Separation and concentration of delta 17-6-keto-PGF1 alpha using monoclonal antibody to omega 3-olefin structure of trienoic prostanoids.

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-prostaglandin F1 alpha (PGF1 alpha) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal antibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for omega 3-olefin structure. The 4G9-12B antibody became more specific for delta 17-6-keto-PGF1 alpha than 6-keto-PGF1 alpha by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, omega 3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating delta 17-6-keto-PGF1 alpha in the human blood or urine.     

The difference in the type of codon-anticodon base pairing at the ribosomal P-site is one of the determinants of the translational rate

By utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNA(Phe) (anticodon; GmAA) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U.S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

Elongation of oligonucleotides in the 3'-direction with activated mononucleotides and their analogs using RNA ligase.

P1-Adenosine 5'-P2-2',3'-ethoxymethylidene nucleosides [A(5')ppN(Em)] from four common nucleosides have been prepared and used for single addition of nucleotides to elongate oligonucleotide chains in the 3'-direction in RNA ligase reaction. U-U-C, T-U-C and A-C-C were used as acceptors. Structural dependence in these acceptors was found to be smaller compared to joining reactions between oligonucleotides. Adenosine analogs including 8-bromo-, 2'-fluoro-, 2'-azido-, 8,2'-O-cyclo-, 8,2'-S-cyclo-adenosine, arabinosyladenine and 2'-deoxyadenosine were added to the 3'-end of A-C-C by adenylation chemically followed by joining with RNA ligase. Symmetrical 5'-pyrophosphates of 8-bromo-, 2'-fluoro- and 2'-azido-adenosine were not recognized as donor substrates.     

Characteristics of Murayama virus in various cell cultures and laboratory animals

Some biological properties of Murayama virus, a new paramyxovirus, were studied. The virus grew well in primary monkey kidney cells as well as embryonated eggs, while the virus yields in primary chick embryo and BHK-21 cells were much lower. The infected BHK-21 cells formed large syncytia and showed typical hemadsorption, but did not produce any detectable amount of hemagglutinin in the culture fluid. The virus yields were very low in Vero. LLC-MK2 and MDCK cells at first passages. The addition of trypsin to the medium enhanced virus growth in Vero and LLC-MK2 but not in MDCK cells. Cell fusion activity of the virus was observed in Molt-4 cells. Hemolytic activity was enhanced by freeze-thawing. Several species of mammals and birds were susceptible to experimental infections with the virus as evidenced by seroconversion and positive virus isolation without showing any clinical signs.     

Large-scale isolation of equine liver alcohol dehydrogenase on a blue agarose gel.

Equine liver alcohol dehydrogenase (EC 1.1.1.1) has been purified by a new scheme using a blue agarose gel (Blue Sepharose) as an affinity sorbent. Starting amounts of 0.6 to 10 kg liver have been processed to enzyme possessing 1.5 U/mg average specific activity, in about three to four days. Some parameters concernining adsorption of enzyme to the blue gel as well as recovery therefrom have been explored.     

Effects of the anti-lipogenic antibiotic cerulenin on growth and fatty acid composition of n-alkane-utilizingCandida lipolytica

Summary The effects of cerulenin, an anti-lipogenic antibiotic, on the growth and cellular fatty acid composition ofCandida lipolytica were investigated by changing the chain length of n-alkane, the growth substrate. The antibiotic inhibited almost completely the growth of the yeast on glucose, n-undecane and n-dodecane, but partly that on n-tridecane. The yeast growth on longer alkanes, e.g., from n-tetradecane to n-octadecane, was not affected by this antibiotic, indicating that a chain elongation system and/or intact incorporation system predominantly operate in the formation of cellular fatty acids from such longer chain n-alkanes. Comparison of the fatty acid profiles between the cells grown on n-alkanes of different chain lengths, especially on n-pentadecane, in the presence and absence of cerulenin, supported the supposition that only the de novo synthesis system of the yeast would be affected by the antibiotic, whereas the chain elongation system would not.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.     

Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications

Nonylphenol (NP) is an important intermediate in the production of various commercial and industrial materials, but is also known as a ubiquitous pollutant in urban aquatic environments. We recently studied the NP-degrading activities of microflora in several aquatic environments, and found a notable degrading activity for wastewater from a sewage treatment plant in Tokyo. This result led us to isolate NP-degrading microbes and identify biodegradation products. Using conventional plate culture techniques and molecular biological methods, Pseudomonas and Sphingomonas species, which are known for their degradation activities of many aromatic compounds, have been isolated. But it has also been found that Sphingomonas sp. (S-strain) is necessary and sufficient for the degradation of NP. Although the role of Pseudomonas sp. (P-strain) remains unclear, P-strain seems to provide some co-nutrients for the growth of S-strain. The degradation products were analyzed by GC/MS and NMR. More than 95% of NP was degraded within 10 days and aromatic compounds other than NP were not found, suggesting that the phenolic part of NP was completely degraded. We also examined the potential of S-strain for bioremedial applications. S-strain cells immobilized on chitosan or alginate beads retain their NP-degrading activity in flask-scale experiments. Furthermore, the chitosan-bound cells in a lab-scale bioreactor have been found to be persistent for repeated use, suggesting that S-strain is applicable to the treatment of NP-contaminated wastewater.