Immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) in the regenerating lizard epidermis indicates a new process for the differentiation of the epidermis in lepidosaurians

The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta‐proteins (beta‐keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen‐, beta‐, mesos‐, and alpha‐layer). Three specific proteins representing the larger families of beta‐proteins, glycine‐rich (HgG5, 28% glycine, 3.6% cysteine), glycine‐cysteine medium‐rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine‐cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta‐cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha‐cells. The protein is located in the pale corneous material forming the compact beta‐layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta‐packets in Oberhäutchen and beta‐cells but disappears in more compact and electron‐pale corneous material. The labeling disappears in mesos‐cells and is present with variable intensity in alpha‐cells, whereas lacunar and clear‐cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta‐cells but is lightly present in the darker corneous material of differentiating and mature alpha‐cells, lacunar‐cells, and clear‐cells. The study suggests that while glycine‐rich proteins (electron‐pale) are specifically used for building the resistant and hydrophobic beta‐layer, cysteine–glycine rich proteins (electron‐denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha‐cells. The differential accumulation of beta‐proteins on the alpha‐keratin cytoskeleton scaffold and not the alternance of beta‐ with alpha‐keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

Granulocytes of reptilian sauropsids contain beta‐defensin‐like peptides: A comparative ultrastructural survey

The ability of lizards to withstand infections after wounding or amputation of the tail or limbs has suggested the presence of antimicrobial peptides in their tissues. Previous studies on the lizard Anolis carolinensis have identified several beta‐defensin‐like peptides that may potentially be involved in protection from infections. The present ultrastructural immunocytochemical study has analyzed tissues in different reptilian species in order to localize the cellular source of one of the more expressed beta‐defensins previously sequenced in lizard indicated as AcBD15. Beta‐defensin‐like immunoreactivity is present in some of the larger, nonspecific granules of granulocytes in two lizard species, a snake, the tuatara, and a turtle. The ultrastructural study indicates that only heterophilic and basophilic granulocytes contain this defensin while other cell types from the epidermis, mesenchyme, and dermis, muscles, nerves, cartilage or bone are immunonegative. The study further indicates that not all granules in reptilian granulocytes contain the beta‐defensin peptide, suggesting the presence of granules with different content as previously indicated for mammalian neutrophilic leucocytes. No immunolabeling was instead observed in granulocytes of the alligator and chick using this antibody. The present immunocytochemical observations suggest a broad cross‐reactivity and conservation of beta‐defensin‐like sequence or steric motif across lepidosaurians and likely in turtles while archosaurian granulocytes may contain different beta‐defensin‐like or other peptides. J. Morphol. 274:877–886, 2013. © 2013 Wiley Periodicals, Inc.     

Immunolocalization of keratin‐associated beta‐proteins in developing epidermis of lizard suggests that adhesive setae contain glycine–cysteine‐rich proteins

The localization of specific keratin‐associated beta‐proteins (formerly referred to as beta‐keratins) in the embryonic epidermis of lizards is not known. Two specific keratin‐associated beta‐proteins of the epidermis, one representing the glycine‐rich subfamily (HgG5) and the other the glycine‐cysteine medium‐rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta‐layer, and disappears in mesos‐ and alpha‐layers. Instead, HgGC10 is present in the Oberhäutchen, beta‐, and also in the following alpha‐layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine–glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta‐layer but also in those forming the alpha‐layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine‐rich beta‐proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta‐layer. This result suggests that the differential accumulation of keratin‐associated beta‐proteins over the alpha‐keratin network determines differences in properties of the stratified layers of the epidermis of lizards. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Immunolocalization of specific beta‐proteins in pad lamellae of the digits in the lizard Anolis carolinensis suggests that cysteine‐rich beta‐proteins provides flexibility

