Fabrication and evaluation of carboxymethylated diethylaminoethyl cellulose microcarriers as support for cellular applications.

Cellulose based microcarriers can be used in biomedical science as supports for cell adhesion and proliferation. However, to facilitate cell attachment to their surface, they require appropriate functional surface charge. Cell function such as adhesion and growth is increased on the modified surfaces with cationic and anionic groups. In this research, diethylaminoethyl cellulose was carboxymethylated to produce soluble multifunctional cellulose with simultaneous presence of cationic and anionic functional groups. Then, carboxymethylated diethylaminoethyl cellulose (CM-DEAEC) were produced by ionic crosslinking. Various instrumental techniques were applied to characterize the microcarriers. Biological tests were also performed to determine cell seeding efficiency, proliferation and attachment on microcarriers. Fabricated CM-DEAEC microcarriers had 1500-1800 µm diameter, +26.0 surface potential, 376% swelling behavior and 233 °C glass transition temperature respectively. The findings showed that CM-DEAEC microcarriers support cellular attachment and proliferation very well and hence are promising materials for cell therapy and tissue engineering applications.

Replication initiation point mapping: approach and implications.

Duplication of eukaryotic chromosomes begins from multiple sites called origins of replication, with DNA synthesis proceeding bidirectionally away from the origin. There is little detailed information available pertaining to whether replication initiates at specific sites or anywhere within a given origin. The development of replication initiation point (RIP) mapping has made it possible to map start sites for DNA synthesis at the nucleotide level. The key step in RIP mapping is the purification of nascent DNA, which is initiated by small RNA primers. For the removal of broken DNA fragments, we utilize lambda-exonuclease, which digests DNA, but leaves nascent strands intact as long as they have the RNA primer still attached. RIP mapping is a sensitive technique and has been successfully applied to single copy loci in both budding and fission yeast, archaebacteria, and human cells. Studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Here, we present a detailed step-by-step protocol for RIP mapping of replication origins in budding yeast.

Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis.

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt conCentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.

High-resolution mapping of points of site-specific replication stalling.

Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.

Mapping origins of DNA replication in eukaryotes.

Methods are described here to map an origin of replication in eukaryotes. Replicating DNA is enriched by BND cellulose column chromatography and by lambda-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation. The general area in which replication begins can be deciphered by neutral/neutral 2D gel electrophoresis: a restriction fragment containing the replication bubble will form a bubble arc on these gels. A more sensitive method employs PCR analysis of nascent strands that are size-fractionated. Once the general area containing the origin of bidirectional replication has been mapped, a finer level of resolution can be obtained by replication initiation point (RIP) mapping, in which start sites of DNA synthesis are identified at the nucleotide level.

The effect of heparin and its neutralisation on functional assays for factor VIIa, factor VII and TFPI.

Recently methods have become available to assay the haemostasis proteins tissue factor pathway inhibitor (TFPI) and activated factor VII (FVIIa). These assays are primarily used in research and in some studies patients may be receiving heparin therapy. We investigated the effect of heparin and its neutralisation by protamine sulphate, hexadimethrine bromide (polybrene) and triethylaminoethyl (TEAE) cellulose, on functional assays for TFPI, FVIIa and also factor VII (FVII). In the clotting assay for FVIIa using truncated recombinant tissue factor, heparin had little effect up to IU/ml, but concentrations higher than this grossly prolonged the clotting time. Protamine and polybrene neutralisation of heparin resulted in some prolongation of the clotting time despite adequate heparin neutralisation, and in addition, in the absence of heparin each of these substances themselves affected the clotting time. TEAE neutralisation of heparin appeared to be effective in the FVIIa assay, although in the absence of heparin we observed a 5% decrease in the clotting time. In the amidolytic substrate assays for TFPI and FVII, heparin with or without neutralisation resulted in only small changes in the optical density at 405nm and hence plasma levels of these factors were not significantly changed.

Determination of soluble nAChR-binding activity of alpha-neurotoxins by an innovative precipitation with DEAE-Sephacel.

A simple and convenient method for determining the binding activity of alpha-neurotoxins toward soluble nicotinic acetylcholine receptor (snAChR) by precipitation with DEAE-Sephacel was established. The determination was carried out by incubation of 125I-neurotoxin with snAChR, followed by precipitation with DEAE-Sephacel. The DEAE-Sephacel particles bind negatively charged snAChR with high affinity and simultaneously precipitate the 125I-neurotoxin bound to the receptors. After centrifugation, the free 125I-neurotoxin in the supernatant was counted, and the amounts of neurotoxins bound to snAChR could be determined. Two alpha-neurotoxins, cobrotoxin and alpha-bungarotoxin, were employed to verify the feasibility of this determination. The different binding characteristics of cobrotoxin and alpha-bungarotoxin to snAChR could be distinguished. This method required only small quantities of DEAE-Sephacel (7 mg), snAChR (0.54 micrograms), and 125I-neurotoxin (90 fmol) for each reaction, and minimized the handling of isotopic materials as compared with the conventional methods. This method is reliable, reproducible, and superior to current methods for the determination of the snAChR-binding activity for alpha-neurotoxins.

Partially phosphorylated glycogen phosphorylase in the lugworm Arenicola marina, its regulatory function during hypoxia.

