Gel Filtration High-performance Liquid Chromatography Research Articles

Primary Structure and Conformation of a Tetrodotoxin-Binding Protein in the Hemolymph of Non-Toxic Shore Crab Hemigrapsus sanguineus

Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding proteins are also found in the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated the TTX-binding protein, HSTBP (Hemigrapsus sanguineus TTX-binding protein), from the hemolymph of H. sanguineus and elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 kDa (subunit-1), 65 kDa (subunit-2), and 26 kDa (subunit-3) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. The open reading frame of the cDNA comprises 5049 base pairs encoding 1683 amino acid residues, and the mature protein contains 1650 amino acid residues from Arg34 to Ser1683. The three subunits are arranged in tandem in the following order: subunit-3 (Arg34-Gln261), subunit-1 (Asp262-Phe1138), and subunit-2 (Val1139-Ser1683). A BLAST homology search showed weak similarity of HSTBP to clotting proteins of crustaceans (29–40%). SMART analysis revealed a von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe1387-Gly1544. We confirmed that the recombinant protein of HSTBP subunit-2 containing the vWF-type (⇒delete hyphen) D domain bound to TTX at a molecular ratio of 1:1.

Profiling of lysophosphatidylethanolamine molecular species in human serum and in silico prediction of the binding site on albumin.

Lysophosphatidylethanolamine (LPE) is a major lysophospholipid produced by phospholipids and binds to human serum albumin (HSA). LPEs may play various roles in vivo depending on the differences in their acyl chains. However, only few reports have been published on the biological functions of LPEs. Hence, we determined the exact relative abundance of the major LPEs in the serum of healthy participants (n= 8) using liquid chromatography-tandem mass spectrometry. Consequently, LPE 18:2 (24.1 ± 5.2%) was found to be the most abundant in serum. To understand the distribution of LPEs, the serum separated via gel-filtration high-performance liquid chromatography was subjected to quantitative measurement. LPEs were more observed in the albumin fraction than the lipoprotein fraction. We also performed a fluorescence displacement assay and an in silico molecular docking experiment using AutoDock to confirm the affinity and binding sites of the LPEs on HSA. The binding affinities of the LPEs for drug sites 1 and 2 on HSA were relatively low, with Ki values of approximately 11 and 3.8μM, respectively. AutoDock analysis revealed the conformation of the LPEs bound to drug sites and the possibility of LPEs binding to other HSA sites. These findings could help to elucidate the biological and pathological functions of LPEs.

Characterization and complete genome analysis of Bacillus velezensis CB6 revealed ATP synthase subunit α against foodborne pathogens.

Given the serious threat of foodborne multidrug-resistant bacteria to animals and humans, finding an effective antibacterial compound has always been an important topic for scientists. Here, from the soil of Changbaishan, we have identified a bacterium that can inhibit the growth of Staphylococcus aureus. Nr genome database analysis and phylogenetic analysis showed that strain CB6 belongs to Bacillus velezensis. We found that the crude extract of strain CB6 has broad-spectrum antibacterial activity against foodborne pathogens. In addition, we showed that the crude extract loses antibacterial activity after treatment with papain. Next, strain CB6 was purified using ammonium sulfate precipitation, a Sephadex G-75 gel filtration column and high-performance liquid chromatography system (HPLC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the antibacterial compound was the protein ATP synthase subunit α (ATP-1), with a molecular weight of 55.397 KDa. Moreover, we reported the complete genome sequence of strain CB6, which is composed of a unique circular 3,963,507bp chromosome with 3749 coding genes and a G + C content of 46.53%. The genome contained 12 gene clusters with antibacterial functions, which constituted over 20.947% of the complete genome. Of note, the amino acid sequence encoding the ATP-1 protein in the strain CB6 genome was identified. In addition to these findings, we speculate that the ATP-1 protein may provide energy for secondary metabolites, which in turn will improve the antibacterial activity of the secondary metabolites. All the above important features make the ATP-1 as a potential candidate for the development of new antibacterial drugs and food preservatives in the future.

