Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.     

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-l-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer ofl-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 g/cm² of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 g/cm² also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004     

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Poly-l-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized byN-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-l-lysine substituted with several mannose derivatives and gluconoyl residues (GlcA_x-, Man_y-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficientin vitro than free MDP in macrophage cytostasis assays.Abbreviations MDP N-acetylmuramyl dipeptide - PLK poly-l-lysine - BSA bovine serum albumin - BOC t-butyloxycarbonyl - DMF dimethylformamide - DCHU N, N-dicyclohexylurea - DCCl N, N-dicyclohexylcarbodiimide - TEA triethylamine - Su succinimidyl - DMSO dimethylsulfoxide - FITC fluoresceinyl isothiocyanate - RPMI Roswell Park Memorial Institute - PBS phosphate buffered saline - Fl fluoresceinyl - GG glycyl-glycyl     

Routine heat inactivation of serum reduces its capacity to promote cell attachment

Summary Heat inactivation (56°C for 40 min) of bovine calf serum was shown to diminish its capacity to promote the attachment of cells to plastic or glass surfaces. This effect was not observed in stationary cultures (culture dishes) but became manifest under conditions in which the cells were subjected to a small amount of liquid shear force, i.e. by growing cells in roller bottles or culture tubes. Of four cell lines tested on bovine calf serum (SV-BHK, BALB-3T3, CV-1, and FS-4) SV-BHK and CV-1 cells showed the greatest sensitivity to loss of attachment-promoting activity. Fetal bovine serum also seemed to be affected by heat inactivation but to a lesser degree than bovine calf serum. Treatment of vessel surfaces with either unheated calf serum or specific attachment factors (gelatin, poly-d-lysine, and fibronectin) greatly increased cell attachment in the presence of heat inactivated serum. Heat inactivation did not seem to affect the ability of cells to grow after attachment. Of the four cell lines tested, the normal human fibroblast line (FS-4) was shown to be most effective at conditioning medium and restoring its capacity to promote the attachment of all four cell lines. This research was supported in part by VECOL, Inc., Bogata, Columbia and by grant SRC 5 U24 RR02557-02 from the National Institutes of Health, Bethesda, MD.     

Effect of polylysine-bound laminin on human retinoblastoma cell lines

Summary We have studied the responses of three human retinoblastoma cell lines (GM1232, Y79, and WERI-Rb1) to substratum-bound laminin (LN), fibronectin (FN), or collagen (CN) in serum-containing medium. About 95% of the cells attached to poly-d-lysine (PN)-pretreated plastic surfaces either unbound or protein-bound within 1 h after plating. With PN-bound LN, GM1232 cells showed an outgrowth of processes and cell spread within 1 d, but very little was seen on PN-bound FN, CN, or unbound PN even by Day 4. Both the percentage of cells with processes and the number of processes/cell were dependent on the amount of LN bound to the surfaces at Day 4. On LN surfaces without PN pretreatment, by Day 2 most cells had formed floating aggregates and were not attached to the surfaces. By Day 4 only a part of the peripheral cells of attached aggregates displayed process outgrowth and cell spreading. Dibutyryl cyclic AMP (dbcAMP) maintained these effects of PN-bound LN, and promoted process branching, cell spreading, and elongation with concomitant inhibition of cell growth. The percentage of cells with processes was 83%. Y79 and WERI-Rb1 cells, passed for several years, showed little and weak response to PN-bound LN either in the absence or presence of dbcAMP. These results indicate that the morphologic differentiation of GM1232 cells is elicited specifically by PN-bound LN, and that dbcAMP maintains and promotes this differentiated status.     

Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis

Inhibitors of proteolytic enzymes were tested for their ability to suppress the clinical signs and CNS lesions produced by injection of purified myelin in complete Freund's adjuvant into Lewis rats. Pepstatin or a series of neutral protease inhibitors including aprotinin, soybean trypsin inhibitor, leupeptin, antipain, transaminomethyl cyclohexane carboxylic acid (AMCA), -amino caproic acid (EACA) nitrophenyl guanidino benzoate (NPGB),d- andl-polylysine, or a new commercial protease inhibitor, dipropionyl Rhein (DPR) were injected daily beginning on day 7 after immunization of rats with myelin. Aprotinin and soybean trypsin inhibitor exacerbated the symptoms and lesions of experimental allergic encephalomyelitis (EAE), leupeptin and antipain had no effect, and the plasminogen activators AMCA, EACA, NPGB, as well as poly-l- and poly-d-lysine and DPR suppressed various aspects of EAE. The measurement of acid protease as a biochemical method for quantitation of the degree of cellular infiltration into the CNS is proposed, and the results with the various treatments presented. AMCA and NPGB may exert their effects at the site of entrance of the lymphoid cells into the CNS.     

