Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.     

Study of the interpolyelectrolyte reaction between chitosan and alginate: influence of alginate composition and chitosan molecular weight

The interpolyelectrolyte reaction between chitosan (CHI) and alginate (ALG) was followed by conductimetry and potentiometry. Five chitosan samples, all with almost the same degree of N-acetylation (DA approximately 0.20) and molecular weights ranging from 5 x 10(3) to 2.5 x 10(5) Da were used. The polyelectrolyte complex was formed using alginate samples with three different M/G values (0.44, 1.31 and 1.96). The composition of the complex, Z (Z = [CHI]/[ALG]) resulted 0.70 +/- 0.02, independently of the molecular weight of chitosan and the composition of the alginate used. The degree of complexation was 0.51 with no dependence on the alginate composition.     

Components in the inoculum determine the kinetics of Azotobacter vinelandii cultures and the molecular weight of its alginate

In cultures of Azotobacter vinelandii inoculated using washed cells (avoiding exhausted broth components) alginates of a higher molecular weight (1200 kDa) than those obtained in cultures conventionally inoculated (350 kDa), were produced. Also, when comparing conventionally inoculated cultures with those inoculated with washed-cells, the alginate lyase activity was delayed and the final polymer concentration decreased from 4.8 to 3.5 g l^–1. This suggests that components in the exhausted inoculum broth play important regulatory roles in alginate biosynthesis and needs to be taken into account when describing polymer biosynthesis.     

Molecular cut-off values of dialysis membranes for alginate and kappa-carrageenan oligosaccharides

The effective molecular weight cut-off values of dialysis membranes for carrageenan and alginate oligosaccharides were evaluated by gel permeation chromatography and nuclear magnetic resonance spectroscopy. For the different membranes tested, i.e. Medicell, Spectra Por 1000D and 3500D, the porous sizes are analogous to tri- and tetrasaccharides. A simple dialysis can be used to recover the majority of the oligosaccharides produced by a carrageenase or an alginate lyase digestion.     

海藻酸钠微胶囊制备及其在微生物包埋中的应用

海藻酸钠微胶囊作为一种包埋系统,因其价廉、无毒、生物相容性好、可生物降解等优点而备受关注.海藻酸钠微胶囊制备的研究一直是微胶囊制备的重要组成部分.本文概述了近年来海藻酸钠微胶囊的研究进展,包括主要制备方法及其影响因素,包埋微生物以改善微生物的应用性能等方面,并展望了海藻酸钠微胶囊在工业微生物等领域的发展.     

The lateral separation of contacts on erythrocytes agglutinated by polylysine

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10–225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 μm as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially peridic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

水杨醛保护法鉴定生物合成聚赖氨酸的单体连接方式

建立了一种有效、方便的分析和鉴定聚赖氨酸结构的方法。采用水杨醛与游离氨基反应生成席夫碱．用NaBH4将席夫碱C=N还原成C-N,在酸性条件下将还原产物水解,用薄层层析法分析了水解产物,根据生成的N保护氨基酸不同,鉴定了生物合成的聚赖氨酸的结构为ε-型结构。此法也可用于蛋白质或多肽的N-末端氨基酸的分析。     

Interfacial instability and the agglutination of erythrocytes by polylysine

Human erythrocytes have been exposed to poylysine of molecular weight range 4 to 220 kDa and concentration range 0.5 to 2,000 /ml at 37°C. Threshold concentrations for cell agglutination by the polycation have been determined for the samples of different molecular weight. Light and electron micrographs show that, in the erythrocyte agglutinates, cell-cell contact is generally made only at discrete, spatially periodic, regions which are distributed over a significant part of the cell surface. The average spacing between contact regions is 0.83 m. The cell membrane has a wavy profile between contact regions. Agglutination occurs only in cell samples whose electrophoretic mobility is significantly altered by polylysine and, in agreement with a previous report, occurs even when the electrophoretic mobility reaches high positive values. The electrophoretic mobility data implies that agglutination requires some protrusion of polylysine from the cell glycocalyx. We discuss how a resulting net attractive intercellular force could act to destabilize the aqueous layer between two cells, allowing surface wave growth which results in spatially periodic contact regions. Examples of situations where cell and membrane contact might be explained by the general concept of interfacial instability are discussed.     

