Ejvis Lamani

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 extracellular 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.

A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase.

The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor‐dependent and a cofactor‐independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor‐independent PGM, genes homologous to those encoding cofactor‐dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor‐dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram‐positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three‐dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.

Insights Into the Catalytic Mechanism of Cofactor-Independent Phosphoglycerate Mutase from X-Ray Crystallography, Simulated Dynamics and Molecular Modeling

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzymes surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

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.

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.

NFI-C2 temporal-spatial expression and cellular localization pattern during tooth formation.

Currently, little is known regarding critical signaling pathways during later stages of tooth development, especially those associated with root formation. Nfi‐c null mice, lacking molar roots, have implicated the transcription factor NFI‐C as having an essential role in root development. Previously, we identified three NFI‐C isoforms expressed in dental tissues with NFI‐C2 being the major transcript. However, the expression pattern of the NFI‐C2 protein is not characterized. In this study we performed in situ hybridization and immunohistochemistry using isoform specific probes. We show the production of a NFI‐C2 peptide antibody, its characterization, the temporal‐spatial expression pattern of the NFI‐C2 protein during odontogenesis and sub‐cellular localization in dental cells. Moderate NFI‐C2 staining, as early as bud stage, was detected mostly in the condensing dental ectomesenchyme. This staining intensified within the dental pulp at later stages culminating in high expression in the dentin producing odontoblasts. The dental epithelium showed slight staining until cytodifferentiation of enamel organ into ameloblasts and stratum intermedium. During root formation NFI‐C2 expression was high in the Hertwigs epithelial root sheath and later was found in the fully developed root and its supporting tissues. NFI‐C2 cellular staining was cytosolic, associated with the Golgi, and nuclear. These data suggest a broader role for NFI‐C during tooth formation than limited to root and periodontal ligament development.

Short root anomaly - A potential “Landmine” for orthodontic and orthognathic surgery treatment of patients

Short root anomaly (SRA) is a poorly understood developmental disorder and can significantly compromise the patients dental treatment. This case report describes the treatment of a 15-year-old girl with SRA and discusses the implication of this disorder on orthodontic and orthognathic treatment of patients.

Maxillary distraction osteogenesis in unilateral cleft lip and palate patients with rigid external distraction system

Aim: Distraction osteogenesis (DO) is a treatment option for patients with maxillary hypoplasia secondary to cleft lip and palate (CLP). Purpose: The aim of this study is to present a technique for maxillary DO using Le Fort I osteotomy with rigid external distraction (RED) system. Subjects and Methods: The patient presented in this paper was an Asian female with CLP aged 13 years and 6 months. She presented with severe midfacial deficiency with a Class III dental malocclusion with a negative overjet and concave facial profile. Cone-beam computed tomography images were recorded preoperatively and the operation performed involved a high Le Fort I osteotomy. The appliance fabricated was banded to upper first molars used for anchorage of the RED system. Distraction of the maxilla was initiated after 7-day latency period. Results: Postoperative cephalometric analysis showed maxillary advancement anteriorly and superiorly, the total distraction treatment period was 10 days. The maxillary advancement was 10.5 mm and the SNA angle increased from 67.5° to 77.9°. Furthermore, the ANB angle changed from −9.8° to 1.6° and the occlusion changed from Class III to Class I. The profile of the face changed from concave to convex and a much better esthetic result was achieved. Conclusion: The study suggests RED system to be a reliable alternative procedure for the treatment of midfacial hypoplasia with or without cleft. Furthermore, it minimizes the risk of the surgical procedure and shortens the operating time.