Knowledge of beta‐protein (beta‐keratin) sequences in Anolis carolinensis facilitates the localization of specific sites in the skin of this lizard. The epidermal distribution of two new beta‐proteins (beta‐keratins), HgGC8 and HgG13, has been analyzed by Western blotting, light and ultrastructural immunocytochemistry. HgGC8 includes 16 kDa members of the glycine‐cysteine medium‐rich subfamily and is mainly expressed in the beta‐layer of adhesive setae but not in the setae. HgGC8 is absent in other epidermal layers of the setae and is weakly expressed in the beta‐layer of other scales. HgG13 comprises members of 17‐kDa glycine‐rich proteins and is absent in the setae, diffusely distributed in the beta layer of digital scales and barely present in the beta‐layer of other scales. It appears that the specialized glycine‐cysteine medium rich beta‐proteins such as HgGC8 in the beta‐layer, and of HgGC10 and HgGC3 in both alpha‐ and beta‐layers, are key proteins in the formation of the flexible epidermal layers involved in the function of these modified scales in adaptation to contact and adhesion on surfaces. J. Morphol. 275:504–513, 2014. © 2013 Wiley Periodicals, Inc.     

Immunocytochemical localization of cysteine‐rich beta‐proteins in the extensible epidermis of the dewlap in the lizard Anolis carolinensis

The dewlap in the lizard Anolis carolinensis is made of scales separated by large interscale regions capable of broad stretching during fan extension. This indicates that the skin contains proteins that allow extension of interscale regions. The immunocytochemical analysis of the epidermis indicates that HgG5, a glycine‐rich hydrophobic beta‐protein poor in cysteine is localized only in the stiff beta‐layer of the outer scale surface, but is completely absent in mesos and alpha‐layers and in hinge regions. HgGC10, a cysteine‐medium‐rich beta‐protein is present in beta‐layers but especially in alpha‐layers of interscale epidermis that presents folds and lacks a beta‐layer. HgGC3 is weakly localized in the alpha‐layer, but is mainly found in hinge regions. HgGC8 and HgG13 are low to absent in the alpha‐ and beta‐layer. The immunolocalization of cysteine‐rich beta‐proteins such as HgGC10/3 in alpha‐layers and interscale epidermis suggests that these small proteins are involved in the formation of a corneous material compatible with dewlap extension. The basement membrane underneath scales is joined to bundles of collagen fibrils in the dermis through anchoring fibrils that likely determine flattening of the epidermis during the extension of the throat fan.     

Immunolocalization of large corneous beta‐proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta‐proteins in the beta‐layer of the epidermis

The distribution of large corneous beta‐proteins of 18–43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta‐proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40–50 kDa suggesting they mix with alpha‐keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha‐layers of most scales and in beta‐cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta‐layer of adhesive pads and in mesos cells. In differentiating beta‐keratinocytes Ac37 is present over 3–4 nm thick filaments located around the amorphous beta‐packets and in alpha‐cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha‐keratins to which it probably associates. Ac39 is seen in the beta‐layer of tail and digital scales, in beta‐cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta‐ and alpha‐layers of the other scales. Ac40 is present in the mature beta‐layer of most scales and dewlap, in differentiating beta‐cells of regenerating scales, but is absent in all the other epidermal layers. The large beta‐proteins are accumulated among forming beta‐packets of beta‐cells and are packed in the beta‐corneous material of mature beta‐layer. Together alpha‐keratins, large beta‐proteins form the denser areas of mature beta‐layer that may have a different consistence that the electron‐paler areas. J. Morphol. 276:1244–1257, 2015. © 2015 Wiley Periodicals, Inc.     

Immunolocalization of beta‐proteins and alpha‐keratin in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material

Immunolocalization of beta‐proteins in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material, Acta Zoologica, Stockholm. The corneous layer of soft‐shelled turtles derives from the accumulation of higher ratio of alpha‐keratins versus beta‐proteins as indicated by gene expression, microscopic, immunocytochemical and Western blotting analysis. Type I and II beta‐proteins of 14–16 kDa, indicated as Tu2 and Tu17, accumulate in the thick and hard corneous layer of the hard‐shelled turtle, but only type II is present in the thinner corneous layer of the soft‐shelled turtle. The presence of proline–proline and proline–cysteine–hinge dipeptides in the beta‐sheet region of all type II beta‐proteins so far isolated from the epidermis of soft‐shelled turtles might impede the formation of beta‐filaments and of the hard corneous material. Western blot analysis suggests that beta‐proteins are low to absent in the corneous layer. The ultrastructural immunolocalization of Tu2 and Tu17 beta‐proteins shows indeed that a diffuse labelling is seen among the numerous alpha‐keratin filaments present in the precorneous and corneous layers of the soft epidermis and that no dense corneous material is formed. Double‐labelling experiments confirm that alpha‐keratin prevails on beta‐proteins. The present observations support the hypothesis that the soft material detected in soft‐shelled turtles derives from the prevalent activation of genes producing type II beta‐proteins and high levels of alpha‐keratins.     

