Bacterial action of fertilization envelope extract from eggs of the fish Cyprinus carpio and Plecoglossus altivelis

The vitelline envelope (VE) and fertilization envelope (FE) in eggs of the fish Cyprinus carpio and Plecoglossus altivelis were purified by homogenization of eggs or embryos in 5 mM Tris-HCl buffer, pH 7.0, containing 2 mM ethylenediamine tetraacetic acid disodium salt (EDTA), except for processing of VEs in Plecoglossus eggs, and by repeated washing wih the same buffer. To extract the outermost layer material, the purified VEs and FEs were processed overnight at 4 degrees C in 5 mM Tris-HCl buffer, pH 7.0, containing 8 mM 2-mercaptoethanol, 2 mM EDTA, 0.3 M alpha-lactose, 0.3 M glucose, and 0.9% NaCl. Since extraction of the outermost layer of the VEs of Cyprinus eggs in this solution was found to be ultrastructurally incomplete, further sonication in the same buffer was necessary. The solution extracted from purified VEs or FEs was dialyzed against 5 mM Tris-HCl buffer, pH 7.0, followed by lyophilization. The extracts from the FEs from both fish species contained two kinds of lectins, one agglutinated human B-type erythrocytes and the other nonspecifically agglutinated fish spermatozoa, and both extracts had a strong bactericidal effect on Vibrio anguillarum that was isolated from diseased cultured fish, but not on Aeromonas hydrophila and Escherichia coli. The extracts of purified VEs from eggs of both fish had no bactericidal effect on the bacteria examined, nor any agglutination effect on human erythrocytes and fish spermatozoa.     

Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide with the estrogen response element.

We studied the effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide (ODN1) harboring the estrogen response element (ERE) by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis. Putrescine and spermidine had no marked effect on the CD spectrum of ODN1. In contrast, spermine provoked and stabilized two characteristic changes in the CD spectrum. The first change was indicated by an increase in the intensity of the CD band at 280 nm at 0.5 mM spermine in Tris-HCl buffer containing 50 mM NaCl. This change appears to be related to changes in base tilt and conformational alterations similar to A-DNA. At 1-2 mM spermine, the CD spectrum was characterized by a loss of positive bands at 220 and 270 nm. This change might have contributions from polyamine-induced condensation/aggregation of DNA. Spectral measurements were also conducted in Tris-HCl buffer containing 150 mM NaCl to minimize contributions from condensation and aggregation of ODN1. Under these conditions, CD spectral changes were retained by (ODN1), although the magnitude of the change was diminished. In contrast, a control oligdeoxyribonucleotide (ODN2) having similar base composition did not show any significant change in the CD spectrum in the presence of 150 mM NaCl and 2 mM spermine. The changes in the CD spectrum of ODN1 were highly sensitive to polyamine structure, as evidenced by experiments using spermine analogs with altered number of -CH2- groups separating the amino and imino groups. Electrophoretic mobility shift analysis further showed ODN1 stabilization by spermine and its analogs. These data demonstrate the ability of an ODN containing ERE to undergo conformational transitions in the presence of polyamines and suggest a possible mechanism for polyamine-mediated alterations in the interaction of estrogen receptor with ERE.     

CD11b is a calcium-dependent epitope in human neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.     

Boron-containing oligodeoxyribonucleotide 14mer duplexes: enzymatic synthesis and melting studies.

