Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

USE OF SENSORY EVALUATION FOR MEASURING DEODORIZING EFFECTS OF AN AIR CONDITIONER

The application of sensory methodology for measuring deodorizing effect of an air conditioner equipped with electric plasma was introduced. Deodorizing effect was measured using chemical and sensory methods at different time (0, 30 and 60 min) and mode (control, blowing and cooling) of an air conditioner. Smoke from a roll of cigarette in a closed room was used as a source of odor and the concentrations of acetic acid and ammonia were measured as odorous chemical components. As one of the sensory methods triangle test was used and as a first step to obtain deodorizing effects by triangle test, the threshold of each panelist was obtained as the log dilution ratio of odor concentration at which the difference from odorless air was detected. The odor concentration at each time and mode was calculated using the threshold of the panel and the deodorizing effect was obtained on the basis of the odor concentration. In addition to a triangle test, scaling methods such as category scaling or magnitude estimation were used to measure deodorizing effect of an air conditioner. Deodorizing effects by scaling methods were calculated based on odor intensity with time at each mode. The regression analysis was done between the efficacy of deodorizing effect by sensory test and those by acetic acid and ammonia, the R² values of the regression equations for triangle test, category scale, and magnitude estimation were 0.84, 0.72 and 0.69, respectively. Deodorizing effect by triangle test explained the decrease of acetic acid and ammonia better than those by category scaling or magnitude estimation while high cost and time consuming labor involved in triangle tests reduced the merit. The results of this study demonstrated that various sensory methods could be used to measure deodorizing effect of air conditioners and further researches on fast and reliable methods are needed to establish the official procedures.     

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.     

Length-dependent formation of parallel-stranded DNA in alternating AT segments

Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.     

The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study

Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit. (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer. (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites. (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1. IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1. An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated. Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA.     

Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

DNA Duplexes Containing Alpha Anomeric Nucleotides and Polarity Reversals: Coexistence of Parallel and Antiparallel DNA

Abstract

Oligodeoxynucleotides that possess alpha anomeric nucleotides and polarity reversals show promise for application in the area of antisense therapy. Here we provide a survey of the spectroscopic, thermodynamic, and enzymatic techniques used in our laboratories to investigate model systems containing such unnatural features with the ultimate goal of designing a new class of more potent and effective antisense therapeutics.