Glycogen phosphorylase (GPase) from the body wall of the lugworm Arenicola marina (Annelida, Polychaeta) probably exists as a phospho-dephospho hybrid (GPase ab). The hybrid was identified by phosphorylation of purified lugworm GPase b (unphosphorylated form) with rabbit muscle GPase kinase and [gamma-32P]ATP. The completeness of phosphorylation was checked on DEAE-Sephacel. Only one GPase form was eluted. Its 32P incorporation was determined to 0.52 +/- 0.08 mol 32P/100,000 x g protein (n = 4). This GPase ab produced by in vitro phosphorylation has shown similar dependences on AMP and caffeine as GPase extracted from the body wall of the lugworm. Its reversible conversion with endogenous phosphatase and kinase to GPase b has also been demonstrated while a completely phosphorylated form (GPase a) was not detected neither in vivo nor in vitro. Lugworm GPase ab has shown a 2.4-fold higher specific activity as GPase b. The Km for P(i) was 16 mmol/l in absence and 13 mmol/l in presence of AMP. Half maximum activation by AMP was reached at 9 mumol/l. IMP up to 10 mmol/l did not activate and ATP up to 4 mmol/l did not inhibit GPase ab in absence of AMP.

Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties.

Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.

Efficient production and isolation of recombinant amino-terminal half-molecule of human serum transferrin from baby hamster kidney cells.

Expression of the amino-terminal lobe of human serum transferrin secreted into the culture medium by transformed baby hamster kidney (BHK) cells has been increased from the levels reported originally of 10-15 micrograms/ml to 55-120 micrograms/ml. Use of the serum substitute, Ultraser G, has facilitated isolation of the recombinant protein, resulting in approximately 80% recovery of expressed hTF/2N from the culture medium. In the three experiments described, 300-750 mg of recombinant protein was collected over a period of 25 days from five expanded surface roller bottles each containing 200 ml of medium (seven to nine collections). The use of alginate beads to encapsulate the transformed BHK cells provided no advantage over normal culturing over 25 days. A lag in production resulting in 30% less recombinant protein over this time period was observed. The production and isolation procedures described are easily handled by one person. The system is amenable to incorporation of isotopically substituted amino acids useful in NMR studies.

Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells.

In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to [3H]thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

Partial purification and properties of Cunninghamella elegans L-alanine dehydrogenase.

L-Alanine dehydrogenase was partially purified from the mycelial extracts of Cunninghamella elegans. The purified enzyme was fractionated by TEAE-cellulose column chromatography into two fractions (subunits) designated as fractions I and II. The activity of both fractions in the aminating reaction is 8 times higher than the activity of the deaminating reaction. pH-Optimal for reductive amination of pyruvate by both fractions were 8 and 10 for oxidative deamination of L-alanine. The Km values of fractions I and II for L-alanine, NAD+, pyruvate, NH4+ and NADH were estimated. Both fractions of L-alanine dehydrogenase were absolutely specific for L-alanine in the oxidative deamination reaction. Maximal activity of both fractions occurred at 40 degrees C. Inhibition by iodoacetate and activation by SH-compounds suggest that sulfhydryl groups may participate in enzyme activity. The 2 fractions were activated with Co2+, Fe2+, Ca2+, Mg2+ and Mn2+ ions, while Zn2+ inhibited both fractions. Stability of the enzyme under different conditions was investigated.

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography.

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

Crosslinked concanavalin A-O-(diethylaminoethyl)-cellulose--an affinity medium for concanavalin A-interacting glycoproteins.

When concanavalin A (Con A) is reacted with a low concentration of glutaraldehyde, the product formed strongly binds to DEAE-cellulose. Thus, the resultant material can be used as an affinity medium for those glycoproteins which interact with Con A. This affinity medium is easy to prepare, has a capacity comparable to that of similar commercially available affinity media, and is stable for up to at least 6 months.

Anticoagulant activity in cell-free peritoneal fluid of an experimental pancreatic ascites tumor.

The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted.

Limitations on the stability of benzoylated DEAE-cellulose.

As used for structural analysis of DNA, benzoylated DEAE-cellulose is subject to deterioration which affects chromatographic performance. This change, which specifically occurs during storage of the resin, has been characterized in terms of anomalous patterns of DNA binding. Subjecting benzoylated DEAE-cellulose to boiling in aqueous suspension, and appropriate storage, ensures maintenance of normal affinity for DNA.

Further evaluation of a heparin neutralizer and its effect on factor IX in normal and coumadin-plasma.

Heparsorb, a commercial anion exchange resin capable of removing large amounts of heparin from heparinized plasma, has recently been introduced into the clinical diagnostic laboratory as a useful reagent for the evaluation of blood coagulation in heparinized blood and the evaluation of an unexpected prolongation of global coagulation tests. In this paper data are presented which indicate that Heparsorb has little or no effect on the activities of blood coagulation factors in heparinized plasma, except for a modest (22%) reduction in factor IX-activity. Maximal loss of factor IX-activity was observed after incubation of plasma with Heparsorb for 15 min at room temperature. Prolonged storage of plasma before or after incubation with Heparsorb and changes in plasma pH had no appreciable effect on the extent of factor IX-activity loss. Evidence is presented to substantiate earlier findings that factor IX-activity is not removed by Heparsorb from plasma of patients on coumadin therapy, and to indicate that this lack of effect of Heparsorb on factor IX-activity in coumadin plasma is due to reduced affinity of hypocarboxy-IX for the heparin neutralizing resin.