96-well Polystyrene Plates Research Articles

Context-dependent Dysregulation of Transcription by Mutant Huntingtin

Huntington disease (HD) is an adult-onset neurodegenerative disease caused by expansion of a polyglutamine (poly(Q) tract in the N-terminal region of huntingtin (htt). Although the precise mechanisms leading to neurodegeneration in HD have not been fully elucidated, transcriptional dysregulation has been implicated in disease pathogenesis. In HD, multiple N-terminal mutant htt fragments smaller than the first 500 amino acids have been found to accumulate in the nucleus and adversely affect gene transcription. It is unknown whether different htt fragments in the nucleus can differentially bind transcription factors and affect transcription. Here, we report that shorter N-terminal htt fragments, which are more prone to misfolding and aggregation, are more competent to bind Sp1 and inhibit its activity. These effects can be reversed by Hsp40, a molecular chaperone that reduces the misfolding of mutant htt. Our results provide insight into the beneficial effects of molecular chaperones and suggest that context dependent transcriptional dysregulation may contribute to differential toxicity of various N-terminal htt fragments.

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen–oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

Plasma-Needle Treatment of Substrates With Respect to Wettability and Growth of Escherichia coli and Streptococcus mutans

In this paper, surface modification of various materials exposed to a nonthermal atmospheric plasma is investigated. The used source is the plasma needle: a radio-frequency-driven nonthermal atmospheric microplasma. A number of substrates (Perspex and polystyrene) were treated with the plasma needle. The modification of materials was subsequently identified as hydrophilization of the surface and was experimentally validated by water-contact-angle measurements. Furthermore, the effect of this modification on the growth of two bacterial species, which are the Escherichia coli and Streptococcus mutans, is studied. The bacteria were cultured on treated and nontreated polystyrene 96-well plates; the growth of E. coli on the treated substrates was enhanced, while for S. mutans, it was reduced. An explanation of these effects is provided

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Differences between single‐ and dual‐species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies toLawsonia intracellularisin Pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.

Fluorescent assay for studying the substrate specificity of neuraminidase

Toyopearl 650 M was purchased from Tosoh (Japan). The 10-~1 pipette tips with filter (cat. no. FI08-96R-I0) and thin-well 0.2-ml PCR microtubes (cat. no. 430-01XNBZ-Q) with caps were obtained from Porex. Black polystyrene 96-well microtiter plates were purchased from Nunc (Denmark, cat. no. 437111). Vibrio cholerae neuraminidase (3.2.1.18) was purchased from Sigma (USA). 3'SiaLac and 6'SiaLac were purchased from Lectinity (Russia). ro-Aminospacered 3'SiaLac, 3'SiaLacNAc (Neu5Ac Cl2-3GaI131-4GlcNAc), and 6'SiaLacNAc (Neu5Ac Cl2-6GaI131-4GlcNAc) were synthesized previously [5-7]. ro-Aminospacered trisaccharide (5 ~ol), followed by triethylamine (5 ~l), was added to BODIPY FLSE (5.5 ~ol) in dimethylformamide (0.5ml). The reaction mixture was stirred at room temperature for 2-3 h and was subjected to gel filtration on a Sephadex LH-20 column equilibrated with acetonitrile/water (1:1, v/v). The yield of BODIPYlabeled trisaccharides was 80-90%. (Complete lH nuclear magnetic resonance data (500 MHz, DzO) are available from www.carbohydrate.ru/docs/nmrdata_OOl.rtf.) Virus NBulgaria/120/82 (HINl) and virus A!FPV/ Rostock/34 (H7Nl) were obtained from the virus repository of the Ivanovsky Institute of Virology in Moscow, Russia, and were propagated in 10-day-old embryonated chick~n eggs. A Madin-Darby canine kidney (MDCK)derived variant of human influenza virus A/Hongkong/ 1143/99 (H3N2) was obtained from the Institute of Applied Microbiology in Vienna, Austria. Primary isolation, propagation of human influenza virus, and purification of all viruses were done as described previously [8]. A titer of viruses was determined in a hemagglutination reaction with human erythrocytes Neuraminidase, or sialidase, is an exoglycosidase that hydrolyzes tX-ketosidic linkage between sialic acid and adjacent sugar residue. There are several methods of neuraminidase activity determination, usually based on fluorescent or chemiluminescent detection of an aglycon formed due to neuraminic acid cleavage [1-3]. Substrate specificity of neuraminidase is usually defined as the enzyme ability to distinguish the type of bond between neuraminic acid and galactose residues. As a rule, 3'SiaLac (Neu5ActX2-3Galf31-4Glc)! and 6'SiaLac (Neu5Ac tX2-6Galf31-4Glc) are used as substrates, and the amount of released neuraminic acid is measured [4]. In this article, we propose a simple and sensitive fluorescent method for determining the neuraminidase specificity using BODIPY -labeled trisaccharides as substrates. The method is based on the quantitative separation of neutral disaccharide-BODIPY from negatively charged uncleaved substrate using an anion exchange microcartridge, with the reaction product being registered by its fluorescence (Scheme 1). BODIPY FLSE (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid) was purchased from Molecular Probes (The Netherlands). All other chemicals used were obtained from Fluka (Switzerland). All chemicals used were of analytical grade. DEAE-

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C.