Factors affecting the assay of histone H1 and polylysine by binding of Coomassie blue G

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.     

Stimulation of enzymatic activity in filament preparations of casein kinase II by polylysine,melittin, and spermine

Summary Casein kinase II (CKII) has been purified from bovine heart tissue. Under conditions of low salt (0.05 M NaCl, 10 MM MgCl₂), CKII forms structured aggregates that appear as filaments similar to results obtained withDrosophila CKII [C.V.C. Glover (1986) J. Biol. Chem. 261:14349]. The aggregates have been analyzed by sucrose density gradients and electron microscopy. Filament preparations of the enzyme have reduced but measurable kinase activity. The addition of salt restores activity. Various modulators of CKII activity have been examined with the enzyme in the low salt, polymerized form. The polyamines spermine or spermidine stimulated CKII activity as much as six fold; putrescine had no effect. Polylysine of varying lengths activated CKII 4–6 fold. Melittin, the basic polypeptide from bee venom, was also an effective activator. Activation of filament preparations was also observed if the CKII specific peptide (RRREEETEEE) was used as the substrate in place of casein. These results with filament preparations provide an alternative in vitro system for the study of possible regulatory aspects of CKII.     

Sustained and therapeutic levels of human factor IX in hemophilia B mice implanted with microcapsules: key role of encapsulated cells

BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein was explored in this study. In order to prevent immune rejection of the delivered cells, they were enclosed in non-antigenic biocompatible alginate microcapsules prior to being implanted intraperitoneally into mice. We have shown that encapsulated C2C12 myoblasts can temporarily deliver therapeutic levels of factor IX (FIX) in mice, but the C2C12 myoblasts elicited an immune response to FIX. In this study we report the use of mouse fetal G8 myoblasts secreting hFIX in hemophilia mice. METHODS: Mouse G8 myoblasts were transduced with MFG-FIX vector. A pool of recombinant G8 myoblasts secreting approximately 1500 ng hFIX/10(6) cells/24 h in vitro were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into immunocompetent C57BL/6 and hemophilic mice. RESULTS: Circulating levels of hFIX in treated mice reached approximately 400 ng/ml for at least 120 days (end of experiment). Interestingly, mice treated with encapsulated G8 myoblasts did not develop anti-hFIX antibodies. Activated partial thromboplastin time (APTT) of plasmas obtained from treated hemophilic mice was reduced from 107 to 82 sec on day 60 post-treatment, and whole blood clotting time (WBCT) was also corrected from 7-9 min before treatment to 3-5 min following microcapsule implantation. Further, mice were protected against bleeding following major trauma. Thus, the FIX delivery in vivo was biologically active. CONCLUSIONS: Our findings suggest that the type of cells encapsulated play a key role in the generation of immune responses against the transgene. Further, a judicious selection of encapsulated cells is critical for achieving sustained gene expression. Our findings support the feasibility of encapsulated G8 myoblasts as a gene therapy approach for hemophilia B.     

褐藻寡糖的制备方法及生物活性研究进展

褐藻胶是一类多糖聚合物,由于其独特的理化性质和有益健康的作用,已被广泛应用于制药和食品工业.然而,由于褐藻胶的水溶性低、黏度大,进而限制了褐藻胶的开发和应用.褐藻寡糖(alginate oligosaccharide,AOS)是褐藻胶的降解产物,由于其分子量低、水溶性高、安全无毒等特点,近年来受到广泛关注.AOS独特的...     

