Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.     

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Background

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?

Results

Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2^Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.

Conclusions

Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.

     

The mitochondrial citrate transport protein: evidence for a steric interaction between glutamine 182 and leucine 120 and its relationship to the substrate translocation pathway and identification of other mechanistically essential residues

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in > or =98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.     

Organization of the Mitochondrial Apoptotic BAK Pore: OLIGOMERIZATION OF THE BAK HOMODIMERS*

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.”     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Identification of the substrate binding sites within the yeast mitochondrial citrate transport protein

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K(m) values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K(m) values (i.e. 5 to >300-fold). Each of these mutants displayed V(max) values that were reduced by > or = 98% and resultant catalytic efficiencies that were reduced by > or = 99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.     

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.