Immunohistochemical detection of urea transporter-A in the tear-producing part of the lacrimal system.

OBJECTIVE: Urea constitutes a physiological and presumably well-regulated constituent of tear fluid. Its lacrimal concentration is significantly decreased in dry eye disease. Urea homeostasis within the tear fluid may also depend on the expression of urea transporters. The present study reports on the expression patterns of urea transporter A (UT-A) in the cells and tissues of the ocular surface and the lacrimal glands. METHODS: UT-A immunohistochemistry was performed on 5 µm paraffin sections of paraformaldehyde-fixed human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). RESULTS: UT-A immunostaining was largely comparable in all three species. UT-A signals were detected in the corneal epithelium and endothelium, in the conjunctival epithelium, in the acinar cells and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The Meibomian glands and the glands of Zeis exhibited a marked UT-A-positive staining in the basal cells of the alveolar epithelia and in the ductal epithelia. CONCLUSION: UT-A shows comparable expression patterns to UT-B (previous study) at the ocular surface and in the lacrimal glands, as determined by immunohistochemistry. The presence of both urea transporters in the lacrimal functional unit suggests that they are essential for the normal function of the lacrimal system and the integrity of the tear film. Potential alterations in urea transporter expression might be associated with the significant reduction of urea found in the tear fluid of dry eye patients. They may thus play an important role in the pathogenesis of dry eye disease.

Urea transporter-B expression on the ocular surface and in the lacrimal glands.

OBJECTIVE: Urea is a component of tear fluid showing a significantly decreased concentration in dry eye disease. The urea content of tear fluid may depend on urea transporters. The purpose of this study was to examine the expression of urea transporter B (UT-B) at the ocular surface and in the lacrimal glands. METHODS: UT-B protein and mRNA expression was investigated in human, porcine, and murine samples. Immunohistochemical staining for UT-B was performed on paraffin sections of human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). Reverse transcriptase polymerase chain reaction was conducted to detect UT-B mRNA in human and murine cornea, conjunctiva, Meibomian gland, and lacrimal gland (n = 5 each). RESULTS: UT-B protein expression was comparable in all three species. It was found in the corneal epithelium and endothelium, in the conjunctival epithelium, in the end pieces and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The glands of Zeis and the Meibomian glands showed intense UT-B signals in the basal layers of the alveolar epithelia and in the cells of the ductal epithelia. UT-B mRNA was detected in all samples analyzed. CONCLUSION: UT-B is expressed by the cells and tissues of the ocular surface and in the lacrimal glands. Potential changes in urea transporter expression might have implications for the pathogenesis of dry eye disease. Since comparable results were obtained for all species investigated, the presented findings may open the door for DED-relevant experimentation on porcine and murine model systems.

Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family.

Multidrug-resistant bacteria pose serious problems in hospital-acquired infections (HAIs). Most antibiotic resistance genes are acquired via conjugative gene transfer, mediated by type 4 secretion systems (T4SS). Although most multidrug-resistant bacteria responsible for HAIs are of Gram-positive origin, with enterococci being major contributors, mostly Gram-negative T4SSs have been characterized. Here, we describe the structure and organization of PrgL, a core protein of the T4SS channel, encoded by the pCF10 plasmid from Enterococcus faecalis. The structure of PrgL displays similarity to VirB8 proteins of Gram-negative T4SSs. In vitro experiments show that the soluble domain alone is enough to drive both dimerization and dodecamerization, with a dimerization interface that differs from all other known VirB8-like proteins. In vivo experiments verify the importance of PrgL dimerization. Our findings provide insight into the molecular building blocks of Gram-positive T4SS, highlighting similarities but also unique features in PrgL compared to other VirB8-like proteins.

Polymerization and shrinkage stress formation of experimental resin composites doped with nano- vs. micron-sized bioactive glasses.

This study investigated the effect of adding bioactive glass 45S5 (BG) of different particle sizes to dental composite on resin polymerization and shrinkage stress formation. Commercial flowable composite was mixed with either 15 wt% BG fillers (nanometric, micrometric, or hybrid BG) or inert barium glass. Real-time linear polymerization shrinkage and shrinkage stress were recorded, and the degree of conversion was measured using FTIR spectroscopy. The commercial (unmodified) composite developed significantly higher linear shrinkage and shrinkage stress than the groups with 15 wt% added inert or BG fillers. After adding inert barium glass, the composite showed significantly higher linear shrinkage than when micrometric BG was added. The addition of bioactive or inert glass fillers did not affect the degree of conversion. Shrinkage stress can be reduced by adding inert or bioactive fillers (nano- and/or microparticulate BG) without affecting monomer conversion.

Polymerization shrinkage and shrinkage force kinetics of high- and low-viscosity dimethacrylate- and ormocer-based bulk-fill resin composites.

The aim of the present study was to investigate polymerization shrinkage, shrinkage force development, and degree of monomer conversion of high- and low-viscosity dimethacrylate- and ormocer-based bulk-fill resin composites. Two flowable bulk-fill composites (SDR, x-tra base), two high-viscosity bulk-fill composites (Bulk Ormocer, SonicFill), and two conventional composite materials (Esthet X flow, Esthet X HD) were photoactivated for 20 s at 1275 mW/cm2. Linear polymerization shrinkage and shrinkage force were recorded in real time using custom-made devices, and the force rate and time to achieve maximum force rate were determined. Degree of conversion was measured using Fourier-transform infrared spectroscopy. Data were analyzed with one-way ANOVA and Tukey's HSD post-hoc test, and bivariate correlations were computed (α = 0.05). The category of high-viscosity bulk-fill resin composites showed the significantly lowest polymerization shrinkage and force development. Within the tested flowable composite materials, SDR bulk-fill generated the significantly lowest shrinkage forces during polymerization and attained the significantly highest degree of conversion. Strong positive correlations were revealed between shrinkage force and both linear polymerization shrinkage (r = 0.902) and maximum force rate (r = 0.701). Linear shrinkage and shrinkage force both showed a negative correlation with filler volume content (r = - 0.832 and r = - 0.704, respectively). Bulk-fill resin composites develop lower shrinkage forces than their conventional flowable and high-viscosity counterparts, respectively, which supports their use for restoring high C-factor posterior cavities. Overall, bulk-fill composites with high filler amount and low force rate showed the most favorable shrinkage force characteristics.

EssC is a specificity determinant for Staphylococcus aureus type VII secretion.

The type VII protein secretion system (T7SS) is found in actinobacteria and firmicutes, and plays important roles in virulence and interbacterial competition. A membrane-bound ATPase protein, EssC in Staphylococcus aureus, lies at the heart of the secretion machinery. The EssC protein from S. aureus strains can be grouped into four variants (EssC1-EssC4) that display sequence variability in the C-terminal region. Here we show that the EssC2, EssC3 and EssC4 variants can be produced in a strain deleted for essC1, and that they are able to mediate secretion of EsxA, an essential component of the secretion apparatus. They are, however, unable to support secretion of the substrate protein EsxC, which is only encoded in essC1-specific strains. This finding indicates that EssC is a specificity determinant for T7 protein secretion. Our results support a model in which the C-terminal domain of EssC interacts with substrate proteins, whereas EsxA interacts elsewhere.

Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus.

The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Structure of the TatC core of the twin-arginine protein transport system.

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2 ) of Escherichia coli.

Under phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 µM and 5.1 ± 0.3 µM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable.

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.