The molecular weight of the extracellular haemoglobin of Lumbricus terrestris determined by electron microscopy

1. The mol. wt of the extracellular haemoglobin of the oligochaete Lumbricus terrestris was determined by counting in negatively stained electron micrographs. 2. The value obtained using apoferritin as a mol. wt standard is (3.8 +/- 0.3) x 10(6), in agreement with recent determinations employing different physical methods. 3. We conclude that all annelid extracellular haemoglobins and chlorocruorins which have the same dimensions as Lumbricus haemoglobin probably have the same mol. wt.     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

Low molecular weight dextran: Immobilization of cells of Leuconostoc mesenteroides KIBGE HA1 on calcium alginate beads

Dextran is a long chain polymer of d-glucose produced by different bacterial strains including Leuconostoc, Streptococcus and Acetobacter. The bacterial cells from Leuconostoc mesenteroides KIBGE HA1 were immobilized on calcium alginate for dextran production. It was observed that dextran production increases as the temperature increases and after reaching maxima (30 °C) production started to decline. It was also observed that at 50 °C free cells stopped producing dextran, while immobilized cells continued to produce dextran even after 60 °C and still not exhausted. It was found that when 10 g% substrate (sucrose) was used, maximum dextran production was observed. Immobilized cells produced dextran upto 12 days while free cells stopped producing dextran only after 03 days. Molecular mass distribution of dextran produced by immobilized cells is low as compared to free cells.     

Characterization of reservoir-type microcapsules made by the solvent exchange method

The purpose of this reseach was to characterize and optimize the properties of microcapsules produced by the solvent exchange method, a new microencapsulation technique. Reservoir-type microcapsules containing lysozyme as a model protein were produced using a coaxial ultrasonic atomizer under various formulation and instrument settings, and characterized with respect to in vitro release kinetics and stability of the encapsulated protein. The solvent exchange method could encapsulate protein drugs with high efficiency under an optimized condition and was mild enough to preserve the integrity of the encapsulated lysozymes during the process. In vitro release studies showed that the microcapsules could release proteins in a controllable manner. The solvent exchange method is a mild and simple microencapsulation method that could encapsulate lysozyme, maintaining its functional integrity.     

Comparison of molecular weight determination of sodium alginate by sedimentation-diffusion and light scattering

The weight average molecular weight, M̄_w, of sodium alginates were determined by the sedimentation-diffusion technique using photon correlation spectroscopy rather than boundary spreading in the analytical ultracentrifuge to determine the translational diffusion coefficients. This enables the diffusion coefficients to be determined easily and accurately. Excellent correlation is found between the observed zero concentration translational diffusion coefficient, D_O and the M̄_W values in a emphirical power law. The M̄_W obtained by sedimentation-diffusion and laser light scattering compare very favourably. The concentration dependence of the photon correlation spectroscopy data allowed determination of the coil overlap concentration, c*. The inverse proportionality of c* to both M̄_W and [η] is demonstrated.     

Receptor of the serpentine-type and heterotrimeric G protein as targets of action of polylysine dendrimers

