Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.     

Analysis of eighteen deletion breakpoints in the parkin gene

Parkin mutations are responsible for the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP). On initial screening of Japanese patients with AR-JP, we had found that approximately half of the parkin mutations are deletions occurring between exons 2 and 5, forming a deletion hot spot. In this study, we investigated the deletion breakpoints of the parkin mutations in 22 families with AR-JP and examined the possible association between these deletion events and meiotic recombinations. We identified 18 deletion breakpoints at the DNA nucleotide sequence level. Almost all these deletions were different, indicating that the deletion hot spot was generated by recurrent but independent events. We found no association between the deletions and specific DNA elements. Recent copy number variation (CNV) data from various ethnic groups showed that the deletion hot spot is overlapped by a highly polymorphic CNV region, indicating that the recurrent deletion mutation or CNV is observable worldwide. By comparing Marshfield and deCODE linkage maps, we found that the parkin deletion hot spot may be associated with a meiotic recombination hot spot, although such association was not found on comparison with recent high-resolution genetic maps generated from the International HapMap project. Here, we discuss the possible mechanisms for deletion hot spot formation and its effects on human genomes.     

Variation in stress resistance patterns among stx genotypes and genetic lineages of shiga toxin-producing Escherichia coli O157

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Local Anesthetics and Antipsychotic Phenothiazines Interact Nonspecifically with Membranes and Inhibit Hexose Transporters in Yeast

Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake, but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt⁰ strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not the HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby mitigating the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects based on lipid theory.     

Extreme habitats that emerged in the Pleistocene triggered divergence of weedy Youngia (Asteraceae) in Taiwan

Weeds with broad distributions and large morphological variation are challenging for systematists and evolutionarily intriguing because their intensive dispersal would likely prevent local morphological differentiation. Study on weeds will help to understand divergence in plants unlikely to be affected by geographical and ecological barriers. We studied Youngia japonica based on nrDNA and cpDNA sequences. This is a widespread native in Asia and invasive worldwide; nevertheless, three subspecies (japonica, longiflora, and formosana) and an undescribed variant occur in Taiwan. Bayesian and the most parsimonious phylogenies revealed that subspecies longiflora is a different linage and independently arrived in Taiwan during the Pleistocene via land connection to the Asian Continent. Bayesian time estimation suggested that Youngia in Taiwan diverged in the lower Pleistocene or more recently. Extreme habitats that emerged in the Pleistocene, i.e., cold mountain ranges for subspecies formosana and xeric, raised coral reefs for the undescribed Youngia variant probably had triggered the divergence. Components of Youngia in Taiwan are not monophyletic; a coalescent-based test suggested incomplete lineage sorting. Nevertheless, the samples within each taxon share unique morphological features suggesting a common gene pool and each taxon has different dominant ITS and/or cpDNA types; these conditions suggest ongoing process toward monophyly via coalescent processes and support the delimitation of intraspecific taxa.     

Individual differences in sensitivity to bitter-tasting substances

Perception of several bitter-tasting compounds was tested in52 subjects. Stable individual differences in the perceivedintensity of the bitterness of suprathreshold concentrationsof quinine sulfate (QSO₄) and urea were found. Whereas 18 subjectsjudged selected concentrations of these compounds to be equallybitter, 17 found QSO₄ to be more bitter than urea, and 17 foundurea to be more bitter than QSO₄. These reliable individualdifferences were significantly related to threshold sensitivityto QSO₄; that is, individuals who perceived QSO₄ to be moreintense than urea at suprathreshold concentrations also hadlower QSO₄ thresholds than did those who perceived urea to bemore intense than QSO₄. There appeared to be no relationshipbetween the relative perceived intensities of these compoundsand rating of the bitterness of PROP (6-n-propylthiouracil).However, QSO₄-sensitive individuals tended to find the bitternessof suprathreshold caffeine and sucrose octaacetate to be greaterthan that of suprathreshold magnesium sulfate, whereas the reversewas true for urea-sensitive individuals. This pattern parallelsthe pattern of cross-adaptation among these compounds reportedby other investigators. These results are consistent with theexistence of multiple bitter transduction sequences and suggestthat individual differences in response to various bitter compoundsmay reflect differences in teh relative availability of specifictransduction sequences.