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.

PH-sensing 96-well microtitre plates for the characterization of acid production by dairy starter cultures.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37 degrees C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

Evaluation of antigen-coating procedures of enzyme-linked immunosorbent assay method for detection of trypanosomal antibodies

Research was undertaken to improve the antigen-coating step of indirect enzyme-linked immunosorbent assay (ELISA) method through the use of polystyrene 96-well plates precoated with antigenically stabile crude trypanosomal antigens. The plates were precoated with antigens, air dried and sealed before being packed in plastic bags with silica gel desiccant packets. Such plates stored at +4 and +37°C provided an assay performance, which was superior to that of plates freshly coated with antigens from a frozen stock. Antigen-precoated plates consistently proved stable after storage up to +50°C for at least 1 year. The accuracy of the assay was not affected, i.e. trypanosomal antibody-positive sera were clearly discriminated from trypanosomal antibody-negative negative sera. In contrast, lyophilized trypanosomal antigens lacked stability on storage at +37°C for longer than 1 month. It was concluded that the routine use of antigen precoated polystyrene plates for the enzyme immunoassay technique will contribute to improved assay robustness at an acceptable diagnostic proficiency. The modified coating procedure will also provide an improved quality assurance and standardization procedure for the assay, which is required to allow the reliable detection of trypanosomal antibodies and comparison of data from different laboratories.

A Novel Carbohydrate-Glycosphingolipid Interaction between a β-(1–3)-Glucan Immunomodulator, PGG-glucan, and Lactosylceramide of Human Leukocytes

The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.

A rapid screening procedure to identify mini-Tn10 insertion mutants of Escherichia coli K-12 with altered adhesion properties

A rapid screening procedure was developed for detection of Escherichia coli mutants with altered adhesion abilities using polystyrene 96-well microtiter plates as attachment surfaces. During this assay, bacterial strains grew and adhered simultaneously, and attached cells were measured after crystal violet staining. Starting with a total of 7000 W3110::Tn10 insertion mutants of E. coli K-12 W3110, 50 adhesion-deficient mutants were isolated which showed less than 40% attachment, and 22 mutants were found with an attachment of 40–75%. Motility assays were performed on these 72 mutants, and 34 displayed altered motility.

A rapid screening procedure to identify mini-Tn10 insertion mutants of Escherichia coli K-12 with altered adhesion properties.

A rapid screening procedure was developed for detection of Escherichia coli mutants with altered adhesion abilities using polystyrene 96-well microtiter plates as attachment surfaces. During this assay, bacterial strains grew and adhered simultaneously, and attached cells were measured after crystal violet staining. Starting with a total of 7000 W3110::Tn10 insertion mutants of E. coli K-12 W3110, 50 adhesion-deficient mutants were isolated which showed less than 40% attachment, and 22 mutants were found with an attachment of 40-75%. Motility assays were performed on these 72 mutants, and 34 displayed altered motility.

Use of Jacalin as a Solid Phase in ABO Reverse Grouping

A major problem of using red cells as the solid phase in assay systems is the difficulty to bind them strongly to appropriate surfaces. We report here on a number of lectins of different specificities which were examined for their ability to bind red cells to polystyrene 96-well microtitre plates. The use of the Thomsen-Freidenreich antigen-specific lectins, jacalin, mushroom and Ma- clura pomifera agglutinin proved the most useful for ABO reverse grouping. Jacalin-coated plates were also compared with plates coated with poly-L- lysine and bovine serum albumin/glutaraldehyde for the binding of erythrocyte membranes and were found to be superior. We also describe the colorimetric detection of the solid phase red cell antibody reaction by using an indicator erythrocyte and peroxidase chromogenic substrate.

Interaction of a 59-kDa Connective Tissue Matrix Protein with Collagen I and Collagen II

We have studied binding to collagen of the 59-kDa protein present in most connective tissues. Collagen fibril formation, measured as increasing turbidity, was markedly retarded and reduced by the presence of small amounts of this protein. This was true for both collagen I and collagen II. The effect was also observed when pepsin-treated collagens were used, indicating that interaction with the telopeptides is not involved. The proportion of collagen precipitated in the assay was not or only marginally reduced. Thus, the altered optical properties indicate that structurally different fibrils are formed in the presence of the 59-kDa protein. The 59-kDa protein bound to collagen I or collagen II that had been insolubilized on polystyrene 96-well microtiter plates, as measured by enzyme-linked immunosorbent assay. Analogously, binding to the collagens was demonstrated for the PG-S2 low Mr proteoglycan, previously shown only to inhibit collagen fibrillogenesis. The two matrix components showed similar strength of binding, i.e. Kd 35 nM for the 59-kDa protein and 16 nM for PG-S2 at 20 degrees C. The results do not reveal if the collagen interaction site of the 59-kDa protein is different from that of PG-S2. Our observations do, however, suggest that the 59-kDa protein, as well as PG-S2, have functions related to the regulation of collagen organization in tissues.