline FGMs (608.5±12.3 ng/g dry feces) approximately 30h post-injection. Reproductive behaviors and veterinary procedures resulted in elevated FGM concentrations from all individuals except for one male. The longitudinal characterization demonstrated that sex and season did not influence (P<0.05) FGM concentrations. Individuals were highly variable with mean FGM concentration of 2010.1±862.4 ng/g dry feces (range, 816.3-7889.1 ng/g dry feces). Mean FGM baseline concentration was 878.5±201.8ng/g dry feces (range, 475.2-1955.5 ng/g dry feces) with a mean elevated FGM concentrations of 2694.3±1111.4 ng/g dry feces (range, 1110.3-10,683.3 ng/g dry feces). This study provides the foundation for future research on how the environment directly affects the adrenocortical activity in this species of armadillo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far., Methods: Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy., Results: The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface., Conclusions: Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.

Reproductive strategies vary considerably among species, but most studies have focused on a very limited number of mammalian species. Knowledge of the reproductive cycle and behavior is essential for developing and implementing in situ and ex situ conservation strategies for threatened and endangered species. This study aimed at characterizing the seasonal reproductive pattern of female pichis Zaedyus pichiy, a threatened small armadillo native to arid regions of Argentina and Chile, through direct observations, histological studies, and by measuring fecal immunoreactive estrogens, progestagens and glucocorticoids in 10 wild-born, captive pichis and in free-ranging individuals. Results suggest that pichis are seasonal breeders that give birth to one yearly litter of 1-2 offspring, which do not leave the burrow until they are weaned at approximately 37 days. Ovarian follicular growth seems to occur throughout the year. Fecal progestagen, estrogen and glucocorticoid concentrations were minimal during the first half of pregnancy, increased to peak concentrations of up to 3500, 200 and 200ng/g dry feces, respectively, and decreased before parturition. Postpartum progestagen concentrations were greater in lactating females than females that aborted or did not raise their offspring (p<0.0001), which is probably related to an elevated corticosteroid synthesis that contributes to maintain lactation, given that fecal glucocorticoid concentrations were of similar pattern. Observations of a second pregnancy after late abortion or death of the newborn litter and sustained follicular growth during pregnancy and lactation suggest that female pichis can become receptive briefly after having lost their litter. Fecal estrogen and progestagen concentrations of non-pregnant, non-lactating females did not have a well-defined hormonal cyclic pattern, and corpora lutea were only observed in pregnant females.

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Hemomonochorial placentation occurs in diverse species. We have examined placental glycosylation in five widely separated mammals with this type of placentation--lesser hedgehog tenrec (Echinops telfairi), spotted hyena (Crocuta crocuta), nine-banded armadillo (Dasypus novemcinctus), human (Homo sapiens) and guinea pig (Cavia porcellus)--in order to assess whether evolutionary convergence to the hemomonochorial state is accompanied by a similar convergence of glycan expression. Placentae from 2 E. telfairi, 3 C. crocuta, 1 D. novemcinctus, 4 womenand 1 C. porcellus were fixed and processed into epoxy resin. Binding of twenty-three lectins was assessed using a semiquantitative ranking system. The trophoblast apical/microvillous membrane of all five species showed marked similarities in glycosylation. In the N-linked series, there were abundant bi/tri-antennary complex chains, while the non-bisected variants were much scarcer. All species had plentiful N-acetyl lactosamine sequences; at chain termini, binding to Galbeta1,4GlcNAc and Galbeta1,3GalNAc sequences was greatly enhanced after neuraminidase treatment. In all species, terminal NeuNAcalpha2,3 residues were detected. The tenrec had unusually abundant terminal N-acetyl galactosamine. The basal plasma membrane/basal lamina showed glycosylation patterns distinct from the microvillous membrane in each case, indicating chemical diversity of the two opposite faces of trophoblast. Similar classes of glycan at the hemochorial interface suggest conservation of function. The observed lectin binding patterns suggest broad similarities of glycosylation that may have arisen by convergent evolution., (Copyright 2007 S. Karger AG, Basel.)

