Three cross leg flaps for lower leg reconstruction of Gustilo type III C open fracture.

A 60 year old male had Gustilo type III C open fracture of the right lower leg. After radical debridement, the large open defect including certain loss of the bone tissue was successfully augmented and covered, by consecutive three cross-leg flaps, which consisted of the free rectus abdominis musculocutaneous flap, the fibula osteocutaneous flap and the conventional sural flap. Although indication for amputation or preservation is decided with multiple factors in each case, a strategic combination of cross-leg flap, free flap, external fixation and vascular delay could increase the potential of preservation of the lower leg with even disastrous Gustilo type III C.

Derivatization with 1-pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry.

Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionization-tandem mass spectrometry (MALDI-MS(n)) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to MS(n) in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method.

Vitamin D3, vitamin K2, and warfarin regulate bone metabolism in human paranasal sinus bones.

Several recent studies have indicated that the paranasal sinus bones undergo pathophysiological changes in patients with chronic sinusitis. We examined the mineralization activity of osteoblasts and the production of osteocalcin and cytokines in cultured human osteoblasts derived from ethmoidal bones treated with vitamin D3, vitamin K2, and warfarin to investigate the metabolic effects of these treatments on paranasal sinus bones. In the bones treated with vitamin D3 plus vitamin K2, osteocalcin production and the ratio of the mineralization of osteoblasts were increased. Warfarin inhibited the promotive effects of vitamin K2 in the presence of vitamin D3. With regard to TGF-beta production, there was quite a difference in response depending on the isoforms. In conclusion, we have demonstrated that these vitamins and warfarin may be useful in improving bone metabolism in paranasal sinus bones, and may additionally improve the pathogenesis of chronic sinusitis.

Beta-galactoside alpha2,6-sialyltransferase I cleavage by BACE1 enhances the sialylation of soluble glycoproteins. A novel regulatory mechanism for alpha2,6-sialylation.

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

pH-dependent formation of membranous cytoplasmic body-like structure of ganglioside G(M1)/bis(monoacylglycero)phosphate mixed membranes.

Membrane structures of the mixtures of ganglioside G(M1) and endosome specific lipid, bis (monoacylglycero) phosphate (BMP, also known as lysobisphosphatidic acid) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering. At pH 8.5-6.5, a G(M1)/BMP (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and G(M1) forms a micelle, the results indicate that both G(M1) and BMP are required to produce closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies in cells derived from patients suffering from G(M1) gangliosidosis. Similar to G(M1) gangliosidosis, cholesterol was trapped in BMP vesicles in G(M1)- and in a low pH-dependent manner. Studies employing different gangliosides and a G(M1) analog suggest the importance of sugar chains and a sialic acid of G(M1) in the pH-dependent structural change of G(M1)/BMP membranes.

Bone-constructing cells from ethmoid bone may have multilineage differentiation potential: preliminary report.

OBJECTIVE: In order to investigate multilineage differentiation in human cultured cells from ethmoid bone, we conducted a morphological study to examine adipogenic and chondrogenic differentiation. MATERIAL AND METHODS: After reaching confluence, cells underwent terminal adipogenic differentiation by treatment with 100 microM indomethacin, 0.5 mM 1-methyl-3-isobutylxanthine, 1 microM dexamethasone (DEX), 10 microg/ml insulin and 0.3% dimethylsulfoxide in a medium supplemented with 10% fetal bovine serum. Chondrogenic differentiation was attempted by centrifuging a pelleted micromass using transforming growth factor-beta3 (TGF-beta3), DEX, ascorbic acid (AA), pyruvate acid, proline, glucose and (ITS)-plus. RESULTS: The cultured cells displayed adipocyte but not chondrogenic lineage under these conditions. Considering the possibility that some differentiation potential may be lost with in vitro culture but maintained using another chondrogenic differentiation medium containing TGF-beta1, it is possible that cultured cells may have multilineage potential, including chondrogenic differentiation ability. CONCLUSIONS: These morphological abilities of human cultured cells may indicate the possibility of the existence of mesenchymal stem cells in sinus bone. If mesenchymal stem cells exist in ethmoid bone, they may play an important role in future research on the regulation mechanisms of human bone tissue.

Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall.

A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion.

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2.

Cell wall materials isolated from Chlorella cells were degraded by the polysaccharide-degrading enzyme vAL-1 encoded by chlorovirus CVK2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses of the degradation products (oligosaccharides) revealed major oligosaccharides contain unsaturated GlcA at the reducing terminus, and a side chain attached at C2 or C3 of GlcA(C4?C5), which mainly consisted of Ara, GlcNAc and Gal. The results indicated that vAL-1 is a novel polysaccharide lyase, cleaving chains of beta- or alpha-1,4-linked GlcAs. The unique structures of Chlorella cell wall were also revealed. Studies on the complicated structures of naturally occurring polysaccharides will be greatly facilitated by using vAL-1 as a tool in structural analysis.