A set of three 14mer oligodeoxyribonucleotides of sequence d(5'-CTATGGCCTCAG*CT-3'/3'-GATACCGGAGTCGA-5') containing G* variants either as 2'-deoxyguanosine phosphate (unmodified), N7-cyanoborane 2'-deoxyguanosine phosphate (base-modified) or 2'-deoxyguanosine boranophosphate (backbone-modified) were synthesized by template-directed primer extension. Both the N7-cyanoborane 2'-deoxyguanosine triphosphate and 2'-deoxyguanosine alpha-boranotriphosphate nucleotides are good substrates for Sequenase. We infer that a single Sp boranophosphate linkage (which has a stereochemistry equivalent to the corresponding Rp thiophosphate analog) is formed in the backbone-modified 14mer. Thermally induced helix-coil transitions were monitored for the hybridized duplexes using UV and circular dichroism (CD) spectroscopy. The CD spectra of the two types of boron-modified hybrids closely resemble the unmodified parent duplex, forming B-type helices in 150 mM NaCl, 1 mM EDTA, 10 mM phosphate, pH 7.4, buffer. UV melting results indicate that both hybrids have stabilities comparable with the parent duplex as measured by Tm or delta G degree 25. These studies indicate that singly modified base- or backbone-boronated DNA are good analogs of normal DNA.     

Salt-induced conformational transitions in chromatin. A flow linear dichroism study

The chromatin structure in solution has been studied by the flow linear dichroism method (LD) in a wide range of ionic strengths. It is found that increasing the ionic strength from 0.25 mM Na2EDTA, pH 7.0 to 100 mM NaCl leads to a strong reduction of the LD amplitude of chromatin and inversion of the LD sign from negative to positive at 2 mM NaCl. Chromatin exhibits a positive LD maximum value at 10-20 mM NaCl. These data enable us to conclude that in very low ionic strength (0.25 mM Na2EDTA) the nucleosome discs are oriented with their flat faces more or less parallel to the chromatin filament axis. Increasing ionic strength up to 20 mM NaCl leads to reorientation of the nucleosome discs and to formation of chromatin structures with nucleosome flat faces inclined to the fibril axis. A conformational transition of that kind is not revealed in H1-depleted chromatin. The condensation of the chromatin filaments with increasing concentration of NaCl from 20 mM to 100 mM slightly influences the orientation of the nucleosomes.     

Differential extraction of isoperoxidases from Pisum shoots

When green or etiolated Alaska pea shoots are extracted with water or 50 mM phosphate buffer, pH 6·5, or macerated without addition of buffer, the elution profile of the resulting supernatant lacks several isoperoxidase components which are readily extracted from the residue with 10 mM EDTA at pH 6·5. Subsequent extraction with 270 mM NaCl does not solubilize any further components. These results cast doubt on the validity of earlier reports on isoperoxidase patterns in Pisum and other plants.     

The low ionic strength form of the sodium salt of poly(dm5C-dG) is a B DNA.

The low-salt Z form of poly(dm5C-dG) reported in the literature was shown to be due to minute amounts of bivalent cations. If the polymer was purified by dialysis against 1 mM EDTA, poly(dm5C-dG) showed the normal B form. No change in this B form was observed up to 0.8 M NaCl. Very small amounts of Mg2+ or Ca2+ (10(-6) to 10(-5) M) induced the characteristic Z form CD spectrum, which could easily and rapidly be reverted to B-DNA by addition of EDTA.     

Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body

Human cathepsin K (EC 3.4.22.38) is a member of the cysteine protease family with high primary sequence homology to cathepsins S, L, and B. It has been shown that cathepsin K plays a major role in the resorption of the bone matrix by osteoclasts. Cathepsin K has a potential as a drug target for the diseases related to bone matrix metabolism such as osteoporosis. We have expressed recombinant human procathepsin K in Escherichia coli as inclusion bodies. Purified procathepsin K had size of 38kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50mM Tris-HCl, pH 8.0 buffer containing 5mM EDTA, 10 mM GSH, 1mM GSSG, 0.7 M L-arginine, 0.5 M NaCl, and 1% CHAPS and further dialysis against 25 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl. Mature active cathepsin K was prepared from refolded procathepsin K by incubating at 40 degrees C in pH 4.0 buffers with or without pepsin or cysteine. The presence of pepsin or cysteine in autocatalysis buffer did not have effect on the degree of conversion of nascent to mature cathepsin K, but reduced the autocatalysis time slightly. Proteolytic activity was confirmed using synthetic substrate, and Western blotting identified mature cathepsin K. Active cathepsin K had type I and II collagenolytic activities which could be inhibited by E-64.     

Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.     

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic, and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of nonequilibrium UV-melting and annealing curves in the range of pH 5.1-6.7 in a 10 mM citrate/phosphate buffer containing 0.1 M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both the association and dissociation process in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability, as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.     

Origin of the heterogeneous distribution of the yield of guanyl radical in UV laser photolyzed DNA

Oxidative guanine lesions were analyzed, at the nucleotide level, within DNA exposed to nanosecond ultraviolet (266 nm) laser pulses of variable intensity (0.002-0.1 J/cm(2)). Experiments were carried out, at room temperature, in TE buffer (20 mM Tris-HCl, pH 7.5; 1 mM EDTA) containing 35 mM NaCl, on 5'-end radioactively labeled double-stranded and single-stranded oligomer DNA at a size of 33-37 nucleobases. Lesions were analyzed on polyacrylamide gel electrophoresis by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. The quantum yields of lesions at individual sites, determined from the normalized intensities of bands, were plotted against the irradiation energy levels. Simplified model fitting of the experimental data enabled to evaluate the spectroscopic parameters characterizing excitation and photoionization processes. Results show that the distribution of guanine residues, excited to the lowest triplet state or photoionized, is heterogeneous and depends on the primary and secondary DNA structure. These findings are generalized in terms of excitation energy and charge-migration mediated biphotonic ionization. On the basis of the changes in the yield of the guanyl radical resulting from local helical perturbations in the DNA pi-stack, it can be assessed that the distance range of migration is <6-8 bp.     

Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells

More than 90% of rapidly-labelled nuclear RNA was associated with a nuclear matrix prepared from mouse leukemia L5178Y cells. The binding was not affected with up to 4 M NaCl; however, these RNAs were released from the nuclear matrix by treatment with a low ionic strength buffer (5 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.4 mM calcium chloride), without destruction of the sphere of the nuclear matrix. Actin filaments in the nuclear matrix were depolymerized with this buffer accompanied with rapidly-labelled RNAs. When the depolymerization was inhibited by slight modifications of the low ionic strength buffer (replacement of ATP by the same concentration of GTP; replacement of calcium ion by the same concentration of magnesium ion; addition of 20 micrograms/ml of phalloidine, which is a specific inhibitor of actin depolymerization), the release of rapidly-labelled RNAs from the nuclear matrix was also inhibited. The complex containing rapidly-labelled RNAs and matrix proteins was solubilized by a sonication from the nuclear matrix, and subjected to cesium chloride equilibrium centrifugation. Rapidly-labelled RNAs were concentrated on the bottom of the gradient accompanied with a small number of proteins (68K, 60K, 43K and 40K). The 43K protein was identified as actin by immunoblotting. By RNase digestion before equilibrium centrifugation, actin in the bottom fractions disappeared. These results suggest that rapidly-labelled RNAs anchor on the actin filaments in the nuclear matrix.     

Hydroxyapatite chromatography of phage-display virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.     

Influence of salts on the activity and the subunit structure of ornithine decarboxylase from rat liver

The influence of salts on the subunit structure and the kinetics of purified rat ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. Salts were found to cause subunit dissociation of the enzyme, producing the monomeric form of molecular weight 55 000 in the presence of 0.25 M NaCl/10 mM sodium phosphate buffer (pH 7.0): the molecular weight was estimated to be 150 000 in 10 mM and 250 000 in 1 mM sodium phosphate buffer. Inclusion of NaCl in kinetic assays of rat ornithine decarboxylase had little effect on maximal velocity. However, the Km value for L-ornithine was dramatically increased with increasing sodium chloride concentration: the presence of 0.25 M NaCl resulted in a 10-fold increase of the Km. Thus, the presence of salts caused dramatic changes both in the subunit structure and in the catalytic property of the enzyme, although a direct correlation between both the changes was not evidenced.