Molecular Size Analysis Research Articles

Ionic polyurethane materials for enhanced platelets adhesion and biocompatibility

Polyurethane ionomers (PUI) are employing as one of the prominent materials in the field of biomedical applications. PUI materials have been synthesized from the reaction of poly(ethylene glycol) (PEG) with diisocyanate reagent followed by the addition of dihydroxyl benzoic acid (DHBA) salt by two-step polymerization process. PUI material generated from NCO terminated polyurethane prepolymers bonded with ionomer fragment exhibit bio and haemocompatibility. All of these PUI films were characterized by FTIR and FT-NMR for structural identifications. Study of molecular weights and size analysis of PUI obtained were determined by Gel permeation chromatography and particle size analysis. Essentially, haemocompatibile nature of these PUI have been studied by thrombogenicity, haemolysis and platelet adhesion test. As such polyurethane linked with ionomer fragment exhibits better platelets adherence to soft or biological tissue.

Effects of improved post-harvest handling on the chemical constituents and quality of carrageenan in red alga, Kappaphycus alvarezii Doty

Red alga Kappaphycus alvarezii Doty is an important commercial species widely cultivated in southeast Asian countries for its polysaccharide, kappa-carrageenan. Common post-harvest handling technique involves sun-drying of harvested seaweed on platforms at the farms. Quantity and quality of carrageenan varies depending on the duration and care taken during the post-harvest handling of the raw seaweed. In this study, dynamics of moisture content, water activity index (aw), carrageenan yield, and carrageenan quality were investigated by subjecting the seaweed to three post-harvest methods: (1) freeze-drying (FD), (2) shade-drying (SD), and (3) direct sun-drying (DSD). Seaweed dried under FD and SD produced high yield (56–58 %), superior gel strength (1,454–1,424 g cm−2), high viscosity (57–58 cPs), and low syneresis (15–17 %). But, carrageenan extracted from DSD seaweed gave 28 % lower yield, 38 % lower gel strength, 27 % lower viscosity, and 8–9 % higher syneresis. In addition, gelling temperature and melting temperature of the DSD carrageenan were lower by 4 and 9 °C, respectively. Molecular size analyses of carrageenan extracted from seaweed dried under FD and SD contained carrageenan of 700 KDa (80 %) and 200 KDa (4–10 %). However, carrageenan extracted from DSD seaweed contained smaller carrageenan molecules, 460 KDa (55 %), 210 KDa (25 %), and <100 KDa (20 %). Further, scanning electron microscope images illustrated the severe effects of DSD on the morphology of seaweed cells. Therefore, SD technique was found to be the best post-harvest processing technique that gave quality carrageenan in a high quantity. Due to its simplicity and low cost, it is a practical approach to be practiced in southeast Asian countries.

Fast and precise method for HPLC–size exclusion chromatography with UV and TOC (NDIR) detection: Importance of multiple detectors to evaluate the characteristics of dissolved organic matter

A new type of high-performance liquid chromatography (HPLC)–size exclusion chromatography (SEC) system with ultraviolet (UV) absorbance detection and non-dispersive infrared (NDIR) detection of total organic carbon is described. The introduction of an online degassing tube and a low-volume HPLC column helped to reduce the analytical time and increase the sensitivity of the SEC system. This study is the first in which linear calibration curves ( R 2 > 0.99) were obtained for both UV absorbance and NDIR data for polystyrene sulfonate standards, which are the most suitable standards for molecular size analysis of aquatic humic substances as well as dissolved organic matter (DOM). Using the calibration curves, the molecular size distribution of DOM in water collected from Lake Kasumigaura and in pore water from lake sediments was estimated. Most of the DOM had a molecular weight less than 4000 Daltons (Da), and the amount of low-molecular-weight DOM (∼2000 Da) with low UV absorbance increased with depth in the sediment pore water. This result shows the importance of combining quantitative analysis by NDIR detection with qualitative analysis by UV detection to determine the chemical and physical properties of DOM. The possible sources and reactivity of DOM in Lake Kasumigaura and its sediment pore water are also discussed.