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

MON-612 Familial Dysalbuminemic Hyperthyroxinemia Cases in Five Unrelated Japanese Families and the Influences on Free T4 Measurement

Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. In Japanese FDH patients with the HSAR218P mutation, not only one-step but also two-step immunoassays for free T4 (FT4) can yield false-positive results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of TSH, even when multiple assays are used. Patient 1 was a 29-year-old pregnant woman. A radiographic examination and hormone stimulation test were not performed. We suspected that the patient had FDH because of her family history such as abnormal thyroid function and hyperthyroxinemia (Total T4 level was greater than the measurement sensitivity). Genetic analysis using direct sequencing for HSA revealed that she was consistent with FDH. She experienced a normal delivery. Other patients were performed the thyroid test with Chief complaints of infertility or depression and their thyroid function abnormalities were pointed out. Thyroid function tests revealed elevated FT4 and FT3 levels; their TSH level was within the reference range in all patients. To investigate T4 to HSA binding, gel filtration high-performance liquid chromatography was used for the biochemical analyses. Furthermore, FT4 was measured in FDH patients using one-step method LUMIPULSE (Fujirebio) using T3 as an antigen and an anti-T4 antibody as a label. The genomic analysis revealed a heterozygous missense mutation in HSA(R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. Mean FT4 level measured with LUMIPULSE was 1.28 ng/dL within the reference value range. In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis. When thyroid function abnormality without clinical findings is observed, it is considered preferable to understand the antigen and antibody to be used and to select measurement method.

Seven Familial Dysalbuminemic Hyperthyroxinemia Cases in Three Unrelated Japanese Families and High-Performance Liquid Chromatography Analysis of the Thyroxine Binding Profile

Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. One- and 2-step immunoassays of serum samples from FDH patients (e.g., Japanese patients) with the HSA R218P mutation can yield false-positive free thyroxine (FT4) results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of thyroid-stimulating hormone (TSH) (e.g., syndrome of resistance to thyroid hormone, TSH-producing pituitary adenoma), even when multiple assays are used. To investigate T4 to HSA binding, we examined serum samples from 7 patients from 3 Japanese families with FDH. Clinically, abnormal thyroid function tests were noted in pregnant Patient 1. Patients 2 and 3 had histories of inappropriate treatment with antithyroid drugs and surgery. All patients and affected family members were diagnosed with FDH using direct sequencing analysis. Gel filtration high-performance liquid chromatography was used for the biochemical analyses. The genomic analysis revealed a heterozygous missense mutation in HSA (R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. The results for the albumin effluent from healthy volunteer and TSHoma patient sera showed no corresponding TT4 peak. In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis. F = free; FDH = familial dysalbuminemic hyperthyroxinemia; HPLC = high-performance liquid chromatography; HSA = human serum albumin; PCR = polymerase chain reaction; SITSH = syndrome of inappropriate secretion of TSH; T = total; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone; WT = wild-type.

Analysis of the causes of discrepancies in troponin I concentrations as measured by ARCHITECT High-Sensitive Troponin I ST and STACIA CLEIA cTnI.

Background Recently developed reagents for the highly sensitive measurement of cardiac troponin I are useful for early diagnosis of acute coronary syndrome. However, differences in measured values between these new reagents and previously used reagents have not been well studied. In this study, we aimed to compare the values between ARCHITECT High-Sensitive Troponin I ST (newly developed reagents), ARCHITECT Troponin I ST and STACIA CLEIA cardiac troponin I (two previously developed reagent kits). Methods Gel filtration high-performance liquid chromatography was used to analyse the causes of differences in measured values. Results The measured values differed between ARCHITECT High-Sensitive Troponin I ST and STACIA CLEIA cardiac troponin I reagents (r = 0.82). Cross-reactivity tests using plasma with added skeletal-muscle troponin I resulted in higher reactivity (2.17-3.03%) for the STACIA CLEIA cardiac troponin I reagents compared with that for the ARCHITECT High-Sensitive Troponin I ST reagents (less than 0.014%). In addition, analysis of three representative samples using gel filtration high-performance liquid chromatography revealed reagent-specific differences in the reactivity against each cardiac troponin I complex; this could explain the differences in values observed for some of the samples. Conclusion The newly developed ARCHITECT High-Sensitive Troponin I ST reagents were not affected by the presence of skeletal-muscle troponin I in the blood and may be useful for routine examinations.

Detection of cheese whey and caseinomacropeptide in fermented milk beverages using high performance liquid chromatography

Cheese whey level and caseinomacropeptide (CMP) index of fermented milk beverages added with four levels of cheese whey (0, 10, 20, and 40%) and stored at 8-10oC for 0, 7, 14 and 21 days were determined by high performance liquid chromatography-gel filtration (HPLC-GF). Additionally, the interference of the starter culture and the storage time on the detection of cheese whey and CMP were investigated. Refrigerated storage up to 21 days did not affect (P>0.05) cheese whey and CMP amounts in milk (0% of cheese whey) and in fermented milk beverages added with 10 and 20% of cheese whey (P>0.05). However, cheese whey and CMP amounts were higher than expected (P<0.05) in fermented milk beverages added with 40% of cheese whey and stored for 21 days.

Conformational changes in human Hsp70 induced by high hydrostatic pressure produce oligomers with ATPase activity but without chaperone activity.

We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.

Coupled affinity and sizing chromatography: A novel in-process analytical tool to measure titer and trend Fc-protein aggregation

The expanding use of monoclonal antibodies in the biopharmaceuticals industry has brought the need for new analytical tools. We have developed a coupled affinity and gel-filtration high-performance liquid chromatography method to simultaneously analyze titer and quality of monoclonal antibodies. Before this assay, available analytical methods for protein aggregation required highly purified proteins. This assay can qualitatively describe a protein from a clarified cell culture solution by trending protein aggregation over time while measuring protein titer. It can be used to assess proteins in both early- and late-stage culture due to its dynamic range and sensitivity. This assay is a sensitive technique that overcomes the time limitations of previous approaches. It provides an essential tool to accomplish process optimization.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

다양한 HPLC Column에서의 IgY(Immunoglobulin Yolk) 분리특성

동물 혈청 중의 IgG (Immunoglobulin G)에 해당되는 난황에 포함된 면역 단백질 IgY (Immunoglobulin Yolk)는 식품 단백질로 장내 면역 물질로 중요하다. IgY를 정제하기 위해 신선란의 노른자에 카리지난이나 아라빅검을 전처리 물질로 사용하였다. 전처리 후 FPLC (Fast Protein Liquid chromatography)의 DEAE (Diethylaminoethyl) Sepharose 칼럼에서 이온교환법에 의해 불순물을 제거하여 IgY를 얻고, GF HPLC (Gel Filtration High Performance Liquid Chromatography)로 IgY의 분자량을 측정하고 표준 IgY와 비교하여 IgY 단백질을 동정하였다. GF HPLC에서 IgY의 다양성을 발견하였고 IgY 단백질 군의 다양성을 IE HPLC (Ion Exchange High Performance Liquid Chromatography)에서 AX, CX, SCX 칼럼을 사용하여 pH, NaCl 농도를 바꾸어 조사하였다. AX를 사용하여 0.5M NaCl, pH=8 조건에서 3개의 IgY 피크를 분리하였고, SCX를 이용했을 때 0.5M NaCl, pH=5 조건에서도 3개의 IgY 피크를 분리할 수 있었다. IgY (Immunoglobulin Yolk) in egg yolk corresponds to IgG (Immunoglobulin G) in animal serum and plays an important role as immunological proteins in intestines. Carrageenan and Arabic gum were used as pretreatment agents to purify IgY from fresh egg yolk. DEAE (Diethylaminoethyl) Sepharose column in FPLC (Fast Protein Liquid chromatography) was an ion exchange tool to remove contaminants as well as to elute IgY from the column. GF HPLC (Gel Filtration High Performance Liquid Chromatography) enables to measure the molecular weights of IgY and to identify the purified IgY by comparing the molecular weight of standard IgY with the purified one. IgY is a heterogeneous group of different molecular weight and ionic properties, which was investigated with various IE HPLC (Ion Exchange High Performance Liquid Chromatography) columns such as AX, CX and SCX. Three peaks of IgY were separated in the AX column under the conditions of 0.5 M NaCl and pH=8. The SCX column also gave the three peaks of IgY at 0.5 M NaCl and pH=5.

Determination of the Oligomer Size of Amyloidogenic Protein β-Amyloid(1–40) by Single-Molecule Spectroscopy

Amyloid diseases are traditionally characterized by the appearance of inter- and intracellular fibrillar protein deposits, termed amyloid. Historically, these deposits have been thought to be the etiology of the disease. However, recent evidence suggests that small oligomers of the amyloidogenic protein/peptide are the origin of neurotoxicity. Although the importance of identifying the toxic oligomeric species is widely recognized, such identification is challenging because these oligomers are metastable, occur at low concentration, and are characterized by a high degree of heterogeneity. In this work, a fluorescently labeled β-amyloid(1–40) is used as a model amyloidogenic peptide to test the effectiveness of what we believe is a novel approach based on single-molecule spectroscopy. We find that by directly counting the photobleaching steps in the fluorescence, we can determine the number of subunits in individual β-amyloid(1–40) oligomers, which allows us to easily distinguish among different species in the mixtures. The results are further analyzed by comparison with Monte Carlo simulations to show that the variability seen in the size of photobleaching steps can be explained by assuming random dipole orientations for the chromophores in a given oligomer. In addition, by accounting for bias in the oligomer size distribution due to the need to subtract background noise, we can make the results more quantitative. Although the oligomer size determined in this work is limited to only small species, our single-molecule results are in good quantitative agreement with high-performance liquid chromatography gel filtration data and demonstrate that single-molecule spectroscopy can provide useful insights into the issues of heterogeneity and ultimately cellular toxicity in the study of amyloid diseases.

Effects of BmKNJX11, A Bioactive Polypeptide Purified from Buthus martensi Karsch, on Sodium Channels in Rat Dorsal Root Ganglion Neurons

A long-chain polypeptide BmKNJX11 was purified from the venom of Asian scorpion Buthus martensi Karsch (BmK) by a combination of gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The molecular mass was found to be 7036.85 Da by electrospray ionization mass spectrometry. The first 15 N-terminal amino acid sequence of BmKNJX11 was determined to be GRDAY IADSE NCTYT by Edman degradation. With whole cell recording, BmKNJX11 inhibited tetrodotoxin-sensitive voltage-gated sodium channels (TTX-S VGSC) in freshly isolated rat dorsal root ganglion (DRG) neurons in a concentration- and voltage-dependent manner. At a concentration of 40 μg/ml BmKNJX11 lowered the activation threshold and produced negative shifting of TTX-S sodium current (I Na) activation curve. In addition, BmKNJX11 induced shifting of the steady-state inactivation curve to the left, delayed the recovery of TTX-S I Na from inactivation, and also reduced the fraction of available sodium channels. These results suggested that BmKNJX11 might exert effects on VGSC by binding to a specific site. Considering that TTX-S VGSC expressed in DRG neurons play a critical role in nociceptive transmission, the interaction of BmKNJX11 with TTX-S VGSC might lead to a change in excitability of nociceptive afferent fibers, which may be involved in the observed peripheral pain expression.

Effects of Proteoglycan on Dextran Sulfate Sodium-Induced Experimental Colitis in Rats

Proteoglycans (PG) are macromolecules composed of glycosaminoglycan chains covalently attached to a protein core. In this study, we examined the effects of PG on dextran sulfate sodium (DSS)-induced experimental colitis in rats. First, to examine whether PG may ameliorate acute established DSS colitis, PG was administered orally for 5 days to the model animals. We evaluated the effects of PG on the basis of clinical symptoms, hematological analysis, macroscopic observation, and microscopic examination. We then examined whether PG administered orally to rats was detectable in their colonic lumen. After administration of PG, the colonic contents were collected, and the molecular weight of PG in the sample was analyzed by gel filtration high-performance liquid chromatography. Furthermore, we examined whether orally administered PG affected the concentrations of short-chain fatty acids (SCFAs) in the colonic feces. Orally administered PG ameliorated the clinical symptoms of bloody stools and diarrhea, and attenuated the increase in the white blood cell count in rats with established DSS colitis. Histologically, orally administered PG reduced the degree of mucosal erosion and inflammatory cell infiltration into the erosive area induced by DSS. Orally administered PG was detected in rat colon, although its molecular weight was slightly decreased. Orally administered PG significantly increased the concentration of total SCFAs and n-butyrate in rat colonic feces. This is the first study to indicate that exogenous PG ameliorates experimental colitis, suggesting the potential usefulness of PG for clinical treatment of colitis.

Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase

A previous study [Yoshida, H. et al., J. Biochem., 140, 813-823 (2006)] revealed that a protein of unknown nature was copurified with PDM phosphatase of Fusarium moniliforme. In this study, the identity of this protein was investigated. The results of homology search for the tryptic peptides derived from the purified preparation of PDM phosphatase strongly suggested that it might be serine carboxypeptidase. In fact, carboxypeptidase activity was demonstrated in the preparation and partial separation of carboxypeptidase from PDM phosphatase was achieved by gel filtration high-performance liquid chromatography. Cloning and sequencing of the full-length cDNA encoding the carboxypeptidase was successfully conducted. The cDNA possessed an open reading frame for a protein of 575 amino acid residues with a molecular mass of 64,650 Da, which was highly homologous to certain fungal serine carboxypeptidases. Comparison of the deduced amino acid sequence with the N-terminal sequence of the separated carboxypeptidase revealed that the mature enzyme starts at serine 56 of the precursor and has a molecular mass of 58,487 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA demonstrated that the gene of carboxypeptidase consists of four exons. A limited number of close homologs of F. moniliforme carboxypeptidase were detected among fungi by homology search and their evolutionary relationship was discussed.

Vitreous collagen metabolism before and after vitrectomy

To assess vitreous metabolism by measuring C-propeptide levels of type II procollagen (pCOL-II-C) and hyaluronan levels in the vitreous and in the vitreous fluid after vitrectomy for macular hole. We obtained 1-ml vitreous samples during vitrectomy from 34 patients (34 eyes) with a macular hole (age range 50-77 years, mean 64 years). After vitrectomy, we performed fluid-air exchange in six eyes because of unresolved macular holes and collected 4-ml fluid samples. Gel-filtration high-performance liquid chromatography (HPLC) was used to determine the molecular weight of pCOL-II-C in the samples. The pCOL-II-C level was measured by sandwich enzyme immunoassay and hyaluronan by sandwich binding protein assay. HPLC showed that pCOL-II-C in the vitreous samples corresponded to purified pCOL-II-C from cartilage. The vitreous pCOL-II-C level (4.7+/-0.3 ng/ml) was similar to reported synovial fluid levels. In six eyes that underwent fluid-air exchange, pCOL-II-C in the fluid samples remained at a level similar to that in the vitreous samples, while hyaluronan levels in the fluid samples were significantly lower than in the vitreous samples. The molecular weight and concentrations of pCOL-II-C in the vitreous are similar to those in joint fluid. In patients with a macular hole, type II procollagen may be secreted persistently into the vitreous cavity before and after vitrectomy.