The X-ray structure of the tetragonal form of apo acyl-CoA-binding protein (ACBP) from the Harderian gland of the South American armadillo Chaetophractus villosus has been solved. ACBP is a carrier for activated long-chain fatty acids and has been associated with many aspects of lipid metabolism. Its secondary structure is highly similar to that of the corresponding form of bovine ACBP and exhibits the unique flattened alpha-helical bundle (up-down-down-up) motif reported for animal, yeast and insect ACBPs. Conformational differences are located in loops and turns, although these structural differences do not suffice to account for features that could be related to the unusual biochemistry and lipid metabolism of the Harderian gland.

The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. Here, we show that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk led to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches led to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediated this signal. Together, our data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells.

The anatomical, histological, histochemical and ultrastructural characteristics of the lacrimal gland (LG) and nictitans gland (NG) of the armadillo Chaetophractus villosus were described. The histochemical and histological features of both glands in male and female adult animals were compared. The tissues were processed with conventional techniques for light and transmission electron microscopy. Fixed specimens were submitted to a battery of tests for glycans, glycosaminglycans, glycoconjugates, proteins, and lipids. The LG of the armadillo may be considered within the set of glandulae lacrimales superior in which primates, carnivores, perisodactyls and artiodactyls are included. The localization of the NG was similar to that of other mammals. Lacrimal and NG were histologically and histochemically identical. The secretory endpieces consisted of three cell types: (1) Mucous cells (MC) with different types of mucous secretory granules with neutral and sialic acid-containing glycoconjugates (GCs). (2)Seromucous cells (SMC) showing a variety of moderately electron dense secretory granules with flocculent material with carboxylated acidic, neutral, and sialic acid-containing GCs. Intercellular canaliculi with junctional complexes and basolateral intercellular spaces were frequent. (3) Serous cells (SC) with electron dense secretory granules. Histochemically, they showed the strongest reaction for proteins and neutral, weakly acid and carboxylated acidic GCs. The epithelium of the intra- and inter-lobular excretory ducts showed secretory activity, junctional complexes, and wide basolateral intercellular spaces with lateral folds. The endpieces and ducts were surrounded by myoepithelial cells. The stroma was characterized by fenestrated endothelium, unmyelinated axons, and abundant plasma cells. MC, SMC, and the duct system were richly innervated by hypolemmal nerve terminals.

A fundamental assumption of the aerobic capacity model for the evolution of endothermy is that basal (BMR) and peak (PMR) metabolic rates are functionally linked at the intraspecific level. The purpose of this study was to use the nine-banded armadillo Dasypus novemcinctus as a model to test this assumption. Measurements of BMR, PMR, mass and rectal temperature were obtained over two summers from wild, adult individuals from a population in Louisiana, USA. BMR and PMR were positively correlated (r=0.62), and both were significantly higher (by 46% for BMR and by 35% for PMR) in 1999 than in 1998. Similar results were obtained whether metabolic rates were expressed in whole-animal or mass-independent units. These results suggest the existence of a functional link between BMR and PMR and are therefore consistent with the aerobic capacity model. In addition, this study confirmed that, compared with most eutherian mammals, the nine-banded armadillo exhibits low and highly variable basal and peak metabolic rates (20-60% the predicted values; 23% and 27% coefficients of variation) and rectal temperatures (range 32.7-35.3 degrees C). Such metabolic traits are, however, consistent with the general pattern previously observed for other members of the order Xenarthra and with the hypothesis that low metabolic rates in armored mammals evolved as a result of unbalanced selection in which, because of low predation risks, selection for a high aerobic capacity was much weaker than the opposing selection for energy conservation.

Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were-stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes.

