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Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and  -Amylase.Hisayori Shigechi, 2004. Conformation of a Bactericidal Domain of Puroindoline a: Structure and Mechanism of Action of a 13-Residue Antimicrobial Peptide. Weiguo Jing, 2003.Puroindoline a, a wheat endosperm-specific protein containing a tryptophan-rich domain, was reported to have antimicrobial activities . We found that a 13-residue fragment of puroindoline a (FPVTWRWWKWWKG-NH2) (puroA) exhibits activity against both gram-positive and gram-negative bacteria . This suggests that puroA may be a bactericidal domain of puroindoline a . PuroA interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the microbicidal effect of puroA may be due to interactions with bacterial membranes . A variety of biophysical and biochemical methods, including fluorescence spectroscopy and microcalorimetry, were used to examine the mode of action of puroA . These studies showed that puroA is located at the membrane interface, probably due to its high content of Trp residues that have a high propensity to partition into the membrane interface . The penetration of these Trp residues in negatively charged phospholipid vesicles resembling bacterial membranes was more extensive than the penetration in neutral vesicles mimicking eukaryotic membranes . Peptide binding had a significant influence on the phase behavior of the former vesicles . The three-dimensional structure of micelle-bound puroA determined by two-dimensional nuclear magnetic resonance spectroscopy indicated that all the positively charged residues are oriented close to the face of Trp indole rings, forming energetically favorable cation-  interactions . This characteristic, along with its well-defined amphipathic structure upon binding to membrane mimetic systems, allows puroA to insert more deeply into bacterial membranes and disrupt the regular membrane bilayer structure .
Molecular Characteristics of Spontaneous Deletions in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius. Dennis W. Grogan, 2003.Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively . The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon . We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected . Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10-8 per cell . Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies . Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures . No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends . The unusually low frequency and low sequence dependence of spontaneous deletions in the S . acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection . Monitoring the Production of Aflatoxin B1 in Wheat by Measuring the Concentration of nor-1 mRNA. Zsuzsanna Mayer, 2003.A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of Aspergillus flavus in wheat . Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR . In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B1 production . The primer-probe system of the real-time PCR was targeted against nor-1, a gene of the aflatoxin biosynthetic pathway . By application of this method the nor-1 transcription was quantified during the course of incubation . After 4 days of incubation nor-1 mRNA could be detected for the first time . The amount of nor-1 mRNA increased rapidly, and the maximum was achieved after 6 days . Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10 . First traces of aflatoxin B1could be detected between the 5th and 6th day of incubation . The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation . To ensure that differences in the nor-1 mRNA concentration were due to different expression levels, the expression of the constitutively expressed ß-tubulin gene (benA56) has also been monitored . The expression of benA56 remained constant during the whole incubation time . As a parameter for fungal growth, the number of nor-1 gene copies was determined during the course of incubation . The numbers of nor-1 gene copies increased at the beginning of the incubation and reached a plateau at day 5 . They correlate well with the viable counts albeit at a higher level .
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