Prognostic impact of perihepatic lymph node metastases in patients with resectable colorectal liver metastases.

BACKGROUND: Although perihepatic lymph node metastases (PLNMs) are known to be a poor prognosticator for patients with colorectal liver metastases (CRLMs), optimal management remains unclear. This study aimed to determine the risk factors for PLNMs, and the survival impact of their number and location in patients with resectable CRLMs. METHODS: Data on patients with CRLM who underwent hepatectomy during 2003-2014 were analysed retrospectively. Recurrence-free (RFS) and overall (OS) survival were calculated according to presence, number and location of PLNMs. Risk factors for PLNM were evaluated by logistic regression analysis. RESULTS: Of 1485 patients, 174 underwent lymphadenectomy, and 54 (31·0 per cent) had PLNM. Ten patients (5·7 per cent) who had lymphadenectomy and 176 (13·4 per cent) who did not underwent repeat hepatectomy. Survival of patients with PLNM was significantly poorer than that of patients without (RFS: 5·3 versus 13·8 months, P < 0·001; OS: 20·5 versus 71·3 months; P < 0·001). Median OS was significantly better in patients with para-aortic versus hepatoduodenal ligament PLNMs (58·2 versus 15·5 months; P = 0·011). Patients with three or more PLNMs had significantly worse median OS than those with one or two (16·3 versus 25·4 months; P = 0·039). The presence of primary tumour lymph node metastases (odds ratio 2·35; P = 0·037) and intrahepatic recurrence requiring repeat hepatectomy (odds ratio 5·61; P = 0·012) were significant risk factors for PLNM on multivariable analysis. CONCLUSION: Patients undergoing repeat hepatectomy and those with primary tumour lymph node metastases are at significant risk of PLNM. Although PLNM is a poor prognostic factor independent of perihepatic lymph node station, patients with one or two PLNMs have a more favourable outcome than those with more PLNMs.

Differential diagnosis of liver tumours using intraoperative real-time tissue elastography.

BACKGROUND: Real-time tissue elastography is an innovative tool that informs the surgeon about tissue elasticity by applying the principle of ultrasonography. The aim of this study was to investigate the accuracy of intraoperative real-time tissue elastography (IORTE) for the detection and characterization of liver tumours. METHODS: Between October 2010 and November 2011, IORTE was performed for liver lesions after the completion of routine B-mode intraoperative ultrasonography (IOUS). The elasticity images of all tumours, except those of cysts, were classified into six categories according to modified criteria (types 1-6), according to the degree of strain contrast with the surrounding liver. The concordance of IORTE with pathological examination of the tumour, B-mode IOUS and clinical diagnosis after follow-up was assessed. RESULTS: Images were obtained from 92 hepatocellular carcinomas (HCCs), 92 adenocarcinomas, 19 other malignant tumours and 18 benign tumours in 158 patients. Using a minilinear probe, 73 of 88 HCCs were classified as having a 'HCC pattern' (type 3, 4 or 5), resulting in a sensitivity of 83·0 per cent, a specificity of 67·2 per cent and an accuracy of 73·7 per cent. Some 66 of 90 adenocarcinomas were classified as 'adenocarcinoma pattern' (type 6), resulting in a sensitivity of 73·3 per cent, specificity of 95·1 per cent and accuracy of 85·9 per cent. IORTE detected seven new lesions (8 per cent). CONCLUSION: IORTE is useful for the detection and characterization of liver tumours.

Glycogen debranching enzyme in bovine brain.

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.

Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan.

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS 14+1G-->T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29-1G-->C), a 1-bp deletion (587delC), a 2-bp deletion (4216-4217delAG), a 1-bp insertion (2072-2073insA), and a 3-bp insertion (4735-4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G-->T, and 4216-4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan.

Structural analysis of N-linked sugar chains of human blood clotting factor IX.

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.

Processing pathway deduced from the structures of N-glycans in Carica papaya.

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B.

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.

A new sugar chain of the proteinase inhibitor from latex of Carica papaya.

The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues.

Analysis of oligosaccharide structures from the reducing end terminal by combining partial acid hydrolysis and a two-dimensional sugar map.

A new sensitive and convenient method for the structural analysis of oligosaccharides was developed, in which partial acid hydrolysis was combined with two-dimensional sugar mapping of pyridylamino (PA)-oligosaccharides. A PA-oligosaccharide was partially hydrolyzed, the acid hydrolysis conditions being optimized so as to obtain various PA-oligosaccharide fragments with high yields from different types of PA-oligosaccharides. The acid hydrolyzates were then fractionated every 1 glucose unit by size-fractionation HPLC, and each fraction was analyzed by reversed-phase HPLC. The structure of each PA-oligosaccharide fragment was identified on a two-dimensional sugar map prepared with standard PA-sugar chains, after which the original PA-oligosaccharide was reconstructed from the reducing end terminal based on the obtained structures of the PA-oligosaccharide fragments. The method was successfully applied to resolving the structure of an N-linked sugar chain.

An assay method for glycogen debranching enzyme using new fluorogenic substrates and its application to detection of the enzyme in mouse brain.

An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives. Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1- 4Glcalpha1-4Glc-PA (FD6) and Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1- 4Glcalpha1-4Glcalpha1-4G lc-PA (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.

Introduction of a new scale into reversed-phase high-performance liquid chromatography of pyridylamino sugar chains for structural assignment.