Molecular processes of the action of polycationic peptides that represent polylysine homo- and heterodendrimers on the functional activity of the biogenic amine- and peptide hormone-sensitive adenylyl cyclase signaling system (AC system) in rat myocardium and brains were studied. An intended use of these peptides is that of highly effective polymer carriers for biologically active substances. The polylysine homodendrimers of the third [(NH₂)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (I), fourth [(NH₂)₃₂(Lys)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (II), and fifth [(NH₂)₆₄(Lys)₃₂(Lys)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (III) generations, as well as polylysine heterodendrimers of the fifth generation, [(NH₂)₆₄(Lys-Glu)₃₂(Lys-Glu)₁₆(Lys-Glu)₈(Lys-Glu)₄(Lys-Glu)₂Lys-Ala-Ala-Lys(ClAc)-Ala-NH₂] (IV), [(NH₂)₆₄(Lys-Ala)₃₂(Lys-Ala)₁₆(Lys-Ala)₈(Lys-Ala)₄(Lys-Ala)₂Lys-Ala-Lys(ClAc)-Ala-Ala-NH₂] (V) and [(NH₂)₆₄(Lys-Gly-Gly)₃₂(Lys-Gly-Gly)₁₆(Lys-Gly-Gly)₈(Lys-Gly-Gly)₄(Lys-Gly-Gly)₂Lys-Gly-Gly-Lys(ClAc)-Ala-Ala-NH₂] (VI), interact with the C-terminal regions of α subunits of the heterotrimeric G proteins, preferably of the inhibitor type, and stimulate its activity in respector-independent manner. The most effective G-protein activators were homodendrimers II and III and heterodendrimer V. The polylysine dendrimers disturbed the functional coupling of receptors of biogenic amines and peptide hormones with G_i proteins and, to a lesser extent, G_s proteins. This was manifested as a decrease in the regulatory effects of the hormones on AC activity and the GTP binding of the G protein, as well as by a decrease in the affinity of receptors to agonists in the presence of polylysine dendrimers, which is a consequence of the dissociation of the receptor-G protein complex. It has also been shown that, based on their molecular mechanisms and selectivity of action on G proteins, polylysine dendrimers are similar to mastoparan and melittin, which are natural toxins of insect venom.     

Strong effect of the interaction potential cut-off on the crystallinity of films grown by simulations

The paper deals with the methodology of film growth simulations using classical molecular dynamics and an empirical interaction potential. We focus on the effect of the cut-off distance (r_C) of the short-range part of the potential. On the one hand, we find that r_C does not affect the qualitative conclusions of the simulations and that its quantitative effect is in the logical direction (better crystallinity at higher r_C). On the other hand, we show that the aforementioned quantitative effect is very strong, and clearly underestimated in the literature. The film crystallinity is affected by (non-)neglecting of as seemingly low energies as several meV per bond. The results are important for the design of growth simulations of crystalline films and for the correct interpretation of their results.     

The immunogenicity of soluble haptenated polymers is determined by molecular mass and hapten valence

T cell-independent Ag are believed to stimulate antibody formation in the relative absence of Ag processing and T cell help. Previous studies on the type 2 T cell independent (TI-2) Ag DNP-polyacrylamide, have shown that when one systematically varies the molecular mass and hapten valence, the immunogenic potential of this type of molecule depends on definable molecular characteristics. It was found that to be immunogenic, these molecules had to exceed a threshold molecular mass of 100,000 Da and a threshold hapten valence of 20. The present study was undertaken to determine whether such findings could be generalized to other molecules belonging to the TI-2 class of Ag. The molecular characteristics of five chemically different fluoresceinated (FL)-polymers were systematically varied, and their ability to stimulate an IgM antihapten immune response was measured. The polymers used as carriers were carefully size-fractionated and consisted of one natural polymer (dextran), one modified natural polymer (carboxymethyl cellulose), and three synthetic polymers (Ficoll, polyvinyl alcohol, and polyacrylamide). The carriers varied in physical structure from the highly cross-linked Ficoll, to the somewhat branched dextran, to the linear polyacrylamide, carboxymethyl cellulose, and polyvinyl alcohol. Polymers were haptenated with FL and size-fractionated so as to yield a panel of molecules with varying molecular mass, hapten valence, and hapten density. Anti-FL IgM response to these haptenated polymers was measured in vivo after i.p. injection of the FL-polymer in saline, and measured in vitro after culture with unfractionated spleen cells from naive mice. In agreement with the previous studies on DNP-polyacrylamide, it was found that to be immunogenic, each of the FL-polymers had to exceed a comparable threshold value of molecular mass and of hapten valence. Optimal immunogenicity occurred when the FL-polymers had values of mass and hapten density lying within a predictable range. Immunogenicity decreased when these optimal parameters were substantially increased or decreased. We conclude that the immunogenicity of soluble haptenated polymers depends on predictable physical molecular characteristics, and is relatively independent of the chemical composition and conformation of the carrier polymer.