Detection of allergen composition and in vivo immunogenicity of depigmented allergoids of Betula alba

Chemical modification of allergen vaccines to reduce IgE binding improves safety while maintaining clinical efficacy. However, this also complicates the characterization of allergoids using techniques as for native allergen extracts. The objective of this study was to analyse the molecular size of Betula alba depigmented allergoids, conservation of major allergens in the allergoids and in vivo antibody response to immunization. The molecular size of depigmented allergoids was evaluated by high performance-size exclusion chromatography and light scattering techniques. Protein composition was compared with native extracts by capillary liquid chromatography-tandem mass spectrometry based peptide mapping. Rabbits were immunized with depigmented allergoid of Betula pollen adsorbed onto aluminium hydroxide (Depigoid). IgG antibodies against individual allergens were determined by ELISA and immunoblot. Depigmented allergoids contained a range of high molecular weight particles, approximately 60% of which had a molecular weight of 1-3 MDa. Peptide sequencing confirmed the preservation of five isoforms of Bet v 1, as well as Bet v 2, Bet v 6 and Bet v 7. Sera from immunized rabbits showed high levels of specific IgG to rBet v 1.0101 and rBet v 2. The mean protein content was 544+/-106 microg per mg of freeze-dried material for depigmented allergoids and 434+/-71 for native extracts. They retain the capacity to induce specific IgG antibodies against individual allergens present in the native extract. These findings confirm the immunogenicity of depigmented allergoids and may explain why patients treated with these vaccines are protected against the native allergens. Analysis of molecular size and allergen content may be useful techniques for characterization and standardization of allergoid products.

Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms

Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients. To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population. A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs. The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity. Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

Heparan Sulfate Regulates Self-renewal and Pluripotency of Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.

Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses

The mechanisms by which the excess genetic material of chromosome 21 results in the dysmorphologic features of Down syndrome (DS) are largely unknown. It has been found that the extracellular matrix of nuchal skin of DS fetuses exhibits an higher content of hyaluronan (HA) compared to that of euploid fetuses. Since HA plays a central role in many morphogenetic processes during embryogenesis, an alteration in its metabolism could be involved in the pathogenesis of several structural defects of DS. The extracellular matrix of umbilical cord (UC) is the mammalian tissue with one of the highest content of HA. Therefore we sought to explore the quantitative HA modifications during gestation, tissue distribution and HA metabolism in euploid and DS UCs. Euploid UCs (n=28) and UCs from DS fetuses (n=13) were obtained after termination of pregnancy, spontaneous abortion, or at delivery. Quantitative and molecular size analysis were performed using HPLC and FPLC. Tissue distribution was visualized by immunohistochemistry. Gene expression for HA synthases (HAS) and hyaluronidases (HYAL) were quantified by real-time PCR techniques and HYAL activity was detected by zymography. In euploid UC only HA of a molecular weight of 1700 kDA was present while in DS UC an additional lower weight HA molecule of 1100 kDA was found. Immunohistochemistry showed a larger amount of Wharton's jelly HA in DS UCs than in euploid UC. Real-time PCR analysis showed that HAS 2 and HYAL 2 were expressed at significant levels in all specimens. A higher expression of HAS 2 and a lower expression of HYAL 2 was found in the Wharton's jelly of DS fetuses compared to that of euploid fetuses at 14 weeks of gestation. On the contrary, at term HYAL 2 expression was higher in DS specimens than in those from euploid fetuses. Zymographic studies showed a similar behavior with a lower HYAL activity at early gestation and a higher HYAL activity at term gestation in DS UCs compared to euploid specimens. Therefore we can conclude that HA is more represented in DS UCs than in euploid UCs. A complex alteration of the HA metabolism characterized by an increased synthesis of lower weight HA molecules is a peculiarity of DS UCs.

ATP-, K+-dependent heptamer exchange reaction produces hybrids between GroEL and chaperonin from Thermus thermophilus.

Chaperonin from Thermus thermophilus (Tcpn6014.Tcpn107) splits at the plane between two Tcpn607 rings into two parts in a solution containing ATP and K+ (Ishii, N., Taguchi, H., Sasabe, H., and Yoshida, M. (1995) FEBS Lett. 362, 121-125). When Escherichia coli GroEL14 was additionally included in the solution described above, hybrid chaperonins GroEL7.Tcpn607 and GroEL7. Tcpn607.Tcpn107 were formed rapidly (<20 s) at 37 degrees C. The hybrid was also formed from Tcpn6014 and GroEL14 but not from a mutant GroEL14 lacking ATPase activity. The hybrid formation was saturated at approximately 300 microM ATP and approximately 300 mM K+. These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction with Thermus chaperonin under nearly physiological conditions. Similar to parent chaperonins, the isolated hybrid chaperonins exhibited ATPase activity that was susceptible to inhibition by Tcpn107 or GroES7 and mediated folding of other proteins. Once formed, the hybrid chaperonins were stable, and the parent chaperonins were not regenerated from the isolated hybrids under the same conditions in which the hybrids had been formed. Only under conditions in which GroEL in the hybrids was selectively destroyed, such as incubation at 70 degrees C, Thermus chaperonin, but not GroEL14, was regenerated from the hybrid. Therefore, the split reaction may not be an obligatory event repeated in each turnover of the chaperonin functional cycles but an event that occurs only when chaperonin is first exposed to ATP/K+.

