An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

Background  

Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Replacement of P1 Leu18 by Glu18 in the reactive site of turkey ovomucoid third domain converts it into a strong inhibitor of Glu-specific Streptomyces griseus proteinase (GluSGP).

Turkey ovomucoid third domain with P1 Leu18 at its reactive site is an excellent inhibitor of chymotrypsin and elastase and of many other serine proteinases with related specificities. Semisynthetic replacement of P1 Leu18 by Lys18 causes the expected change into a trypsin inhibitor. Strikingly, semisynthetic replacement P1 Leu18 to Glu18 changes turkey ovomucoid third domain into a powerful inhibitor of Glu-specific Streptomyces griseus proteinase, GluSGP. Of the 131 natural avian ovomucoid third domains we have sequenced none have P1 Glu18, but several avian ovomucoid first domains have P1 Glu24. They are weak to moderate inhibitors of GluSGP.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Stimulation of mouse connective tissue-type mast cells by hemopoietic stem cell factor, a ligand for the c-kit receptor.

The proliferative capacity of mouse connective tissue-type mast cells (CTMC) was analyzed by using a newly discovered c-kit ligand, termed stem cell factor (SCF). More than 90% of CTMC in the peritoneal cavity responded to recombinant rat SCF (rrSCF) and were able to give rise to pure mast cell colonies in methylcellulose culture. Serial observation (mapping) of growth of individual CTMC in culture containing rrSCF confirmed their striking proliferative ability. No serum but accessory cells (non-CTMC cells) in the peritoneal population were required for the clonal growth of CTMC induced by rrSCF in our methylcellulose culture of whole peritoneal cells. The rrSCF-induced mast cell colony formation from peritoneal CTMC was completely inhibited by the addition of anti-c-kit antibody, which can block the binding of SCF to c-kit, to the culture. When IL-3 was combined with rrSCF, mast cell colonies dramatically increased in size. Mapping studies revealed that the combination of the two factors augmented the proliferative rate of CTMC. Approximately 60% of the constituent cells of the mast cell colonies which were formed from peritoneal CTMC in the culture containing rrSCF alone were stained with berberine sulfate, which is a characteristic of CTMC. However, most mast cells which were induced by rrSCF+IL-3 from peritoneal CTMC contained berberine(-)-safranin(-)-Alcian blue(+) granules. Although IL-4 exhibited little synergism with rrSCF in the induction of CTMC proliferation, the addition of IL-4 to the culture containing rrSCF+IL-3 resulted in an increase in mast cells which retained CTMC characteristics.     

General base-catalyzed ester hydrolysis as a model of the “charge-relay” system

The validity of the “charge-relay” system in serine esterases was examined by use of the general base-catalyzed hydrolysis of ethyl chloroacetate (I) as a model system. The general base catalytic rate for 2-benzimidazoleacetic acid (II) exhibited an eightfold positive deviation from the Brønsted plot including benzimidazole, imidazole, N-methylimidazole (V), and acetate ion, though in nucleophilic catalysis of the hydrolysis of p-nitrophenyl acetate, the point for II conformed to the Brønsted relationship together with imidazole and benzimidazole derivatives. The positive deviation of II from the Brønsted plot for the general base-catalysis was attributed to the cooperativity of the carboxyl group of II, the imidazolyl group of II, and the hydroxyl group of water. The present result provides support for the “charge-relay” system. Furthermore, the (essentially) total loss of the enzymatic activity due to N-3 methylation of histidine-57 in α-chymotrypsin is discussed in comparison to the general base-catalysis of V in the hydrolysis of I, which is also favorable for the “charge-relay” system.     

The use of cycloamylose to probe the “charge-relay” system

The effect of the combination of imidazolyl and carboxyl groups on the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin was examined to shed light on the role of the “charge-relay” system in serine esterases. 2-Benzimidazole-acetic acid, which has both the imidazolyl and carboxyl groups in the same molecule, accelerates the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin. On the other hand, neither benzimidazole (which has only an imidazolyl group) nor 2-naphthaleneacetic acid (which has only a carboxyl group) exhibited measurable acceleration. The cleavage of m-t-butylphenyl acetate by the α-cyclodextrin-2-benzimidazolecetic acid system takes place through inclusion complex formation between m-t-butylphenyl acetate and α-cyclodextrin, followed by catalysis associated with the combination of the carboxyl anion, the neutral imidazolyl group, and the alkoxide anion. The most probable explanation for the combination of the three groups in the catalysis involves nucleophilic attack by the imidazolyl group, assisted by the carboxyl and alkoxide anions. The mechanism of the combination of the imidazolyl, carboxyl, and hydroxyl groups is apparently different from those shown by the “charge-relay” system in enzymatic reactions.     

Characterization by pyocine typing and serotyping of oral and sputum strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients

Oral and sputum isolates of Pseudomonas aeruginosa in patients with cystic fibrosis were investigated. Of the 17 patients studied, 12 patients (71%) yielded both mucoid and nonmucoid variants of Pseudomonas aeruginosa from sputum and (or) various oral ecological sites, such as buccal mucosa, tongue dorsum, dental plaques, and saliva. A total of 51 strains of mucoid and nonmucoid Pseudomonas aeruginosa were isolated from these patients and were phenotypically characterized by both pyocine typing and serotyping. Five patients (42%) were colonized or infected by a single strain of Pseudomonas aeruginosa, whereas 7 patients (58%) were cocolonized or coinfected by two or more phenotypically different strains of Pseudomonas aeruginosa. To understand the mechanisms involved in Pseudomonas aeruginosa colonization, it may be necessary to identify multiple isolates of Pseudomonas aeruginosa not only from the sputum but also from the various oral ecological sites and to further explore the role of the oral cavity in this colonization.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

A conditionally immortalized epididymis caput cell line, MEPC5, was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and the cells grew continuously. However, the downregulation of T-antigen at a nonpermissive temperature of 39 degrees C and the upregulation of cell density at 33 degrees C were associated with growth arrest and the increased protein expression of p21(waf1), a cell cycle inhibitor. The cells expressed epididymal caput-expressed genes such as phosphatidylethanolamine binding protein, polyoma enhancer activator 3, ME1, sulfated glycoprotein-2 (SGP-2), androgen receptor, and retinoic acid receptor alpha. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions. The established mouse epididymis caput epithelial cell line MEPC5 retains some characteristics of differentiated epididymis epithelial cells, and should prove an excellent model for studies of gene expression and the physiological functions of epididymis caput epithelial cells.