The sex differentiation in crustaceans is known to be controlled by a peptide hormone called androgenic gland hormone (AGH). AGH was extracted and purified from the androgenic glands (AGs) of the male isopod Armadillidium vulgare by high-performance liquid chromatography. AGH consisted of two peptide chains and their N-terminal amino acid sequences were determined. A cDNA encoding AGH was cloned by PCR and sequenced. The cDNA had an open reading frame of 432 bp, which encoded a preproAGH consisting of a signal peptide (21 residues), B chain (44 residues), C peptide (46 residues), and A chain (29 residues). Through processing, the A and B chains might form a heterodimer interlinked by disulfide bonds. The A chain possessed a putative N-linked glycosylation site. A Northern blot analysis using the cDNA as a probe detected a hybridization signal with 0.8 kb in the RNA preparation only from the AGs., (Copyright 1999 Academic Press.)

Conventional histochemistry and the binding patterns of 22 biotinylated lectins were examined for characterisation of glycoconjugates in the components of the olfactory mucosa of the armadillo Chaetophractus villosus. The mucous lining the olfactory epithelium showed binding sites for DSL, WGA, STL, LEL, PHA-E and JAC. Only the basilar processes of the supporting cells stained for Con-A and S-Con A. The olfactory receptor neurons stained with LEL, LCA, Con A, S-Con A, JAC and PNA. The layer of basal cells did not react with any of the lectins studied. Bowman's glands in the lamina propria showed subpopulations of acinar cells reacting with SBA, S-WGA, WGA, STL, Con A, PSA, PNA, SJA, VVA, JAC and S-Con A, but in our optical studies with lectins we were unable to differentiate between mucous and serous cells in the way that is possible on electron microscopy. The ducts of Bowman's glands were labelled with S-WGA, STL, LEL, PHA-E, BSL-I and JAC. This histochemical study on the glycoconjugates of the olfactory mucosa in the order Xenarthra provides a basis for further experimental investigations.

During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell movements which probably involve adhesive interactions between cells. In the present study, we examined the dynamic aspects of cadherin-based cell-cell adhesion in the morphogenetic events to assess its contribution to morphogenesis. DE- and DN-cadherin show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. We found that switching of cadherin expression from the DE- to the DN-type in the mesodermal germ layer occurred downstream of the mesoderm-determination genes twist and snail. However, examination of their protein expression patterns showed that considerable amounts of DE-cadherin remained on the surfaces of mesodermal cells during invagination, while DN-cadherin did not appear on the cell surfaces at this stage. Further immunocytochemical analysis of the localizations of DE-cadherin and its associated proteins Armadillo (beta-catenin) and Dalpha-catenin revealed dynamic changes in their distributions which were accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Simultaneously, adherens junctions (AJs), based on the cadherin-catenin system, were shown to change their location, size, and morphology. These dynamic aspects of cadherin-based cell-cell adhesion appeared to be associated with the following: (1) initial establishment of the blastoderm epithelium, (2) acquisition of cell motility in the neuroectoderm, (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of the DE-cadherin-catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Moreover, the processes responsible for loss of epithelial cell polarity and elimination of preexisting DE-cadherin-based epithelial junctions during early mesodermal morphogenesis are discussed., (Copyright 1998 Academic Press.)

The fatty acid-binding protein (FABP) from armadillo liver was purified to homogeneity by a procedure involving gel filtration and two anion exchange chromatography steps. The purified protein proved to have a pI between 5.0 and 5.2 and migrated by sodium dodecyl sulfate-polyacrilamyde gel electrophoresis as a single entity of approximately 14 kDa. The armadillo FABP cross-reacted with antiserum against rat liver FABP but not against rat intestinal FABP. The same as other members of the family, it has a blocked N-terminus. Amino acid sequencing of peptides obtained by cyanogen bromide cleavage and in-gel tryptic digestion shows that the armadillo, despite being one of the less evolved mammals, has a liver FABP of the same type as that of highly evolved mammals.