Subunit Structure of Rod cGMP-Phosphodiesterase

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.

Absolute on-line molecular mass analysis of basic fibroblast growth factor and its multimers by reversed-phase liquid chromatography with multi-angle laser light scattering detection

The multi-angle laser light scattering (MALLS) detection method was combined with reversed-phase high-performance liquid chromatography to analyze multimerization of basic fibroblast growth factor (bFGF) formed by oxidation of bFGF with air or with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). This analysis provided the absolute molecular mass and the mean square radius for each eluted protein fraction of each slice of the chromatogram. It was shown that depending on the oxidation conditions, bFGF forms different multimetric forms, from dimers to hexamers. It was found that these multimers have varied conformations of the same molecular mass, but different structure. Molecular mass and size analyses provided molecular conformation of the aggregates; the results indicated the formation of rod-like rigid structures. The MALLS analysis confirmed that, during oxidation, each bFGF monomer bound sequentially to form the extended multimer. The proposed scheme of bFGF oxidation with DTNB revealed that the difference in the aggregate structural forms was probably due either to the presence of covalently bound residues of nitrobenzoic acid in the products of oxidation, or to the participation of sulfhydryl groups in disulfide bond formation.

Characterization of a Novel α-Tocopherol-Binding Protein from Bovine Heart Cytosol

We previously reported the identification of a new α-tocopherol-binding protein (∼ 15 kDa) in the cytosol of rat liver and heart and in rabbit heart (A, K, Dutta-Roy et al., J, Nutr, Biochem. 5, 562-570, 1994). This protein specifically binds α-tocopherol and enhances its transfer between separate membranes, In the present paper we have purified and characterized the α-tocopherol-binding protein from bovine heart cytosol and compared its various structural and functional properties with the similar size (∼ 15 kDa) cytosolic fatty acid-binding protein of this tissue. α-Tocopherol-binding protein was purified to electrophoretic homogeneity from bovine heart cytosol by a procedure involving precipitation with 70% ammonium sulfate, followed sequentially by gel filtration chromatography and chromatofocusing, The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 16 kDa, Isoelectric focusing of the purified protein showed that the pI value is around 4.5. The binding of α-tocopherol to the purified protein was rapid, reversible, and saturable. The α-tocopherol-binding protein did not bind oleate as assessed by direct radiolabeled fatty acid binding and fluorescence enhancement assay, Amino acid analysis showed the presence of a large number of Ala, Gly, Ser, Lys, and Pro residues and a lesser number of aromatic residues in this protein. Anti-bovine heart fatty acid-binding protein antibody did not recognize the α-tocopherol-binding protein in the Western blot. The Western blot, ligand affinity, molecular size, and amino acid analysis data suggest that the α-tocopherol-binding protein is different from the cytosolic fatty acid-binding protein and that it may be involved in intracellular transport and metabolism of α-tocopherol.

Evaluation of sodium dodecyl sulfate non-acrylamide, polymer gel-filled capillary electrophoresis for molecular size separation of recombinant bovine somatotropin

Sodium dodecyl sulfate (SDS) non-acrylamide, polymer gel-filled capillary electrophoresis was examined as an alternative to the high-performance size-exclusion chromatographic (HPSEC) method for the analysis of recombinant bovine somatotropin (bovine growth hormone, rbSt). A calibration curve for the molecular mass of protein standards ( M r 14 000 to 97 000) was linear ( r 2= 0.998) when the molecular mass of the proteins and their peak migration time were plotted on a logarithmic (log-log) scale. Relative standard deviation (R.S.D.) of the molecular man determination was approximately 2–3%. A pre-production as well as a production lot of the SDS gel-filled capillary columns were examined. Performance of both of these columns were equivalent. Peak migration time remained relatively constant beyond 140 sample injections. Gradual loss of theoretical plates was noted over the course of the assay; however, the peak resolution remained adequate for the analysis of rbSt. Peaks corresponding to monomer, dimer, trimer, and tetramer of rbSt were base-line resolved by the SDS gel-filled capillary electrophoresis. Theoretical plate of the monomer peak was approximately 28 000–30 000 per 40 cm column. Both the monomer and the dimer recovery studies indicated that the calibration curves are linear ( r 2 > 0.993) and the slopes are no different from one. Amounts of the components in rbSt determined by the SDS gel-filled capillary electrophoresis compared well with those of the HPSEC method. The results of this study indicated that the SDS non-acrylamide gel-filled capillary electrophoresis may be a viable alternative to the HPSEC method for the molecular size separation and analysis of recombinant proteins.

