Separation of D-amino acid-containing peptide phenylseptin using 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns.

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (-), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (-) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.

Self-assembly of tripeptides into γ-turn nanostructures.

Self-assembling phenylalanine-based peptides have garnered interest owing to their potential for creating new functional materials. Here, we designed four diastereomers, l-Phe-l-Phe-l-Phe (FFF), d-Phe-l-Phe-l-Phe (fFF), l-Phe-d-Phe-l-Phe (FfF) and l-Phe-l-Phe-d-Phe (FFf), to analyze the effect of the d-isomer on the self-assembly. Using SEM, TG, VCD, and solid-state NMR measurements, we found that only FFf forms a γ-turn conformation and self-assembles into a nanoplate with higher thermal stability. The supramolecular structure of FFf consists of intra- and intermolecular hydrogen bonds and π-π stackings. From our results, we have discovered that FFf forms a new type of self-assembling γ-turn conformation, clarifying the structural role of a d-amino acid residue in supramolecular formation.

Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5 × 10-7 to 5 × 10-3 M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5 × 10-7 to 5 × 10-4M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5 × 10-7 and 5 × 10-3 M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Effect of CO2 laser irradiation on hormesis induction in cultured oral cells.

BACKGROUND: Many drugs (including toxicants) and radiation therapy have been reported to exert bi-phasic hormetic effects on cultured cells, but only when both the concentration and treatment time were optimal. Most previous studies have been carried out with multiple laser modalities other than CO(2) laser, and there has been no comparison of the hormetic response between normal and tumor cells. We investigated here whether CO(2) laser treatment induces hormesis in human gingival fibroblast (HGF) and oral squamous cell carcinoma (HSC-2) cells. MATERIALS AND METHODS: Cells were cultured for 24, 48 or 72 hours after exposure to various irradiation powers, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. RESULTS: CO(2) laser irradiation stimulated cell growth at low and inhibited it at high irradiation power. Among three dispatch modes, super pulse (SP)2 most effectively induced growth stimulation in HGF, at an irradiation dose slightly lower than that which induced cytotoxicity. Higher irradiation doses were comparably cytotoxic against both normal (HGF) and tumor (HSC-2) cells, reaching a plateau of cytotoxicity within 24 hours. CONCLUSION: Since both the range and magnitude of hormetic response in HGF cells were very narrow and small, it is crucial to establish the optimal conditions for hormesis induction for clinical application in dentistry.

Surgical management of retinal detachment associated with myopic macular hole: anatomic and functional status of the macula.

PURPOSE: To evaluate the postoperative status of the macula after vitreous surgery with internal limiting membrane removal for macular hole related retinal detachment in patients with severe myopia. DESIGN: Interventional case series. METHODS: We prospectively examined 10 eyes with retinal detachment associated with a myopic macular hole from 10 consecutive patients, and performed pars plana vitrectomy with internal limiting membrane peeling. Macular buckling was performed in one eye during the initial treatment and in three eyes during subsequent operations. The main outcome measures were the anatomic reattachment rate and the postoperative status of the macular hole. We examined the macular area pre- and postoperatively with slit-lamp biomicroscopy and with a scanning laser ophthalmoscope. Cross-sectional imaging of the macular area was conducted with optical coherence tomography. RESULTS: Successful retinal reattachment was achieved in seven eyes (70%) after the initial surgery and in three eyes (30%) after additional procedures. Visual acuity remained unchanged in two eyes (20%), and improved by two or more logarithmic units of minimum angle of resolution (logMAR) measurement in eight eyes (80%). The macular hole was anatomically closed in only one eye (10%). Postoperative enlargement of the macular hole was observed in seven eyes. CONCLUSIONS: In highly myopic eyes with macular hole related retinal detachment, closure of the macular hole is difficult to attain despite the complete relief of tangential traction by internal limiting membrane peeling. Results indicate the presence of a possible imbalance between the retina and the choroid-sclera complex associated with axial elongation and posterior staphyloma in highly myopic eyes.

Severe hypotony after macular translocation surgery with 360-degree retinotomy.

PURPOSE: To report a case of severe hypotony after macular translocation with 360-degree retinotomy. DESIGN: Interventional case report. METHODS: A 50-year-old woman with myopic neovascular maculopathy underwent macular translocation with 360-degree retinotomy in her left eye. RESULTS: After the second procedure of silicone oil removal, severe hypotony developed. No clear sign of leakage was found. Pure perfluoropropane gas tamponade was then performed, which resulted in temporal resolution of severe hypotony, but the hypotony recurred as the gas bubble was absorbed. Ten weeks after the second surgery, the hypotonous eye was refilled with silicone oil. No apparent cyclitic membrane was observed intraoperatively. After this procedure, the choroidal and retinal folds regressed; intraocular pressure has been between 5 and 7 mm Hg for more than 4 months thereafter. CONCLUSION: Severe hypotony can occur as a complication of otherwise uneventful macular translocation with 360-degree retinotomy.