Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Non-syndromic hypodontia of maxillary lateral incisors and its association with other dental anomalies

Abstract Background Tooth agenesis (TA) is the developmental absence of one or more teeth and is the most common craniofacial disorder in humans. Maxillary lateral incisor agenesis (MLIA) is a specific subtype of TA and can have esthetic, functional, and psychosocial implications for patients. The aim of this study was to evaluate the prevalence of MLIA amongst patients with non-syndromic tooth agenesis, as well as its association with other dental anomalies. Materials and methods The dental records of 240 patients with non-syndromic congenitally missing teeth treated at the University of Alabama at Birmingham Department of Orthodontics were reviewed. Dolphin Imaging software was used to identify missing teeth, microdonts, peg laterals, impactions, and transpositions. Data were analyzed using chi-square or Fisher’s exact test. All the tests were two-sided at the significance level of 0.05 (SAS 9.4). Results In the patient cohort, MLIA prevalence was 37.5% (second most common) and no gender or ethnic differences were identified. We also observed the bilaterally missing lateral incisors more frequently than the unilateral presentation (p = 0.0006). Additionally, 62.5% of patients with unilateral MLIA displayed a contralateral tooth that was a peg (p = 0.0001); however, no association was found with other microdonts. Furthermore, of the 90 patients missing at least one maxillary lateral incisor, 42.2% were missing another tooth type and 10% of MLIA patients also had an impacted tooth (mainly maxillary canines). However, these were not statistically significant. Finally, no transposed teeth were found in our patients. Conclusions This study found that maxillary lateral incisors were the second most frequently missing teeth. When clinicians diagnose congenital absence of a maxillary lateral incisor, the patient should be evaluated for other missing teeth, peg lateral incisors, or potential impactions, especially maxillary canines

Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extra– cellular matrix of connective tissues, through an enzymatic β–elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 Å resolution crystal structure, substrate modeling and mutagenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C–terminal β–sheet domain is to modulate enzyme activity through binding to calcium ions

Tissue- and Cell-Specific Alternative Splicing of NFIC

Alternative splicing is an important mechanism for increasing genetic complexity leading to multiple transcripts from single genes and gene regulation through alternative promoters. Splicing often leads to unique tissue-specific patterns of mRNAs with specific biological functions. Nuclear factor I-C (NFI-C), a member of the NFI gene family, is expressed in numerous tissues including brain, liver, spleen and heart. However, the unique dental phenotype of Nfic–/– mice lacking molar roots demonstrates a critical role for this transcription factor in root formation. In humans, the NFI-C gene is alternatively spliced producing 4 isoforms. However, different spliced variants have not been studied in association with tissue specificity. The main objective of this study is to identify the NFI-C isoforms expressed in dental cells/tissues, comparing them to the spliced variants in nondental cells/tissues and to analyze their relative expression levels in various cell types. Using bioinformatics, we analyzed the NFI-C gene structure, identifying 2 potential alternative promoters driving expression of selective mRNA transcripts. Our studies show the expression of 3 NFI-C transcripts with the overall splicing pattern conserved between dental and nondental cells tested. Furthermore, by quantitative real-time PCR analysis, we found that although the relative levels of these transcripts were similar in dental and nondental cells, significant differences were observed within the dental cells tested. These are the first studies to analyze the expression of NFI-C isoforms in dental versus nondental cells/tissues, finding subtle cell-/tissue-specific expression patterns that could explain the dental phenotype of Nfic–/– mice

Design of new benzoxazole-2-thione derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole.

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties

The Acid-Activated Ion Channel ASIC Contributes to Synaptic Plasticity, Learning, and Memory

AbstractMany central neurons possess large acid-activated currents, yet their molecular identity is unknown. We found that eliminating the acid sensing ion channel (ASIC) abolished H+-gated currents in hippocampal neurons. Neuronal H+-gated currents and transient acidification are proposed to play a role in synaptic transmission. Investigating this possibility, we found ASIC in hippocampus, in synaptosomes, and in dendrites localized at synapses. Moreover, loss of ASIC impaired hippocampal long-term potentiation. ASIC null mice had reduced excitatory postsynaptic potentials and NMDA receptor activation during high-frequency stimulation. Consistent with these findings, null mice displayed defective spatial learning and eyeblink conditioning. These results identify ASIC as a key component of acid-activated currents and implicate these currents in processes underlying synaptic plasticity, learning, and memory

Schematic representation of the Ad5 fiber proteins carrying intact and modified C-terminal knob domains.

<p>Fiber modifications are indicated in the corresponding vector names: Ad5 (G/L) has unmodified fiber knob and possesses the native CAR tropism; Ad5-RGD contains a peptide ligand with an “RGD motif” in the HI loop (red loop) of the fiber knob; Ad5-pK7 contains a stretch of seven lysine residues (green oval) fused to the C-terminus of the Ad5 knob via a (GS)₅ linker (green hook); Ad5-pK7/RGD incorporates both modifications in the corresponding locales of the same fiber molecule; Ad5/3 contains a chimera fiber with Ad5 fiber “knob” domain (gray) replaced with the Ad serotype 3 (Ad3) knob (blue), which retargets the vector to Ad3 receptor(s).</p