Tumour Necrosis Factor α Augments the Release of an Endothelium-Dependent Vasoconstrictor from Human Polymorphonuclear Leukocytes

Summary: We have studied the effect of cytokine priming by recombinant human tumour necrosis factor α (rhTNFα) on the release of vasoactive mediators from human polymorphonuclear leukocytes (PMNs). Supernatants of human PMNs( 100 µl,5 x 107 ml, from 12 donors) relaxed precontracted rabbit aortic rings. The relaxations were preceded by a transient contraction in four experiments. In contrast, supematants of PMNs incubated with rhTNFα (0.3 n M, 30 min, 37°C) elicited variable vascular responses that consisted of either a sustained contraction (n = 4), a transient contraction followed by a sustained relaxation (n = 2), or a sustained relaxation only (n = 6). The vascular tone produced by the rhTNFα-treated PMN supernatant was significantly greater over 30 min than that produced by the control supernatant (p < 0.01). Endothelium removal prevented the PMN-induced contractions and significantly decreased the vascular tone produced by all rhTNFα-treated PMN supematants (p < 0.05). The PMN supematants had no vasoactive effect on aortic rings at resting tension. Molecular size analysis indicated that the PMN-derived contractile mediator is > 30,000 Da. These results suggest that human PMNs, primed by rhTNFα, release a stable endotheliumdependent vasoconstrictor that opposes the action of a stable, spontaneously released direct vasodilator

Structures of sugar chains of the subunits of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

The structures of asparagine-linked oligosaccharides in the subunits of an alpha-amylase inhibitor from the white kidney bean (Phaseolus vulgaris) were determined. Glycopeptides obtained from each subunit were treated with hydrazine, then N-acetylated. The oligosaccharides thus liberated were labeled with 2-aminopyridine at their reducing ends and purified by gel-permeation, reverse-phase, and size-fractionation HPLC. The structures of seven oligosaccharides from the alpha-subunit and eight oligosaccharides from the beta-subunit were determined by a combination of composition and molecular size analyses, exo- and endoglycosidase digestions, partial acetolysis, and 1H-NMR spectroscopy. The major glycan chains in the alpha-subunit were Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc and (Man alpha 1-2)Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6 (Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc, while a glycan chain Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4GlcNAc comprised more than 70% of the sugar moiety of the beta-subunit.

Congenital myopathy associated with abnormal accumulation of desmin and dystrophin

We studied a 5-yr-old boy clinically presenting congenital myopathy. Muscle biopsy showed sarcoplasmic accumulation of desmin filaments leading to diagnosis of desmin storage myopathy. An immunohistochemical study of other cytoskeletal proteins (actin, alpha-actinin, vimentin and dystrophin) was performed. Desmin positive areas reacted strongly with anti-midrod and C-terminus dystrophin antibodies. Probed with the same antibodies by Western blot, desmin and dystrophin showed normal molecular size but densitometric analysis demonstrated a parallel increase of both proteins. Our results indicate that intrasarcoplasmic desmin storage is associated with an abnormal accumulation of dystrophin. Since no other cytoskeletal proteins are accumulated this finding seems to be specific and suggests a possible structural and functional association between these two proteins in striated muscle.

Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.

A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.