Addition of a monosaccharide residue to a pyridylaminated (PA)-N-linked sugar chain results in an increment or decrement in the elution time on reversed-phase HPLC, the difference being defined as the partial elution time of the residue. Based on this principle, an empirical rule was deduced, which states that the elution time is roughly equal to the sum of the partial elution times of the component sugar residues [Anal. Biochem., 167 (1987) 321-326]. In practice, however, some partial elution times obtained from different pairs of mother PA-sugar chains are found to deviate, and consequently the closeness of the elution times of PA-sugar chains calculated therefrom to the observed times is reduced in such cases. To improve the reliability of the additivity rule and to generalize elution times so that they are less dependent on minor alterations in the elution conditions, we have devised a new scale for elution time, which we have named a reversed-phase scale. The elution times on the reversed-phase scale (the R values) are read from a conversion curve constructed using the elution times of eight selected standard PA-sugar chains. The partial elution times on the reversed-phase scale of 22 monosaccharide residues were calculated from the R values of 93 PA-sugar chains. The R values obtained by summing the partial elution times of all the component monosaccharide residues became much closer to the R values obtained from the reversed-phase scale, compared to the results obtained using the previous method. In addition, the R values were less influenced by minor change in the elution conditions. These features of the new scale allow more accurate structural assignment of sugar chains.

Presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-D-xylosyltransferase involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins in the human hepatoma cell line HepG2.

HepG2 cells were examined for the presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-xylosyltransferase activity by using 2-[(2-pyridyl)amino]ethyl beta-D-glucopyranoside (Glc beta-R) as a fluorogenic acceptor. UDP-D-xylose and the acceptor were incubated with the HepG2 microsomal fraction, and the enzymatic reaction mixture was analyzed by HPLC. Structural analysis of the fluorogenic product by alpha-D-xylosidase digestion, FAB-MS, and Smith degradation revealed that it was Xyl alpha 1-3Glc beta-R, thus demonstrating the existence of beta-D-glucoside alpha-1,3-xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP-D-xylose: alpha-D-xylodie alpha-1,3-xylosyltransferase of the HepG2 microsomal fraction producing Xyl alpha 1-3Xyl alpha 1-3Glc-R' [R', (2-pyridyl)amino-] from UDP-D-xylose and Xyl alpha 1-3Glc-R' [Minamida, S., Aoki, K., Natsuka, S., Omichi, K., Fukase, K., Kusumoto, S. & Hase, S. (1996) J. Biochem. (Tokyo) 120, 1002-1006]. The newly detected enzyme appears to be involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins such as human blood coagulation factors VII and IX.

Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.

We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

Preparation of neoglycolipids with a definite sugar chain moiety from glycoproteins.

A method for preparing neoglycolipids possessing a sugar chain with a definite structure using two N-linked sugar chains as model compounds was developed. Pyridylaminated sugar chains obtained from glycoproteins were purified by reversed-phase HPLC and a pyridylaminated sugar chain thus obtained was converted to a 1-amino-1-deoxy derivative. The product was conjugated by reductive amination with phosphatidylglycolaldehyde prepared by periodate oxidation of dipalmitoyl phosphatidylglycerol. Neoglycolipids formed were purified by gel filtration from the reaction mixture. The structures of the purified neoglycolipids were confirmed by composition analysis for sugar, fatty acid, phosphorus, and 2-acetamido-1,2-dideoxy-1-[(hydroxyethyl)amino]-D-glucitol, the linkage region of the sugar chain, and lipid moieties. The method provides a convenient means of preparing neoglycolipids having a definite structure.

Classification of sugar chains of glycoproteins by analyzing reducing end oligosaccharides obtained by partial acid hydrolysis.

Sugar chain types were classified on the basis of reducing end di- and trisaccharide structures. Sugar chains liberated from glycoproteins by the hydrazinolysis-N-acetylation method were pyridylaminated, and pyridylamino (PA-) sugar chains were purified by HPLC. The PA-sugar chains thus purified were partially hydrolyzed with 1 M trifluoroacetic acid. The acid hydrolysis conditions were investigated with the object of obtaining PA-di- and trisaccharides with high yields for different types of PA-sugar chains. The acid hydrolysates were separated by size-fractionation HPLC into PA-mono-, PA-di-, and PA-trisaccharides, and each fraction was analyzed by reversed-phase HPLC. The structures were then identified by comparing the HPLC elution positions with those of authentic PA-oligosaccharides derived from N-linked sugar chains and 12 types of O-linked sugar chains.

Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

A new disaccharide Fuc alpha 1-2Man found in human urine.

Human urine collected from healthy individuals was ultrafiltered and the filtrate was gel-filtered. The fraction including disaccharides was pyridylaminated to convert the reducing sugars to fluorescent pyridylamino (PA)-derivatives. A PA-disaccharide consisting of Fuc and Man was purified by gel filtration, reversed-phase HPLC, and size fractionation HPLC. Structural analysis revealed that the disaccharide was Fuc alpha 1-2Man-PA. The disaccharide is considered to be a metabolite of unknown glycoconjugates.

Linkage position analysis of pyridylamino-disaccharides by HPLC of fluorogenic Smith degradation products.

Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0 degrees C for 15 min or at 4 degrees C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4 degrees C for 40 h were purified by reversed phase HPLC, and the fraction collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0 degrees C for 15 min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney alpha-L-fucosidase.

Identification of trisaccharide Xyl alpha 1-->3Xyl alpha 1-->3Glc in human urine.

Oligosaccharides in human urine were converted to pyridylamino (PA)-derivatives. From the PA-oligosaccharides, a saccharide that was chromatographically identical with a synthetic standard, Xyl-alpha 1-->3Xyl alpha 1-->3Glc-PA, was isolated by gel filtration and HPLC. Structure analysis showed that the saccharide was Xyl alpha 1-->3Xyl-alpha 1-->3Glc-PA. It is likely that Xyl alpha 1-->3Xyl alpha 1-->3Glc originated from such glycoconjugates as blood coagulation factors VII and IX, and protein Z.