Immunogold labeling shows that glycine‐cysteine‐rich beta‐proteins are deposited in the Oberhäutchen layer of snake epidermis in preparation to shedding

Shedding in snakes is cyclical and derives from the differentiation of an intraepidermal shedding complex made of two different layers, termed clear and Oberhäutchen that determine the separation between the outer from the inner epidermal generation that produces a molt. The present comparative immunocytochemical study on the epidermis and molts of different species of snakes shows that a glycine‐cysteine‐rich corneous beta‐protein in a snake is prevalently accumulated in cells of the Oberhäutchen layer and decreases in those of the beta‐layer. The protein is variably distributed in the mature beta‐layer of species representing some snake families when the beta‐layer merges with the Oberhäutchen but disappears in alpha‐layers. Therefore, this protein represents an early marker of the transition between the outer and the inner epidermal generations in the epidermis of snakes in general. It is hypothesized that specific gene activation for glycine‐cysteine‐rich corneous beta‐proteins occurs during the passage from the clear layer of the outer epidermal generation to the Oberhäutchen layer of the replacing inner epidermal generation. It is suggested that in the epidermis of most species glycine‐cysteine‐rich corneous beta‐proteins form part of the dense corneous material that rapidly accumulates in the differentiating Oberhäutchen cells but decreases in the following beta‐layer of the inner epidermal generation destined to be separated from the previous outer generation in the process of shedding. The regulation of the synthesis of these and other proteins is, therefore, crucial in timing the different stages of the shedding cycle in lepidosaurian reptiles. J. Morphol. 276:144–151, 2015. © 2014 Wiley Periodicals, Inc.     

Low‐cysteine alpha‐keratins and corneous beta‐proteins are initially formed in the regenerating tail epidermis of lizard

During tail regeneration in lizards, the stratified regenerating epidermis progressively gives rise to neogenic scales that form a new epidermal generation. Initially, a soft, un‐scaled, pliable, and extensible epidermis is formed that is progressively replaced by a resistant but non‐extensible scaled epidermis. This suggests that the initial corneous proteins are later replaced with harder corneous proteins. Using PCR and immunocytochemistry, the present study shows an upregulation in the synthesis of low‐cysteine type I and II alpha‐keratins and of corneous beta‐proteins with a medium cysteine content and a low content in glycine (formerly termed beta‐keratins) produced at the beginning of epidermal regeneration. Quantitative PCR indicates upregulation in the production of alpha‐keratin mRNAs, particularly of type I, between normal and the thicker regenerating epidermis. PCR‐data also indicate a higher upregulation for cysteine‐rich corneous beta‐proteins and a high but less intense upregulation of low glycine corneous protein mRNAs at the beginning of scale regeneration. Immunolabeling confirms the localization of these proteins, and in particular of beta‐proteins with a medium content in cysteine initially formed in the wound epidermis and later in the differentiating corneous layers of regenerating scales. It is concluded that the wound epidermis initially contains alpha‐keratins and corneous beta‐proteins with a lower cysteine content than more specialized beta‐proteins later formed in the mature scales. These initial corneous proteins are likely related to the pliability of the wound epidermis while more specialized alpha‐keratins and beta‐proteins richer in glycine and cysteine are synthesized later in the mature and inflexible scales. J. Morphol. 278:119–130, 2017. ©© 2016 Wiley Periodicals,Inc.     