Genotypic and cytogenetic study of acute myelocytic leukemia and chronic myelocytic leukemia in blast crisis: Specific δ rearrangement pattern does not involve J δ gene locus

We have analysed the configuration of immunoassociated genes and the karyotypes of 30 patients with acute myelocytic leukemia (AML) and 10 with chronic myelocytic leukemia in blast crisis (CML-BC). In AML, the frequencies of T-cell receptor (TcR) β, γ, and δ chain and immunoglubulin heavy and light chain gene rearrangements were 4.2%, 19%, 8%, 10.7% and 10.5%, respectively. In CML-BC, they were 10%, 20%, 40%, 50% and 0%, respectively. Nine patients had abnormalities in chromosome 2, 7 or 14, upon which immunoassociated genes are located. There seems to be no apparent relationship between these chromosome abnormalities and gene rearrangements. In all patients but one (5/6), the δ rearrangement was accompanied by other immunoassociated gene rearrangements. Molecular size analysis revealed specific δ rearranged band(s) (19.5 kb-BamHI and/or 6.9 kb-EcoRI), as commonly detected in B-acute lymphocytic leukemia (ALL). All the patients with the δ rearranged band, however, had a germline configuration of J δ gene loci, suggesting a DD or V(D)D (probably V δ2(D)D) pattern. This study also indicates that the δ rearrangement is specific in AML or CML-BC and distinct from that in early T leukemia/lymphoma.

Interleukin-1 stimulates proteoglycan and hyaluronic acid production by human gingival fibroblasts in vitro.

AbstractThe effect of recombinant interleukin 1β [IL‐1β] on proteoglycan and hyaluronic acid synthesis by human gingival fibroblasts has been investigated. It was found to stimulate gingival fibroblast proliferation in a dose dependent fashion with the midpoint of this response being in the 10−11 mol/L range. At a concentration of 10−11 mol/L, IL‐1β stimulated proteoglycan synthesis by 40 per cent. Although IL‐1β can stimulate cell proliferation and prostaglandin synthesis, its effect on proteoglycan synthesis was independent of these parameters. The kinetics of proteoglycan degradation in the presence or absence of IL‐1β was monitored by pulse chase experiments and were found not to differ between treated and untreated cultures. The molecular size and carbohydrate composition of the proteoglycans was not affected by IL‐1β. Additional studies revealed the synthesis of hyaluronic acid was also stimulated by IL‐1β. As for the proteoglycans, inhibition of cell proliferation did not affect the stimulatory effect of IL‐1β. However, blockage of prostaglandin synthesis abolished the stimulatory effect of IL‐1β on hyaluronic acid synthesis. The effect of IL‐1β on hyaluronic acid synthesis was found to be related to elevated levels of the enzyme hyaluronate synthetase. Molecular size analysis of newly synthesized hyaluronic acid revealed that cells treated with IL‐1β synthesized more large molecular mass hyaluronic acid. Taken together, these findings are considered to reflect the ability of gingival fibroblasts to respond to inflammatory mediators in a manner indicative of early tissue repair.

Fetomodulin: Marker surface protein of fetal development which is modulatable by cyclic AMP

A novel cell surface marker of fetal development was identified in both in vivo and in vitro systems of the mouse using monoclonal antibodies against a glycoprotein of an apparent size of 133,000 Da. Two independent clones of hybridomas were isolated by fusing murine myeloma cells, NS-1, with spleen cells of a rat which was immunized with murine 3T3 fibroblast. The analysis of molecular size and tryptic peptides of the immunoprecipitate indicated that fibroblast and putative parietal endoderm cells, which were derived by induced differentiation of F9 embryonal carcinoma cells with retinoic acid and cyclic AMP, expressed apparently the same protein. Undifferentiated F9 cells and F9 cells which were treated with retinoic acid or cyclic AMP alone had little or no immunoprecipitable proteins. Analogously, parietal endoderm of in vivo embryos tested positive for this protein but visceral endoderm and embryonic ectoderm did not. The amount of this surface protein was increased in fibroblast and differentiated F9 cells by elevation of intracellular cyclic AMP concentrations. These results are consonant with a hypothesis that this surface protein plays a role in fetal development via a quantitative modulation by cyclic AMP.

Ultrathin‐layer sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis and silver staining of urinary proteins

AbstractUltrathin‐layer sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis in combination with silver staining is a highly sensitive and rapid method for molecular size analysis of urinary proteins. First results are achieved after 4 h (electrophoretic run, 100 min, followed by Coomassie Brilliant Blue staining, 2 h). After additional silver staining, including a recycling step, the results are ready for evaluation within 7 h. Urine samples do not need to be concentrated. Within a single gel, 25–27 samples can be run side by side under identical conditions and thus compared without ambiguity. By coelectrophoresis of pure proteins and immunological analysis 13 urinary proteins could be identified and for 7 of them the detection limits were determined. Glomerular protein patterns in samples of urine with a protein concentration in the normal range are compared with the concentration of albumin and transferrin measured by a sensitive enzyme immunoassay. Typical renal and extrarenal protein patterns are shown.