Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.     

Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR

Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motifs: polyalanine, (GA)(n), GPGXX, and GGX (X = G,A,S,Q,L,Y) will be considered in this study. Although synthetic mimetics of many of these amino acid motifs have been produced in several biological systems, the source of spider silk's mechanical integrity remains elusive. Mechanical robustness may be a product not only of the amino acid structure but also of the tertiary structure of the silk. Historically, solid state nuclear magnetic resonance (ssNMR) has been used to reveal the crystalline structure of the polyalanine motif; however, limitations in amino acid labeling techniques have obscured the structures of the GGX and GPGXX motifs thought to be responsible for the structural mobility of spider silk. We describe the use of metabolic pathways to label tyrosine for the first time as well as to improve the labeling efficiency of proline. These improved labeling techniques will allow the previously unknown tertiary structures of major ampullate silk to be probed.     

Identification of major clonal complexes and toxin producing strains among Staphylococcus aureus associated with atopic dermatitis

Staphylococcus aureus promotes the onset and severity of atopic dermatitis (AD), which is exacerbated by superantigen toxins SEB and SEC. The genetic identity of these isolates, and their relationship to common hospital- or community-associated methicillin resistant S. aureus (HA-MRSA and CA-MRSA) has not been defined. We conducted spa typing, partial multi-locus sequence typing (MLST), and toxin profiling (seb, sec, lukS-PV) of S. aureus from 119 pediatric and 40 adult AD patients. MLST clonal complexes CC45, CC5, CC15, CC1, CC8 and CC30 accounted for 79% of isolates, representing the same major groups reported for nosocomial S. aureus in hospital intensive care units. The highest disease severity was associated with CC1, which was significantly greater relative to CC15 (p?=?0.017) or CC30 (p?=?0.040), but with no significant difference relative to CC45, CC5 or CC8. Although there were two few lukS-PV, seb or sec isolates to infer a role in disease severity, CC45 was identified as a source of SEC producing strains, and lukS-PVL was associated with a small number of CC5 pediatric isolates. CC1 harbored the only CA-MRSA that was identified, and was a source of isolates that expressed both seb and sec, and closely resembled the USA400 strain of CA-MRSA.     

Chronic apocynin treatment attenuates beta amyloid plaque size and microglial number in hAPP(751)(SL) mice

Background

NADPH oxidase is implicated in neurotoxic microglial activation and the progressive nature of Alzheimer''s Disease (AD). Here, we test the ability of two NADPH oxidase inhibitors, apocynin and dextromethorphan (DM), to reduce learning deficits and neuropathology in transgenic mice overexpressing human amyloid precursor protein with the Swedish and London mutations (hAPP(751)_SL).

Methods

Four month old hAPP(751)_SL mice were treated daily with saline, 15 mg/kg DM, 7.5 mg/kg DM, or 10 mg/kg apocynin by gavage for four months.

Results

Only hAPP(751)_SL mice treated with apocynin showed reduced plaque size and a reduction in the number of cortical microglia, when compared to the saline treated group. Analysis of whole brain homogenates from all treatments tested (saline, DM, and apocynin) demonstrated low levels of TNFα, protein nitration, lipid peroxidation, and NADPH oxidase activation, indicating a low level of neuroinflammation and oxidative stress in hAPP(751)_SL mice at 8 months of age that was not significantly affected by any drug treatment. Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)_SL mice. To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.

Conclusions

Together, this study suggests that while hAPP(751)_SL mice show increases in microglial number and plaque load, they fail to exhibit elevated markers of neuroinflammation consistent with AD at 8 months of age, which may be a limitation of this animal model. Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.     

Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion; however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.