Cell immobilization in k-carrageenan with tricalcium phosphate

The objectives of this research were to investigate the growth of immobilized yeast cells in k-carrageenan gel and study the effect of trapping hydroxyapatite (tricalcium phosphate) crystals into the matrix. Using k-carrageenan, the final number of cells per milliliter of gel is at least an order of magnitude higher than free cells per milliliter of medium. A "cell retention" theory explaining this cell concentration difference was proposed. Coexistence of yeast cells and an additional agent such as tricalcium phosphate results in sustained viability through internal pH control, increased cell loading, greater settling velocity, and enhanced ethanol production.     

Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety

Extracts of the vine-like plant Tripterygium wilfordii (TW) have been widely used in China as an immunosuppressant and anti-inflammatory drug for the treatments of rheumatoid arthritis, lupus erythematosus and other inflammatory disorders. In this study the molecular mechanisms of action of three TW extracts (ethanol, aqueous, polysaccharide) on the expression of inflammatory cytokines and adhesion molecules were investigated by RT-PCR and immunofluorescence binding techniques. The lipopolysaccharide (LPS)-mediated stimulatory effects of tumor necrosis factor-alpha (TNF-alpha) cytokine production and cell adhesion molecule (CD11c, CD18, CD14, CD54) expression in human monocytic THP-1 cells were modulated by treatments of the TW extracts or tacrolimus (FK506). The TW polysaccharide moiety exhibited more profound immunosuppressive properties than the aqueous and ethanol extracts. Biochemical characterization of the polysaccharide moiety revealed a major molecular weight of 22 kDa (viz. PSP22). The PSP22 was found to be a potential immunosuppressant that manifests the necessary immunomodulating properties.     

Depolymerization of chondroitin C Sulfate by immobilized Proteus vulgaris cells

Chondroitinase ABC catalyzing the depolymerization of chondroitin sulfate was induced by incubating the Proteus vulgaris cells in a medium containing chondroitin C sulfate as an inducer. Incubation of P. vulgaris cells for 12 h in the presence of 0.3% inducer was optimal to obtain the cells with highly active chondroitinase ABC. Such cells were immobilized in k-carrageenan gel lattice, and some properties of chondroitinase ABC in immobilized cells were studied in comparison with those of the enzyme without immobilization (free enzyme). The stabilities of the enzyme toward heat and storage were remarkably improved by immobilizing the cells in k-carrageenan gel lattice. Optimal pH and temperature for activity of the enzyme were slightly shifted to the alkaline region and higher temperature by immobilization and were 9.0 and 35 degrees C, respectively.     

In vitro facilitating role of polygonatum sibiricum polysaccharide in osteogenic differentiation of bone marrow mesenchymal stem cells from patients with multiple myeloma

Background

Bone marrow mesenchymal stem cells (BMMSCs) were proved to play a vital role in multiple myeloma (MM). Polygonatum sibiricum polysaccharide (PSP) was found to have anti-tumor pharmacological effects, yet its interaction with BMMSCs remained poorly understood. Therefore, we explore the effect of PSP on osteogenic differentiation of BMMSCs.

Methods

BMMSCs were isolated by density gradient centrifugation. CD90 and CD34 were detected by flow cytometry (FCM). Osteogenic marks were detected by quantitative real-time PCR (qRT-PCR) and Western blotting (WB). The vitality of cells treated with different concentrations of PSP was observed by Cell Counting Kit-8 (CCK-8). ALP staining kit was used to detect the activity of alkaline phosphatase (ALP). Alizarin red staining detected the formation of mineralized nodules. Osteoblast-associated genes were evaluated by qRT-PCR and WB. The phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) signaling pathways were tested by WB.

Results

The BMMSCs showed good growth under an inverted microscope. FCM showed that CD34 and CD45 was low-expressed, whereas CD44, CD90 and CD105 was highly expressed. Compared with the Control group, the expressions of Runx2 and ALP in cells were significantly increased. CCK-8 showed that different concentrations of PSP had no significant effect on the viability of BMMSCs. BMMSCs treated with 25 mg/l PSP were stained the most deeply by ALP. Mineralized nodules in PSP groups dramatically increased, and hit a peak under the action of 25 mg/l PSP. PSP up-regulated p-PI3K, p-AKT, and p-mTOR, but had no significant effect on PI3K, AKT, and mTOR.

Conclusion

PSP induced osteogenic differentiation of BMMSCs from MM patients.

     

Recombinant PSP94 (prostate secretory protein of 94 amino acids) demonstrates similar linear epitope structure as natural PSP94 protein

PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer. Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen. Since GST-PSP94 was expressed in E. coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure. This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent. The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences. Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini. © 1996 Wiley-Liss, Inc.     