Cell contacts of the adherens type are organized around transmembrane proteins of the cadherin family. Whereas the extracellular domains mediate homophilic interactions between cadherins of neighbouring cells the cytoplasmic domains organize a set of proteins into the junctional plaque. Among these junctional plaque proteins are members of the armadillo gene family, beta-catenin, plakoglobin (gamma-catenin), B6P/plakophilin and p120. These proteins are assumed to play a key role in cell cell signalling through intercellular junctions. Here we report cloning of a cDNA encoding a new armadillo family member, p0071, closely related to p120 and B6P/plakophilin and more distantly related to armadillo, plakoglobin, beta-catenin and other members of the gene family. The deduced amino acid sequence encodes a basic protein of 1,211 amino acids with a central armadillo repeat region which is conserved in sequence and organization of its ten individual motifs between p120, B6P/plakophilin and p0071. In contrast the end domains of the three proteins are variable in size and sequence. The RNA coding for p0071 is expressed in all tissues examined. Using antibodies generated against the armadillo repeat region of the protein we show that p0071 is localized at cell-cell borders and is expressed in the desmosomal plaque of some cultured epithelial cells. The protein seems to be an accessory component of the desmosomal plaque as well as of other adhesion plaques and might be involved in regulating junctional plaque organization and cadherin function. Our data provide evidence for a subfamily of armadillo related proteins that share not only structural features but also have in common their localisation in the junctional plaque. We therefore suggest that family members exert similar functions and might be involved in cell signalling through cell contacts.

The conformational free energy of armadillo metmyoglobin was examined over a pH range of 4.4-8.0 and a guanidinium chloride concentration of 0-2.3 M. For isothermal unfolding at 25 degrees essentially the same value was obtained for the conformational free energy from all the data: 27 +/- 2 kJ/mol. These data suggest that the armadillo has the least stable metmyoglobin of any mammal thus far examined. The cooperativity of the unfolding with respect to denaturant is considerably less than for other mammalian myoglobins. On unfolding only three to four side chains with a pKA of 6 in the unfolded protein are protonated instead of the six found for horse and sperm whale myoglobins.

An acidic protein fraction with an apparent molecular weight of 34 000 has been isolated from the Cetavlon-treated, mucin-free supernatant of the armadillo submandibular gland 0.01 M NaCl extract. This purified material, which was obtained in a yield of 0.45%/g wet gland, contains 24 mol % acidic amino acids and 4 mol % basic amino acids. Hexosamines, sialic acid, and neutral sugars represent 7% of the dry sample weight. In polyacrylamide gel and cellulose acetate electrophoresis, a single protein band was observed. The acidic protein fraction is highly reactive with the Lowry phenol reagent, giving a protein value 83% higher than that obtained by summation of its anhydrous amino acids, and is explained by the occurrence of peptide linkages peculiar to this material. The presence of other basophilic components besides mucus glycoproteins within the salivary gland of the armadillo may have physiological significance.

Lactate dehydrogenase (LDH) was detected in extracts of untreated and NaOH-treated Mycobacterium leprae. Since armadillo liver LDH isoenzymes with a similar electrophoretic mobility were shown to be considerably more sensitive to inhibition by oxamate than LDH in M. leprae extracts, it was confirmed that M. leprae grown in armadillo liver has its own LDH. Neither the activity of LDH in M. leprae nor its electrophoretic mobility supported the tentative suggestion that an "anomolous" LDH isoenzyme in infected skin biopsies was in fact an M. leprae-derived enzyme.

Superoxide dismutase has been identified and peroxidatic activity demonstrated in Mycobacterium leprae. The superoxide dismutase, shown indirectly to be a manganese-containing enzyme, was present at low activity in the cell-free extract. Peroxidatic activity was detected in a haemoprotein on polyacrylamide gels, but quantitative assay was not possible. Catalase, although present in a cell-free extract, appeared to be a host-derived enzyme, thus emphasizing the importance of establishing the authenticity of enzyme activities in host-derived M. leprae. The implications for the growth of M. leprae in vivo and its non-cultivability are discussed in the light of these findings.

