Case 1: A 66-year-old man was transported to the emergency room with malaise and anorexia. Emphysematous cholecystitis was suspected based on abdominal CT, and emergency percutaneous transhepatic ...gallbladder drainage (PTGBD) was thus performed. The patient was discharged on hospital day 11 with the PTGBD tube clamped. However, the clamp had to be released because of relapse of cholecystitis, and open cholecystectomy was performed on day 37. Acute cholecystitis was confirmed by pathological findings.Case 2: A 60-year-old man was referred to our clinic with upper abdominal pain. Emergency PTGBD was performed for emphysematous cholecystitis identified by abdominal CT. The PTGBD tube was clamped on hospital day 11, but cholecystitis relapsed. Thus, the patient underwent laparoscopic cholecystectomy on day 19. Acute necrotic cholecystitis was identified based on pathological findings.The bile culture detected Clostridium perfringens in these two cases. Both patients had a good postoperative course and were discharged 15 days and 8 days, respectively, after the cholecystectomy.Emphysematous cholecystitis, which is a variant of acute cholecystitis according to the guidelines, frequently leads to a serious condition. PTGBD may also be useful in high-risk patients with this form of cholecystitis, which is classified as a severe complication.
As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4‐methylumbelliferone (MU), ...although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane‐rich fraction as an enzyme source in a cell‐free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose‐dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU‐treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.
After partial hepatectomy, the liver is capable of complete restoration to its normal size. The extracellular matrix, which surrounds the cells, plays important roles in this regeneration. ...Glycosaminoglycans (GAGs), which are components of the extracellular matrix, interact with several other matrix components and growth factors, and are involved in hepatocyte growth. In this study, the content of heparan sulfate, a major GAG in rat liver, reached a minimum at 12 hours after partial hepatectomy. Galactosyltransferase-I activity, related to the synthesis of GAGs, and sialyltransferase activity, related to the synthesis of glycoconjugates, reached a minimum at 6 hours. The serum and liver contents of hyaluronic acid reached a maximum at 1 day and returned gradually to their preoperative levels. These results suggest that polysaccharide synthesis was decreased in the Golgi apparatus of hepatocytes at the beginning of regeneration, and that hyaluronic acid degradation decreased in the lysosomes of hepatocytes. The ability to synthesize polysaccharides recovered ahead of the ability to degrade hyaluronic acid. The changes in these GAGs with time in the early regeneration period might play an important role in organ regeneration.
When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main ...even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.
Human colorectal cancer cells were incubated with medium containing 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU). The cells synthesized Xyl-MU-derivatives which were detected in the culture medium ...by gel-filtration high-performance liquid chromatography. These included a Xyl-MU-induced glycosaminoglycan and its biosynthetic intermediates, Galbeta1-4Xylbeta1-MU and Galbeta1-3Galbeta1-4Xylbeta1-MU, and other Xyl-MU-induced oligosaccharides, not related to Xyl-MU-induced glycosaminoglycan, were also synthesized. One of these oligosaccharides, sulfate-O-3GlcAbeta1-4Xylbeta1-MU, reacted with HNK-1, a mouse monoclonal antibody raised against human natural killer cells. Human neural cells and skin fibroblasts have also been reported to synthesize HNK-1-reactive sugar chains. Since HNK-1-reactive sugar chains are known to be involved in cell adhesion in the nervous system, the present results suggest that epithelium-derived colorectal cancer cells might also be able to utilize them in cell adhesion.