Formation of adherens and communicating junctions coordinate the differentiation of the shedding‐layer and beta‐epidermal generation in regenerating lizard epidermis

In the lizard epidermis, the formation of a stratified alpha‐ and beta‐layer, separated by a shedding complex for molting, suggests that keratinocytes communicate in a coordinated manner after they leave the basal layers during the shedding cycle. I have therefore studied the localization of cell junctional proteins such as beta‐catenin and connexins 43 and 26 during scale regeneration in lizard using immunocytochemistry. Beta‐catenin is also detected in nuclei of basal cells destined to give rise to the Oberhäutchen and beta‐cells suggesting activation of the Wnt‐pathway during beta‐cell differentiation. The observations show that cells of the entire shedding layer (clear and Oberhäutchen) and beta‐layer are connected by beta‐catenin (adherens junctions) and connexins (communicating junctions) during their differentiation. This likely cell coupling determines the formation of a distinct shedding and beta‐layer within the regenerating epidermis. The observed pattern of cell junctional stratification suggests that after departing from the basal layer Oberhäutchen and beta‐cells form a continuous communicating compartment that coordinates the contemporaneous differentiation along the entire scale. While the beta‐layer matures the junctions are lost while other cell junctions are formed in the following mesos‐ and alpha‐cell layers. This process determines the formation of layers with different texture (harder or softer) and the precise localization of the shedding layer within lizard epidermis. J. Morphol. 275:693–702, 2014. © 2014 Wiley Periodicals, Inc.     

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae,Serpentes, Reptilia), with emphasis on the formation of the shedding complex

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.     

Timing of neurodegeneration and beta‐amyloid (Aβ) peptide deposition in the brain of aging kokanee salmon

Brains of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages (sexually immature, maturing, sexually mature, and spawning) were stained with cresyl violet and silver stain to visualize neurodegeneration. These reproductive stages correlate with increasing somatic aging of kokanee salmon, which die after spawning. Twenty‐four regions of each brain were examined. Brains of sexually immature fish exhibited low levels of neurodegeneration, whereas neurodegeneration was more marked in maturing fish and greatest in spawning fish. Neurodegeneration was present in specific regions of the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Pyknotic neurons were observed in all regions previously reported to be immunopositive for Aβ. Regions that did not exhibit neurodegeneration during aging included the magnocellular vestibular nucleus, the nucleus lateralis tuberis of the hypothalamus, and Purkinje cells of the cerebellum, all of which also lack Aβ; perhaps these regions are neuroprotected. In 14 of 16 brain areas for which data were available on both the increase in Aβ deposition and pyknosis, neurodegeneration preceded or appeared more or less simultaneously with Aβ production, whereas in only two regions did Aβ deposition precede neurodegeneration. This information supports the hypothesis that Aβ deposition is a downstream product of neurodegeneration in most brain regions. Other conclusions are that the degree of neurodegeneration varies among brain regions, neurodegeneration begins in maturing fish and peaks in spawning fish, the timing of neurodegeneration varies among brain regions, and some regions do not exhibit accelerated neurodegeneration during aging. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 21–35, 2002     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.     

Expression of chemosensory proteins in hairs on wings of Locusta migratoria (Orthoptera: Acrididae)

The hairs on the wings of Locusta migratoria were observed and mapped using light microscopy, as well as by scanning and transmission electron microscopy. Based on their ultrastructure, we can distinguish four main types of hairs on the wings of adult L. migratoria , viz, short, medium and long hairs, and sensilla chaetica. The long hairs are located only on the ventral surface of the hindwing, whereas the other three types are present both on the dorsal and ventral surfaces of forewing and hindwing in both sexes. Medium hairs and sensilla chaetica are significantly more abundant on the dorsal surface of forewings in both females and males, than on the ventral surface, whereas the opposite was observed for short hairs (P < 0.01). No significant difference between males and females was observed in the density of any type of hairs (P > 0.1). Several dendritic branches, enveloped by a dendrite sheath, are situated in the lymph cavity of sensilla chaetica. Instead, no dendritic structure was observed in the cavity of the other three types of hairs. Immunocytochemical localization of chemosensory proteins (CSPs) was performed on ultrathin sections of hairs on wings. The antiserum against chemosensory proteins from L. migratoria (LmigCSP-II) strongly labelled sensilla chaetica, with gold granules only found in the outer sensillum lymph. In addition, the epidermal cell membrane of the wing was stained by the antiserum against LmigCSP-II. The other three types of hairs were never labelled. The results indicate that the wings might involve in contact chemoreception process.     

