Crystallization and preliminary X-ray analysis of cellobiose phosphorylase from Cellvibrio gilvus.

A recombinant cellobiose phosphorylase from Cellvibrio gilvus has been prepared and crystallized by the sitting-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.

Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate.

Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases.

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N-acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2-UMB (1).

Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens.

The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity.

Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195.

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE.

Crystallization and preliminary X-ray analysis of xylanase B from Clostridium stercorarium.

Recombinant mature xylanase B from Clostridium stercorarium has been prepared and crystallized by the sitting-drop vapour-diffusion method using 4 mg ml(-1) purified enzyme, 10.3%(w/v) polyethylene glycol 1500, 8.6%(v/v) glycerol and 0.

Successful treatment of primary pulmonary angiosarcoma.

Angiosarcoma in the lung is an uncommon disorder and is usually attributable to metastasis from a primary site. Primary pulmonary angiosarcoma is extremely rare, and the prognosis of affected individuals is dismal, with most patients dying within months of presentation. Indeed, there have been no reported instances of successful treatment of this condition.

A thermostable non-xylanolytic alpha-glucuronidase of Thermotoga maritima MSB8.

A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80 degrees C.

A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195.

Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein.

Enzymatic synthesis of a library of beta-(1-->4) hetero- D-glucose and D-xylose-based oligosaccharides employing cellodextrin phosphorylase.

Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1-->4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.

Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase.

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium.

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct alpha(IV) chains. In this study the expression of these six alpha(IV) chains was demonstrated immunohistochemically. In addition, the alpha2(IV) and alpha5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM.

Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan.

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C.

Percutaneous calcitriol injection therapy (PCIT) for secondary hyperparathyroidism: multicentre trial.

A multicentre trial of percutaneous calcitriol injection therapy (PCIT) was designed to evaluate its clinical usefulness. During a 12-week period, measurement of intact PTH concentration, and other parameters, and ultrasonography were carried out in conjunction with PCIT in 19 haemodialysis patients with secondary hyperparathyroidism and enlarged parathyroid glands (PTGs) that were resistant to vitamin D pulse therapy. Calcijex was injected directly into the PTG three times per week on the patient's non-dialysis days: eight patients received a 2 microg/ml preparation (group A) and 12 received 1 microg/ml (group B).

Guidelines for percutaneous ethanol injection therapy of the parathyroid glands in chronic dialysis patients.

Percutaneous ethanol injection therapy (PEIT) of the parathyroid was originally introduced as an alternative to surgical parathyroidectomy. After the recent elucidation of the pathogenesis of parathyroid hyperplasia in uraemia, 'selective PEIT of the parathyroid glands' was developed, in which enlarged parathyroid glands with nodular hyperplasia are 'selectively' destroyed by ethanol injection, and other glands with diffuse hyperplasia are then managed by medical therapy. The 'Guidelines for percutaneous ethanol injection therapy of the parathyroid glands in chronic dialysis patients' proposed by the Japanese Society for Parathyroid Intervention are presented, including indications, techniques, and post-PEIT management.

Ultrasonographic diagnosis of parathyroid glands and percutaneous ethanol injection therapy.

The first choice for imaging diagnosis of parathyroid gland (PTG) abnormalities is ultrasonography with a high-frequency probe. The patient must be positioned correctly when performing either imaging or percutaneous ethanol injection (PEIT) of the PTG. The enlarged PTGs are examined by ultrasonic tomography using 3D measurements, and it is important to evaluate blood flow; the PTGs are hypervascular in comparison with a nodular lesion of the thyroid.

Kinetic studies on the hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside by the family 18 chitinases ChiA and ChiB from Serratia marcescens.

Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another.

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8.

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C.

General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease.

Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC).

Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase.

The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85 degrees C) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35 degrees C) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.

Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli.

A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P(i) bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P(i).

The role of the N-terminal propeptide of the pro-aminopeptidase processing protease: refolding, processing, and enzyme inhibition.

Pro-aminopeptidase processing protease (PA protease) is an extracellular zinc metalloprotease produced by Aeromonas caviae T-64 and it is classified as M04.016 according to the MEROPS database. The precursor of PA protease consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature PA protease.

Evidence that the putative alpha-glucosidase of Thermotoga maritima MSB8 is a pNP alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase.

The gene (agu) encoding p-nitrophenyl alpha-D-glucuronopyranoside (pNP-GUA) hydrolyzing alpha-glucuronidase of the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified and characterized. The gene previously designated as putative alpha-glucosidase was found to code for a protein that had no alpha-glucosidase activity.

Increased expression of monocyte chemoattractant protein-1 in atherectomy specimens from patients with restenosis after percutaneous transluminal coronary angioplasty.

The plasma concentration of monocyte chemoattractant protein-1 (MCP-1) antigen is higher in patients with restenosis after coronary angioplasty than in those who do not restenose. In this study the MCP-1 expression of coronary atherectomy specimens was investigated by immunohistochemistry. Samples were obtained from 12 patients with restenosis and 15 with de novo lesions by directional coronary atherectomy.

In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64.

PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding.