Development of LC-MS method for detection of mutant uromodulin protein.

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 --> 381, 471, 567, and 679 for GWHWE and m/z 688 --> 381, 445, 541, and 653 for GWHC*E) were detected.

Increased antibody levels to desmogleins 1 and 3 after administration of carbamazepine.

Desmoglein (Dsg) 1 and Dsg3 are recognized as the autoantigens in pemphigus foliaceus and pemphigus vulgaris. Pemphigus-like syndromes have been reported to occur in individuals after exposure to a variety of drugs, but pemphigus caused by carbamazepine is not common. We found that anti-Dsg1 and anti-Dsg3 antibody titres were increased in three individuals administered carbamazepine. Antibody titres against Dsgs 1 and 3 were measured by enzyme-linked immunosorbent assay (ELISA). Of 42 serum samples (25 patients administered carbamazepine, eight patients administered valproic acid and nine healthy volunteers) tested by ELISA, three patients administered carbamazepine showed positive reactivity against both Dsg1 and Dsg3. The patient with the highest titre against Dsg1 and Dsg3 (the index values of anti-Dsg1 and anti-Dsg3 were 79.3 and 86.4, respectively) was a 23-year-old woman (Case 1). The other two patients with positive reactivity were a 5-year-old boy (Case 2) and a 65-year-old man (Case 3). In addition, indirect immunofluorescence study showed intercellular antibodies to the cell surface of the whole epidermis with a titre of 1 : 64 in Case 1 and 1 : 2 in Cases 2 and 3. However, no skin or mucosal involvement was found in any of these cases. There was no difference in the serum concentrations of carbamazepine between the three positive cases and the 22 negative cases of carbamazepine administration. From these facts, the lack of skin diseases may be explained by relatively low values of anti-Dsg 1 and 3 antibodies in Cases 2 and 3. However, it cannot be excluded that undefined exogenous and/or endogenous factors are involved in an outbreak of pemphigus. Furthermore, these findings might be helpful for preventing susceptible individuals from exposure to the suspect drugs.

Towards molecularly imprinted polymers selective to peptides and proteins. The epitope approach.

In this paper, we describe the epitope approach to molecular imprinting. The applicability of molecular imprinting, a method that allows the preparation of biomimetic compounds (artificial receptors and antibodies), is extended by this approach. Our approach makes it possible to obtain imprinted polymers selective to peptides and proteins whereas, to date, molecular imprinting has been used primarily for the preparation of polymers that selectively bind to relatively low molecular weight substances. The epitope approach is based on using (as a template) a short peptide that represents only part of a larger peptide or protein (as an epitope represents an antigen), which in turn can be recognized by the synthesized polymer. It is demonstrated that although other parts of peptides can influence the process of molecular recognition, the polymers imprinted with a short peptide efficiently recognize both the template and larger peptides (for example, oxytocin) that possess the same C-terminal part of the structure.

Recognition of oxytocin and oxytocin-related peptides in aqueous media using a molecularly imprinted polymer synthesized by the epitope approach.

An artificial polymeric receptor prepared by the epitope approach of molecular imprinting was shown to recognize the peptide hormone, oxytocin, in aqueous media. The proposed approach is based on using (as a template) a compound, whose structure represents a small exposed fragment of a larger molecule (as an epitope represents an antigen). A HPLC study has demonstrated the important role of ionic interactions and the N-terminal amino group of oxytocin and oxytocin-related peptides in the process of their recognition by the molecularly imprinted polymer in the aqueous-rich media. However, the specificity of the process is considered to be defined by hydrophobic interactions and hydrogen bonding. Moreover, it was shown that the selectivity of the molecularly imprinted polymer can be attenuated by water content, ionic strength and pH of the chromatographic mobile phase: depending on these factors the template, Tyr-Pro-Leu-Gly-NH2, or, for example, oxytocin, a larger peptide, which possesses the same three amino-acid C-terminal parts of the structure, can be preferentially retained by the molecularly imprinted polymer.

Comparison of Surface and Net Charge Densities of Poly(acrylonitrile) Membranes Grafted with Ionic Monomers: Hydrophilic and Hydrophobic Effects of Graft Chain.

The apparent surface charge densities of grafted poly(acrylonitrile) (PAN) membranes (determined by measurement of zeta potential) were compared with net charge densities (determined by potentiometric titration) in order to examine the effects of hydrophilic and hydrophobic graft chains at the pore surface. Membranes with hydrophilic graft chains showed much smaller net charge densities than membranes with hydrophobic graft chains. However, the apparent surface charge densities of the membranes with hydrophilic graft chains were much larger than those of the membranes with hydrophobic graft chains. This fact can be explained by the formation of ion pairs between charge groups and counterions. Dissociation behaviors for the two types of membranes, in which electrostatic interactions of the charge groups play a significant role were distinctly different. These results confirm the occurrence of ion-pairing effects between the charge groups and the counterions for hydrophobic graft chains in which dissociation of some of the charge groups is suppressed. Copyright 1998 Academic Press.

Oscillations of membrane potential across a polypeptide membrane, induced by an electrical current.

Oscillation of membrane potential across a tri-block copolypeptide membrane composed of (Glu)x-(Leu)y-(Glu)x (x = 0.18 and y = 0.64) was observed under an electrical current, when the membrane was placed between equimolar aqueous salt solutions. The amplitude of the oscillation was influenced by the type of cation and anion in the external salt solution, and the amplitude was in the sequence: K+ > Na+ > Cs+ > Ca2+ and Cl- > Br-. The frequencies of the oscillations were in the range 0.1 to 5 Hz, and were also slightly influenced by the type of cation and anion.

Effect of the chemical modification of the arginyl residue in Bombyx mori silk fibroin on the attachment and growth of fibroblast cells.

We prepared matrices of Bombyx mori silk fibroin (SF) with different degrees of modification of arginyl residues by reaction between 1,2-cyclohexanedione (CHD) and SF. Two kinds of SF, namely native SF (NSF), obtained from the silk gland of silkworm larvae, and regenerated SF (RSF), prepared from cocoons of the same silkworm, were used in this study because their amino acid compositions were slightly different from each other. The attachment and growth of mouse fibroblast (L-929) cells on the matrices of the NSF and RSF, in which half or almost all of the arginyl residues were modified (NSF50, RSF50, NSF100, and RSF100), were studied using a cell culture method. Both NSF50 and NSF100 exhibited higher cell attachment than did the unmodified NSF. While the cell growth on NSF50 was not significantly different from that on NSF and NSF100, the growth on NSF100 was higher than that on NSF. The cells attached to NSF50 and NSF100 were extensively spread out and their filopodia were visible by SEM. The cell attachment and growth on RSF were comparable to those on NSF100. Although RSF50 exhibited almost the same cell attachment as did the unmodified RSF, RSF100 exhibited a lower cell attachment than did the unmodified RSF and RSF50. There were no significant differences in the cell growth among RSF series. The cells attached to RSF50 and RSF100 aggregated to form masses, and their filopodia could not be found. The relationship of cell attachment to the basicity of the substrate is considered because the modification of the positively charged arginyl residue changed the basicity of the substrate and the cell attachment on the substrate.

Attachment and growth of cultured fibroblast cells on PVA/chitosan-blended hydrogels.

Hydrogels were prepared from poly(vinyl alcohol) and chitosan in various blend ratios. The water contents of the hydrogels were in the range of 65 to 75 wt %. The attachment and growth of fibroblast cells (L-929) on the hydrogels were studied with a cell culture method. On the hydrogels with more than 15 wt % chitosan content, the attached cells were able not only to remain viable but also to proliferate. The relative cell attachment after incubation for 30 h increased with increasing chitosan content in the hydrogels. Cell attachment and growth on the hydrogel with 40 wt % chitosan content exceeded those on collagen, a widely-used mammalian cell culture substrate. The morphology of the cells attached onto the hydrogels with a lower chitosan content was spherical, but in hydrogels with more than 15 wt % chitosan content, the number of spindle-shaped cells increased with increasing chitosan content.

Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates.

Poly(ethylene glycol) (PEG)-silk fibroin (SF) conjugates (PEG2-SF) were prepared by the chemical modification of solubilized SF with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (actPEG2) in borate buffer at 37 degrees C. The IR spectra and DSC curves of PEG2-SF and SF suggested the introduction of PEG into SF by the modification and the beta-sheet structure of both SF and PEG2-SF induced by the treatment with methanol aqueous solutions. The content of the PEG component in PEG2-SF was evaluated to be 67% by weight from the melting enthalpy change of PEG observed on the DSC thermogram of PEG2-SF. Water content and contact angle measurements of SF before and after the modification indicated that the hydrophilicity of the PEG2-SF surface increased compared with that of SF. The attachment and growth of fibroblast cells (L-929) on the matrix of PEG2-SF were studied by a cell culture method. PEG2-SF exhibited very low cell attachment and growth, though SF exhibited high cell attachment and growth. The filopodium of the cells attached to PEG2-SF could not be found, and the cells aggregated to form masses in scanning electron microscopy images. These results could be explained in terms of the increased hydrophilicity of the PEG2-SF surface.

Molecular imprinting: synthesis of polymer particles with antibody-like binding characteristics for glucose oxidase.

A phosphate buffer containing a mixture of glucose oxidase, acrylic acid derivatives, N,N'-1,2 dihydroxy-ethylene-bis(acrylamide), N,N'-(methylene)-bisacrylamide and surface-modified silica was radically polymerized with (NH4)2S2O8. The polymer formed a thin layer around the silica beads. After sieving of these polymer particles, the surface bound protein was eluted. In rebinding assays and enzyme activity tests a specific binding capacity for glucose oxidase of up to 0.557 microgram GOD/100 mg dry weight of polymer particles could be determined. These polymer particles have the potential to be used as specific separation or detection material.

Chemical modification of the arginyl residue in silk fibroin: 2. Reaction of 1,2-cyclohexanedione in aqueous alkaline medium.

The arginyl residue of solubilized silk fibroin was chemically modified with 1,2-cyclohexanedione in aqueous alkaline medium to form a stable imidazolidinone ring, and its positive charge was masked. CD spectra of the modified silk fibroin in aqueous solution showed an increase in the fraction of random coil conformation. The increase may be caused by the exposure to alkaline medium in the modification reaction. FT-IR and CD spectra of the silk fibroin films before and after the modification indicated that the conformational change in the modified silk fibroin in the solid state did not occur by the modification of its arginyl residue with 1,2-cyclohexanedione. The chemical stability of the modified silk fibroin film was investigated in vitro with phosphate-buffered saline solution. The modified arginyl residue in the film was stable in the phosphate-buffered solution.

Chemical modification of silk fibroin with N-acetyl-chito-oligosaccharides.

N-Acetyl-chito-oligosaccharides (NACOS)-silk fibroin (SF) conjugates (NACOS-CY-SF) were prepared by the reaction of solubilized SF and cyanuric chloride (CY)-activated NACOS modifier (NACOS-CY). N-Acetyl-D-glucosamine (NAG), as a model compound, was reacted with CY to clarify the chemical structure of the modifier. The 1H- and 13C-NMR spectra of the reactant suggest that the anomeric hydroxyl group of NACOS reacted with the chlorine atom of CY. The content of NACOS in the NACOS-CY-SF conjugates was calculated by comparing the integral values of the signals in the 1H-NMR spectra of the conjugates and the mixture of NACOS and SF. As the 1H-NMR spectrum of the conjugates showed a downfield shift of the aromatic protons of the tyrosine residue, the tyrosine residue in SF reacted with another chlorine atom of the triazine ring of the modifier. The result of the amino acid analysis of the conjugates suggests that lysine residues also reacted with the modifier.

Attachment and growth of cultured fibroblast cells on silk protein matrices.

The attachment and growth of L-929 cells on films made of Bombyx mori silk proteins--fibroin and sericin and their mixtures--was studied by a cell culture method. Both cell attachment and growth were dependent on a minimum of around 90% sericin in the mixture. The results from electron micrography as well as from the DSC measurements supported the notion that the mixture of the two proteins fibroin and sericin has a phase-separated structure in the solid state. The observed minimum of sericin in the cell attachment and growth is thought to be a result of this phase-separated structure. Films of pure component proteins (i.e., 100% fibroin or sericin) exhibited as high a cell attachment and growth as collagen, a widely used mammalian cell culture substrate. However, a morphological study of the attached cells revealed that the cells attached to silk fibroin were extended and had a spindle shape, just like the cells attached to collagen, while the cells attached to the silk sericin had a different shape. It is concluded, therefore, that the attachment condition on silk fibroin is ideal for the viability, growth and function of the cells.

Attachment and growth of fibroblast cells on silk fibroin.

The attachment and growth of fibroblast cells (L-929) on matrices of silk fibroin from Bombyx mori domestic silkworm (DSF) and Antheraea pernyi wild silkworm (WSF) were studied by a cell culture method. The performance of the two kinds of silk fibroin was compared to that of collagen. DSF exhibited as high a cell attachment and growth as collagen did. The cells attached to DSF were extensively spread out and their filopodia were visible in the SEM pictures. WSF, which contains the tripeptide sequence Arg-Gly-Asp (believed to be a specific interaction site for cell-attachment), displayed much higher cell attachment and growth compared to DSF. The cells attached on WSF became virtually flat and their filopodia could be seen, indicating that the cells were very strongly held on the surface.

Cell growth on poly(vinyl alcohol) hydrogel membranes containing biguanido groups.

A cationic, high-water-content hydrogel membrane composed of poly(vinyl alcohol) (PVA) and poly(ally-biguanido-co-allylamine) hydrochloride (PAB) with positively charged biguanido groups that resemble arginine residues was developed. The PAB was prepared by reacting poly(allylamine) hydrochloride (PAA) with guanyl-O-methyl isourea. PAB/PVA hydrogel membranes were prepared by repeated freezing and thawing. For comparison, hydrogel membranes composed of PAA and PVA were also prepared. The interaction between these hydrogel membranes and mouse fibroblast (L929) was studied by a cell culture method. The PAB hydrogel blend had a relatively low percentage of initial cell attachment. The cell growth on the PAB hydrogel membranes showed a maximum at 5 mol % PAB content that was as high as commercially available plastic films. However, cells on hydrogel membranes with 50 mol % PAB content and 0 mol % PAB content (only PVA) did not seem to grow; neither did the 5/95 PAA/PVA membranes. Water contact angles of hydrogel membranes did not vary with the PAB content. Morphology of the cell attachment was observed by SEM. On the PAB blend hydrogel surfaces, cells were not spindle-shaped and monolayers, but rather cells aggregated in spherical clusters.

Chemical modification of silk fibroin with cyanuric chloride-activated poly(ethylene glycol): analyses of reaction site by 1H-NMR spectroscopy and conformation of the conjugates.

Solubilized silk fibroin (SF) in 0.1 M borate buffer (pH 9.4) was modified with 2-O-[methoxy(polyethylene glycol)]-4,6-dichloro-s-triazine (actPEG1) at 4 degrees C. The weight of the modified SF (PEG1-SF) was at least 3.2 times that of the starting material SF. Amino acid analysis of PEG1-SF suggested that the nucleophilic epsilon-amino group of the lysine residue and the nucleophilic imidazole group of the histidine residue in SF reacted with actPEG1. The 1H-NMR spectrum of PEG1-SF showed a downfield shift of the aromatic protons of the tyrosine residue from the corresponding protons of SF. The 1H-NMR spectrum of the SF reacted with cyanuric fluoride (CyF), whose fluorine atoms are known to react with the phenolic hydroxyl group of the tyrosine residue, also showed the downfield shift. These results suggested that the reaction site of SF with actPEG1 was the phenolic hydroxyl group of the tyrosine residue in addition to the lysine and histidine residues. The conformation of PEG1-SF in a solid state was examined by means of IR and X-ray measurement. The IR spectrum of PEG1-SF revealed a change in secondary structure from random coil to beta-sheet due to the coexistence of PEG molecules. The X-ray diffraction pattern of PEG1-SF indicated that the PEG molecules covalently bonding to SF narrowed the spacing of the interchain periodicity and promoted the formation of the interchain beta-sheet.

Identification of novel singly spliced pX mRNA transcripts common to all human T-cell leukemia virus type 1-related retroviruses.

Human T-cell leukemia virus type 1 (HTLV-1) and its related viruses, that is, human T-cell leukemia virus type 2 (HTLV-2), simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV), encode a doubly spliced pX mRNA transcript in addition to the singly spliced env and unspliced gag-pol mRNAs common to the prototypic simple retroviruses, such as murine and avian leukemia viruses. In HTLV-1-infected cells, we recently identified the novel singly spliced pX mRNA responsible for expressing the smaller rex gene product, p21X. In the present study we demonstrate that the novel singly spliced pX mRNA is also expressed in cells infected with HTLV-2, STLV, and BLV, respectively. This finding indicates that all members of the HTLV-1-related virus group have the common ability to express the singly spliced pX mRNA. This novel mRNA in the HTLV-1-related virus group may be analogous to the two-exon nef specific mRNA in human immunodeficiency virus type 1.

Human T cell leukaemia virus type 1 p21X mRNA: constitutive expression in peripheral blood mononuclear cells of patients with adult T cell leukaemia.

Although the p21X protein of human T cell leukaemia virus type 1 (HTLV-1) is generally thought to be expressed from a doubly spliced mRNA transcript (tax/rex mRNA) that encodes the p40tax, p27rex and p21X proteins, we have shown previously that a novel, alternatively spliced mRNA transcript (p21X mRNA) is responsible for p21X production in HTLV-1-infected cell lines. In the present study, we analysed expression of p21X mRNA and tax/rex mRNA in uncultured and cultured peripheral blood mononuclear cells (PBMCs) from eight patients with adult T cell leukaemia by using a quantitative polymerase chain reaction coupled to reverse transcription. The results demonstrated that the expression of p21X mRNA occurs constitutively in all uncultured and cultured PBMCs, whereas the expression of tax/rex mRNA is inducible in the cultured PBMCs, as described previously. In uncultured and cultured PBMCs from the one specimen in which p21X mRNA was highly expressed, the p21X protein was detectable by Western blotting. On the other hand, p27rex protein was detectable only after cultivation. These findings indicate that p21X mRNA is constitutively expressed in vivo and is responsible for production of p21X protein.

Chemical modification of arginyl residues in silk fibroin: 1. Reaction of 1,2-cyclohexanedione in borate buffer.

Chemical modifications of silk fibroin were attempted in order to add new properties and functions to silk fibroin. The arginyl residue in solubilized silk fibroin was chemically modified with the reaction of 1,2-cyclohexanedione in borate buffer. FT-i.r. and c.d. spectra of the silk fibroin before and after the modification indicated that the fraction of random coil conformation increased with the modification. The chemical stability of the modified silk fibroin membrane was investigated in vitro with phosphate buffer. The modified arginyl residue in the membrane was considerably regenerated with the treatment in phosphate buffer.

Physico-chemical properties of silk fibroin membrane as a biomaterial.

A water-insoluble silk fibroin membrane was prepared by immersing a silk fibroin membrane as cast in 50 vol% aqueous methanol solution for different periods of time at 25 degrees C. To use the membrane as a biomaterial, oxygen and water vapour permeability, transparency, mechanical property and enzymatic degradation behaviour in vitro of the membrane in the wet state were investigated. These physico-chemical properties changed according to the condition of the methanol treatment. The membrane had oxygen permeability, water vapour permeability, transparency and biodegradability.