Small-angle x-ray scattering of κ-carrageenan based systems: Sols,gels, and blends with carob galactomannan

Small-angle x-ray scattering using a synchroton x-ray monochromatic radiation was carried out to investigate the structure of different polysaccharides in aqueous medium: carob galactomannan, κ-carrageenan in the sol and in the gel states, and κ-carrageenan-carob galactomannan mixed systems. Experiments performed on a 0.2% carob galactomannan solutions confirmed that this polysaccharide behaved as a neutral polymer in a good solvent. For K-carrageenan in the / state, either in the sodium form or in the cesium form, a maximum in the scattering curve was evidenced. Position and height of this maximum changed with K-carrageenan concentration in close agreement with what is expected for wormlike polyelectrolyte in semidilute solution. In the case of k-carrageenan in the gel state, in the cesium form, scattering curves also exhibited a maximum at an intermediate Q value. The position of this correlation peak did not change with concentration while its intensity increased. This effect was ascribed to a packing of rodlike structures by analogy with a suspension of colloidal elongated particles. This local structure could be viewed as bundles of parallel double helices. Addition of carobgalactomannan in κ-carrageenan gels induced dramatic structural changes. As the galactomannan concentration increased, the correlation peak tended to vanish. In contrast, no change in the cross-sectional radius of gyration was noticed. This phenomenon suggested a screening effect of the galactomannan, resulting in a loss of the correlation between the κ-carrageenan double helices. © 1995 John Wiley & Sons, Inc.     

Pancreatic spasmolytic polypeptide (PSP): I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas

A novel polypeptide, named Pancreatic Spasmolytic Polypeptide (PSP), was discovered in a side-fraction from the purification of porcine insulin. PSP was prepared by two different purification methods based on combinations of precipitations, anion-exchange and cation-exchange chromatography. The highest yield obtained, 52 mg PSP/kg pancreas, indicates that the content of PSP in porcine pancreas is about half the content of insulin. Both preparations appeared to be very pure as judged by basic disc electrophoresis, isoelectric focusing, analytical gel filtration and radioimmunoassays for various polypeptides known to be present in pancreas. The PSP molecule contains 106 amino acids (MW about 11 700). PSP is an acidic (pI 4.4), non-glycosylated protein without free N-terminal amino groups, and with high contents of proline and cystine. The high content of S-S bridges (7 per molecule), an unexpected low apparent MW determined by gel filtration, and a remarkable resistance towards treatment with trypsin and chymotrypsin, point to a compact structure of the PSP molecule.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Physiological and chemical characteristics of antibacterial activity of pancreatic juice.

Attempts were made to find and characterize an antibacterial activity (ABA) factor in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor was found to be heat resistant, the first step was heating for 30 min at 65 degrees C. Afterwards column chromatography, ethanol precipitation and polyacrylamide gel electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with antibacterial activity against Escherichia coli strain AB1157. In the presence of this fraction the number of living bacterial cells in overnight culture decreased about 10,000 fold and a spot-test gave clearly positive results. The results of analysis suggest that the antibacterial factor is a polypeptide active in a pH range 8.0-8.5, that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass spectroscopy analysis of active fraction showed high concentration of porcine pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling bacterial homeostasis has been found in the porcine pancreatic juice.     

Immobilization of enzymes and microbial cells using carrageenan as matrix.

Conditions for the gelation k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shape such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.     

松子壳多糖对小鼠主要免疫细胞功能的影响

目的探讨松子壳多糖（pine nut shell polysaccharide,PSP）对小鼠主要免疫细胞的影响。方法应用MTT法测定PSP对小鼠脾淋巴细胞的毒性和对ConA或LPS诱生小鼠脾T、B淋巴细胞的转化,用中性红吞噬试验测定腹腔巨噬细胞的吞噬功能,应用乳酸脱氢酶释放法测定NK细胞的杀伤活性。结果 PSP对脾细胞毒性很低,各种浓度对小鼠脾淋巴细胞的增殖均有较强的促进作用（P〈0.01）;PSP在浓度50～300μg/mL时,明显促进T淋巴细胞的转化（P〈0.01）,但是当浓度达到300μg/mL时表现出一定的抑制作用（P〉0.05）;PSP在25～200μg/mL时显著促进了小鼠脾B淋巴细胞的转化（P〈0.05）,但是当浓度达到300μg/mL时,抑制作用极显著（P〈0.01）;不同浓度均可以增强小鼠腹腔巨噬细胞吞噬中性红的能力及其代谢功能,当浓度在100μg/mL时能显著的促进巨噬细胞吞噬中性红的能力（P〈0.01）;PSP在浓度100～300μg/mL时,能极显著的促进NK细胞对Yac-1的杀伤作用（P〈0.01）,当浓度达到400μg/mL,对NK细胞杀伤性的促进作用开始减弱。结论 PSP对小鼠脾淋巴细胞的毒性较低,能增强免疫细胞活性,有望成为新一代免疫调节剂。     