Prognostic factors for breast cancer include axillary lymph node status, tumor size, histology, nuclear grade, presence of estrogen and progesterone receptors, HER2/neu status, and mean microvessel ...density (MVD). In this study, we evaluated the usefulness of a new marker, D2-40, by investigating lymph vascular invasion of the tumor immunohistochemically in 132 patients with breast cancer and compared it with those of well-known prognostic indicators. Positive immunostaining of lymphatic endothelium with D2-40 outlining tumor emboli in the lumen of lymphatics was defined as D2-LVI, and lymphatic invasion following conventional hematoxylin and eosin staining was defined as HE-LVI. Significant correlation was observed between HE-LVI and D2-LVI (p<0.001), between lymph node status and HE-LVI (p=0.005), and between recurrent status and D2-LVI (p=0.008) by univariate analysis. Based on multivariate analysis, lymph node status (p<0.001, OR=6.993), tumor size (p=0.005, OR=5.504), D2-LVI (p=0.006, OR=4.740), and MVD (p=0.002, OR=4.484) were independent prognostic factors of disease recurrence. A significant difference in disease-free survival was also found between patients with and without D2-LVI (p=0.0067), but not with or without HE-LVI. Even in node-positive cases, D2-LVI had prognostic meaning. D2-LVI may play a crucial role for predicting recurrence of breast cancers much more than expected. Our data identifying D2-LVI expression in tumors of patients with a poor disease-free survival prognosis provides an easier and more accurate prognostic method than identifying HE-LVI.
The effect of proteoglycan (PG) on colitis was examined in animal experiments using mice. The PG used was extracted from nasal cartilage of salmon head with 4% acetic acid and prepared by ...precipitation with ethanol followed by dialysis. The PG contained about 7% protein, and had a molecular mass of 344 kDa on SDS/PAGE. The glycosaminoglycan (GAG) sugar chains of the PG were composed of hexosamine, uronic acid and sulfate at a molar ratio of 1.0:1.0:0.7. The mice were divided into a control group and an administration group. The control group was given free access to drinking water containing dextran sulfate sodium salt (DSS) to induce colitis. On the other hand, the administration group was given free access to drinking water containing DSS and PG. Then, the time course of survival rates in both groups were measured. In the administration group, the survival rate increased significantly in comparison with that of the control group. The difference in the survival rates indicated that the onset of mouse colitis induced by DSS was inhibited by administration of the PG.
Oligosaccharides from hyaluronic acid and chondroitin 6-sulfate were prepared by digestion with testicular hyaluronidase and separated according to their degree of polymerization by gel-permeation ...chromatography. These materials were successively analyzed by negative-mode ion-spray mass spectrometry with an atmospheric-pressure ion source. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of molecular species carrying multiple charges. Using two adjacent multiply charged molecular ions, the exact molecular weights up to the tetradecasaccharide were calculated with a precision of +/- 1 dalton. This type of mass spectrometry was also demonstrated to be feasible for the analysis of mixtures of oligosaccharides, including tetra-, hexa-, octa- and decasaccharides, from hyaluronic acid or chondroitin 6-sulfate without separation. Ion-spray mass spectrometry was thus shown to be applicable to the structural analysis of oligosaccharides from glycosaminoglycans.
COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar ...chain elongation. However, when p-nitrophenyl-β-N-acetylglucosamine (β-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of β-GlcNAc-PNP and 4-methylumbelliferyl-β-N-acetylglucosamine, then the number of living cells was measured in a time course. In β-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from β-GlcNAc-PNP induced the cell death. Activity of β-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.
A pyridylamination method was applied to glycosaminoglycans and the characteristics of the resulting pyridylamino glycosaminoglycans were examined. First, glycosaminoglycan chains, which uniformly ...possess a xylose residue at their reducing termini, were liberated from proteoglycan by successive digestion with protease and endo-β-xylosidase. Then the glycosaminoglycan chains were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride for 15 h according to the method of Hase, S. et al. J. Biochem. 95,197–203 (1984). The pyridylamination reaction caused neither depolymeriza-tion, de-N-acetylation, nor de-N - or de-O-sulfation. The pyridylamino glycosaminoglycan chains had an intact linkage region (GlcA-Gal-Gal-Xyl) between the carbohydrate chain and the peptide core of the proteoglycan. These pyridylamino glycosaminoglycans should be useful as substrates for endo-type glycosidases that act on glycosaminoglycan chains and as markers for studies of glycosaminoglycan metabolism.
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