Effect of pyruvate dehydrogenase complex deficiency on <Emphasis Type="SmallCaps">l</Emphasis>-lysine production with <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway. This study is dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday.     

In vitro study of alginate–chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure

The application of alginate–chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate–poly-L-lysine (APA) microcapsules for the application of cell therapy.     

Breeding anl-isoleucine producer by protoplast fusion ofCorynebacterium glutamicum

Summary Corynebacterium glutamicum R-18 is a strain forl-isoleucine production. Polyethylene glycol (PEG)-induced protoplast fusion was applied to improve the strain of thisl-isoleucine producer. Strain R-18 was fused with anl-lysine producerC. glutamicum S-37, becausel-isoleucine andl-lysine are synthesized from a common intermediate, aspartate--semialdehyde. Two thousand fusants were checked for their phenotypes. Most of the fusants accumulatedl-lysine, and only 0.9% of the fusants accumulatedl-isoleucine. Two strains, F-28 and F-91, were selected and cultivated in production medium. Fusant F-28 accumulated 12.1 g/l ofl-isoleucine and 4.8 g/l ofl-lysine, and fusant F-91 accumulated 4.8 g/l ofl-isoleucine and 13.0 g/l ofl-lysine, while the parental strains R-18 and S-37 accumulated 9.5 g/l ofl-isoleucine and 26.8 g/l ofl-lysine, respectively. Sugar consumption activity was improved by protoplast fusion, and thel-isoleucine production rate of F-28 was 2.4 times higher than that of R-18.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

l-[3H]lysine binding to rat retinal membrane: II. effect of kainic acid,d,l-α-aminoadipic acid,iodoacetic acid,and modification by dark-exposure

The rat retina and the different brain regions contain membranes sites that bindl-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: I) intraocular injection of kainic acid induces a reduction of the density ofl-lysine binding sites, II)d,l--aminoadipic acid injected into the eye enhances both kinetic parameters (B _max andK _d) ofl-[³H]lysine binding sites, III) the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and IV) the exposure to darkness of the rats reducesl-[³H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites forl-lysine after the exposure to darkness. The results show thatl-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

The deposition and stability of pectin/protein and pectin/poly-l-lysine/protein multilayers

The sequential deposition of pectin and protein – bovine serum albumin (BSA), β-lactoglobulin (BLG) and gelatin – to form multilayer structures was examined by Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR) and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited there was a progressive increase in mass deposited, with a more substantial deposition of protein. Pectin deposition led to a relatively hydrated, open structure which permitted binding of protein within the layer when the biopolymers carried an opposite net charge. On increasing the pH, disassembly of the structures occurred within the vicinity of the isoelectric point of the globular proteins. No disassembly was observed for the pectin/gelatin multilayer. When a globular protein was substituted for a poly-l-lysine layer in a pectin/poly-l-lysine multilayer it was displaced by the subsequent deposition of a poly-l-lysine layer, the more highly charged polycation displacing the relatively low charged polyampholyte. The pectin/poly-l-lysine/protein multilayers remained intact upon titration to pH 8.0.     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Differential structural requirements for the induction of cell attachment, proliferation and differentiation by the extracellular matrix

The subendothelial extracellular matrix (ECM) mediates the attachment of both human Ewing's sarcoma and colon carcinoma cells. Attachment and flattening of the sarcoma cells was sensitive to heat treatment but not to periodate oxidation of the ECM, whereas the colon carcinoma cells attached and flattened over heated but not periodate-treated ECM. Such differential sensitivity to heat treatment and periodate oxidation was also observed using purified fibronectin and laminin, respectively, but the inhibition of cell attachment was greater than with a similarly treated ECM. It is therefore conceivable that fibronectin and laminin specifically mediate the attachment and flattening of Ewing's sarcoma and colon carcinoma cells to the ECM, but that other constituents may support this attachment either directly or via interaction and stabilization of adhesive glycoproteins in the ECM. The ECM-mediated morphological differentiation of adult rat oligodendrocytes was sensitive to periodate oxidation to a much higher extent than to heat treatment of the ECM. In contrast, both treatments had only a small effect on the ECM-induced proliferation of vascular endothelial cells. These results indicate that different constituents of the ECM may be held responsible for its effects on different parameters of cell behavior, and that various cell types respond differently to a given modification of the ECM.