The structure of carbohydrate units of the major glycoprotein fraction of armadillo submandibular gland was investigated. Alkaline borohydride reductive cleavage of the glycoprotein resulted in the release of O-glycosidically linked mono- and disaccharide units. The monosaccharide was identified as N-acetylgalactosaminitol, whereas disaccharide contained of N-acetylneuraminic acid and N-acetylgalactosaminitol. Treatment of the native and desialyzed glycoprotein with alpha-N-acetylgalactosaminidase resulted in the removal of 60% and 96% of N-acetylgalactosamine, respectively. No cleavage of this sugar was affected by the action of beta-N-acetylhexosaminidase. Both N-acetylgalactosamine and N-acetylneuraminic acid were susceptible to oxidation with periodate. Analyses of the partially methylated N-acetylgalactosamine derivatives, obtained from the permethylated native glycoprotein, showed the presence of 3,4,6-tri-O-methyl-N-methylacetamidogalactose and 3,4-di-O-methyl-N-methylacetamidogalactose in a ratio of 1 : 0.4. Only 3,4,6-tri-O-methyl-N-methylacetamidogalactose was found in the hydrolysates of permethylated desialyzed glycoprotein. These results together with our previous data on chemical composition of the glycoprotein suggest that about 30% of the oligosaccharide chains consist of NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr(Ser) and 70% of GalNAc alpha leads to O-Thr(Ser).

Adult female armadillos were injected with tritiated estradiol. The pituitary gland was removed and processed for autoradiography. Nuclear uptake and retention of the steroid was found in 28.8% of the cells in the pars distalis and 3.7% of the pituicytes.

Recent improvements in the sensitivity of assay methods for superoxide dismutase (SOD) have enabled the detection of this enzyme in 18 cell-free extracts of purified Mycobacterium leprae. By converting back to units of SOD obtained in the cytochrome c-based method previously used in work on this enzyme in mycobacteria, it was shown that extracts of M. leprae had 0.15-3.84 U SOD/mg protein (this study). A mean value of 1.31 U/mg protein was calculated. It was not possible to find any factors which could explain the very high levels in some extracts, although correlation with the period of tissue storage at -80 degrees C suggested that M. leprae in freshly killed tissue would have 1.77 U SOD/mg protein. The possibility of contamination by SODs from host and other organisms was unlikely since on gel electrophoresis extracts of M. leprae with high levels of SOD showed only a single band of activity characteristic of manganese-dependent SOD previously demonstrated.

The histochemistry of armadillo skin has been studied. The dendritic cells are extremely large, very sharply outlined by methods for alkaline phosphatase and alpha-naphthyl-acetate esterase, and they are dopa-negative. The mastocytes, however, are dopa-oxidase-positive, probably due to peroxidase rather than tyrosinase activity. The giant cells of the granulomas normally seen in the dermis of the armadillo are strongly beta-glucuronidase-positive. These giant cells are evidently foreign body cells reacting to the crystals always present in the dermis of the armadillo. The centre of these crystals, which are cholesterol and fat-negative, is alkaline phosphatase-positive. Further study of the mastocytes and dendritic cells is necessary to elucidate their nature.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.

The uptake and retention of radiolabeled estradiol by the spinal cord were examined in the baboon and the armadillo and compared to previous observations in the rat. Four females of each species were injected intracardially with 1.0-1.4 micrograms/kg body weight of 3H-estradiol and two females, one baboon and one armadillo, were injected with both labeled and 100-140 micrograms/kg body weight of unlabeled estradiol. One hour after the injections, the animals were killed and segments from the cervical, thoracic, lumbar and sacral cord were removed and processed for autoradiography. In the armadillo, labeling of neuronal nuclei were noted in laminae I & II and in alpha motor neurons. In addition, nuclei of the ependymal cells of the ventral portion of the central canal in the cervical cord concentrated radioactivity. In contrast, the baboon demonstrated only sporadic labeling of neurons in lamina II in all levels of the spinal cord. The comparison of our observations with that of the rat suggest that estrogen mediated sensations are probably coordinated at higher brain centers in the primate as opposed to the more primitive mammals.