Extraction of nontype-specific antigens of group A Streptococcus by potassium thiocyanate

A method for extraction of nontype-specific antigens of the group A streptococcus cellular wall from whole microbial cells is described. Potassium thiocyanate was used as an extracting agent. Nontype-specific antigens of the thiocyanate extract purified by ammonium sulfate precipitation were examined by immunodiffusion in agar gel. The thiocyanate extracts were found to contain several nontype-specific protein antigens part of which were absent from the HCl-extracts. No group A streptococcal polysaccharide was found in the thiocyanate extracts.     

Growth and sporulation of entrapped Bacillus subtilis cells

Growing Bacillus subtilis cells were immobilized in k-carrageenan gel beads. Growth and sporulation of entrapped cells were studied by two different methods: cell enumeration and transmission electron microscopy. Immobilized growing cells had a shorter generation time than free cells did, whereas sporulation timing was unchanged. When steady state was reached, cell density was very high in gel beads.     

黄精多糖组成及其抗氧化活性分析

分离纯化湖南新化产黄精多糖,并对其组分、结构特征和抗氧化活性进行研究。本研究采用碱提法、Sevag法脱蛋白得水溶性粗多糖(PSP),并经DEAE-52和Sepharose CL-6B色谱柱分离纯化得黄精多糖分级组分。通过紫外、红外光谱和GC-MS进行初步结构分析,并检测其体内外抗氧化能力。结果 PSP经纯化得1个组分PSP-1a,不含蛋白质和核酸,具有典型多糖吸收峰,主要由半乳糖、阿拉伯糖、鼠李糖、木糖、葡萄糖组成,其摩尔百分含量分别为0.18%、1.50%、97.25%、0.77%和0.3%。体外抗氧化活性研究表明:PSP和PSP-1a均具有一定抗氧化性能力,且随浓度(1~10 mg/L)增大而增强,PSP-1a的抗氧化能力优于PSP,但弱于VC。体内实验显示,PSP-1a改善了高脂膳食肥胖小鼠结肠匀浆组织的氧化应激状况。     

Mapping of the binding sites involved in PSP94–CRISP-3 interaction by molecular dissection of the complex

Background

Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known.

Methods

In order to identify the residues and/or regions involved in PSP94–CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP).

Results

For PSP94, amino acids Y³, F⁴, P⁵⁶ and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C³⁷A–C⁷³A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94–CRISP-3 complex has been proposed.

Conclusion

The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3.

General significance

Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.     

The polysaccharide component of Pseudomonas pseudomallei slime

The polysaccharide component contained in the slime of the causative agent of melioidosis was obtained. This component was found to be a biopolymer, mainly of the carbohydrate nature, consisting of galactose, glucose, mannose, rhamnose and two unidentified carbohydrates. The slime polysaccharide component contained two thermostable and acid-resistant antigens. The action of alkali led to the loss of one of these antigens. Rabbit antisera to the preparations of the slime polysaccharide component with titers of 1:64 to 1:256, determined in the immunodiffusion test, were obtained. In the precipitation test the slime polysaccharide component reacted with antisera from sick experimental animals, thus confirming the suggestion of its secretion in the process of the development of melioidosis infection.     

Microencapsulation of nanoparticulate complexes of DNA with cationic lipids and polymers in pectin beads for targeted gene delivery

Nanoparticulate complexes of plasmid DNA (pDNA) with cationic liposomes/polymer, of approx 200 nm diameter, were encapsulated with a high degree of efficiency within calcium pectinate gel beads. Electron microscopy showed the DNA nanocomplexes to be evenly distributed throughout the gel matrix. Controlled release of pDNA-lipid nanocomplexes was achieved by the action of pectinase enzymes, whereas release of naked and polymer-complexed DNA was found to be more greatly influenced by the swelling behavior of the polysaccharide matrices in buffer alone. Physical degradation of pDNA within pectin beads was found to be accelerated during bead drying, most probably as a result of shear forces generated within the gel matrices by the evaporation of water. Plasmid complexation with cationic liposomes provided a greater degree of protection for the DNA during bead drying than complexation with cationic polymer, and was shown to successfully transfect cultured cells after release from the beads, via the action of pectinase. Observations concerning the physical stability of nanocomplexed pDNA, and its encapsulation within and release from pectin gel beads, are discussed with reference to the electrostatic interactions existing between the various components.     

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.