CHARACTERIZATION OF DECORIN OF THE HUMAN YELLOW LIGAMENT

Decorin, a component of the low-molecular-weight proteoglycans (PG) of yellow Iigaments was purified, and the structures of the core protein and the glycosaminoglycan (GAG) chains were analyzed. The crude PG fraction extracted under dissociative conditions was subjected to DEAE-Sephacel ion-exchange chromatography, gel-filtration on Sepharose CL-4B, and Octyl-Sepharose CL-4B chromatography, and decorin was obtained. The amino-terminal amino acid sequence of decorin was found to be identical to those of human bone PG-II and human fetal membrane PG-II. The analyses of the GAG chains using two-dimensional electrophoresis on cellulose acetate membranes, affinity high-performance liquid chromatography (HPLC) with a hydroxyapatite column and gel-filtration HPLC after chondroitinase digestion, showed the presence of dermatan sulfate (DS) chains and chondroitin sulfate (CS). Further, it was suggested that CS located in the reducing termini and DS in the non-reducing termini in the hybrid chain. Hirosaki Med. J. 56: 45-54, 2005 ~~ g~~~0~~~0h~~m~~~ ~~0~~.~.*~GM~G, ~.~0~~~57~-~M~G~ ~, ~0~ 7 -3l J\~:BetU!f I) -!t -2 J ~ 0 fJ (GAG) mt~ji0t~m~~~fJT Gt.::. 7 -3l~)\~ 0N*jJiM7 -2 J ~~2:71J~J: e: l-~PG-II:B etUe: ~ij~ij~PG-II0~hC :-3G)t.::. GAGM~ji0-t)vD-;Z 7-t~-~ij~=jzjI;~%r*J} ), /\-1 ~0:::f:~7/\-3l-1 ~fJ ~~fJT~ ~J:, GAGM~ji~J:JijI;*j/iM{WH::~ 0 -1T ~1Jj1t~ (CS), ~FJijI;*j/iM{J!lH::~)v~ -3l ~~1rE~ (DS) i6~~tt9~/\-1 :1' I) 'Y ~~M 1) ~b*tk~~~ff~~*H1. *4~~fiJ~ 2) %iW*~~~ff~~-j::1 t;~~fiJ~

Determination of High and Low Molecular Weight Molecules of Icodextrin in Plasma and Dialysate, Using Gel Filtration Chromatography, in Peritoneal Dialysis Patients

The aim of this study was to apply high performance liquid chromatography (HPLC) with modern gel filtration media to determine high molecular weight (HMW) icodextrin fractions and low molecular weight (LMW) icodextrin metabolites in dialysate and plasma in peritoneal dialysis (PD) patients on treatment with icodextrin, and to explore the potential relationships between these compounds, alpha-amylase activity, and glomerular filtration rate. Retrospective study of dialysate and plasma samples from PD patients. Samples were collected at one PD center. Blood and dialysate samples were obtained from PD patients who were subdivided into three groups: patients using only glucose-based peritoneal dialysis fluid (GPDF; GLU group, n = 23), patients studied after the first long dwell with icodextrin-based peritoneal dialysis fluid (IPDF; 1st ICO group, n = 24), and patients who were regular users of IPDF for the long dwells (ICO group, n = 9). LMW icodextrin metabolites [i.e., maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and maltoheptaose (G7)] and HMW fractions were determined in plasma and dialysate using two different gel filtration HPLC methods. Enzymatic hydrolysis with amyloglucosidase to glucose yielded the total carbohydrate content and this was used to validate the HPLC results. alpha-Amylase activity was determined using a routine method. The results obtained by gel filtration HPLC yielded values of LMW metabolites and HMW fractions in plasma and dialysate in agreement with results obtained with enzymatic hydrolysis. HMW fractions were not detectable in plasma. Absorption of icodextrin from the peritoneal cavity during the long dwell (10 - 16 hours) was, on average, 39% of the amount instilled. During the long dwell, there was a relative decrease in the dialysate concentration of the largest HMW fractions (>21.4 kDa). Plasma concentration of the LMW icodextrin metabolites G2-G7 was highest in the ICO group (2.65+/-0.54 mg/mL) but also higher in the 1st ICO group (1.97+/-0.57 mg/mL) compared with the GLU group (0.52+/-0.23 mg/mL). Plasma alpha-amylase activity was significantly lower in the 1st ICO group and in the ICO group compared with the GLU group. Accurate analysis of HMW icodextrin fractions in dialysate and LMW icodextrin metabolites in plasma and dialysate in PD patients can be achieved by gel filtration HPLC with two different columns. This method can be used to study the complex pattern of changes in icodextrin and its metabolites in plasma and dialysate. The finding that HMW icodextrin fractions were not detected in plasma was unexpected, and differs from results of previous studies by other researchers.

Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides.

For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.