Distinctive structural motifs co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole in the alpha/beta‐hydrolase fold enzymes

The alpha/beta‐hydrolases (ABH) are among the largest structural families of proteins that are found in nature. Although they vary in their sequence and function, the ABH enzymes use a similar acid–base‐nucleophile catalytic mechanism to catalyze reactions on different substrates. Because ABH enzymes are biocatalysts with a wide range of potential applications, protein engineering has taken advantage of their catalytic versatility to develop enzymes with industrial applications. This study is a comprehensive analysis of 40 ABH enzyme families focusing on two identified substructures: the nucleophile zone and the oxyanion zone, which co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole, and independently reported as critical for the enzymatic activity. We also frequently observed an aromatic cluster near the nucleophile and oxyanion zones, and opposite the ligand‐binding site. The nucleophile zone, the oxyanion zone and the residue cluster enriched in aromatic side chains comprise a three‐dimensional structural organization that shapes the active site of ABH enzymes and plays an important role in the enzymatic function by structurally stabilizing the catalytic nucleophile and the residues of the oxyanion hole. The structural data support the notion that the aromatic cluster can participate in co‐ordination of the catalytic histidine loop, and properly place the catalytic histidine next to the catalytic nucleophile.     

Ischemia reduces inter‐alpha inhibitor proteins in the brain of the ovine fetus

Hypoxic‐ischemic (HI) brain injury is a major cause of neurological abnormalities in the perinatal period. Inflammation contributes to the evolution of HI brain injury. Inter‐alpha inhibitor proteins (IAIPs) are a family of proteins that are part of the innate immune system. We have reported that endogenous IAIPs exhibit developmental changes in ovine brain and that exogenous IAIP treatment reduces neuronal death in HI neonatal rats. However, the effects of HI on endogenous IAIPs in brain have not been previously examined. In this study, we examined the effects of ischemia‐reperfusion on endogenous IAIPs levels in fetal sheep brain. Cerebral cortex, cerebellum, cervical spinal cord, choroid plexus, and CSF were snap frozen from sham control fetuses at 127 days gestation and after 30‐min of carotid occlusion and 4‐, 24‐, and 48‐h of reperfusion. IAIP levels were determined by Western immunoblot. IAIP expressions of the 250 kDa Inter‐alpha inhibitor (IaI) and 125 kDa Pre‐alpha inhibitor (PaI) in cerebral cortex and PaI in cerebellum were reduced (p < 0.05) 4‐h after ischemia compared with controls and returned toward control levels 24‐ and 48‐h after ischemia. CSF PaI and IaI were reduced 48 h after ischemia. We conclude that IAIPs in cerebral cortex and cerebellum are reduced by brain ischemia, and return toward control levels between 24 and 48 h after ischemia. However, changes in CSF IAIPs were delayed, exhibiting decreases 48 h after ischemia. We speculate that the decreases in endogenous IAIPs reflect increased utilization, potentially suggesting that they have endogenous neuroprotective properties. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 726–737, 2017     

Immunocytochemical localization of sulfhydryl oxidase in mammalian epidermis suggests that the enzyme cross‐links keratins in the granular and transitional corneous layers

The epidermis of representative mammalian species including humans has been examined for the presence of sulfhydryl oxidase, an enzyme likely involved in the oxidation of corneous proteins containing sulfhydryl groups in the epidermis. A database search indicates that the enzyme shares common sequences in numerous mammalian species so that an antibody against the human sulfhydryl oxidase 2 has been utilized on other species. The immunofluorescent study on the epidermis of the platypus (monotreme), red kangaroo (marsupials), hamster and human (placentals) reveals a prevalent labelling in the granular, transitional and lowermost part of the stratum corneous layer. The detailed ultrastructural immunogold study of the human epidermis reveals a diffuse and uneven labelling in the paler component of the composite keratohyalin granules or among keratin filaments of the transitional layer while the labelling disappears in the corneous layer. The study supports the hypothesis of the participation of the enzyme in the oxidative process that determines the formation of stable disulphide groups among keratins and other corneous proteins of the stratum corneum. This process gives rise to the resistant cell corneous envelope of keratinocytes in addition to the isopeptide bonds that derive from the catalytic action of epidermal transglutaminase on several corneous proteins.