Two-dimensional peptide mapping by reversed-phase column chromatography, applied to the sequence determination of cytochrome c from the wild type and a mutant of the butterfly, pieris brassicae

Two-dimensional peptide mapping has been very effective in the characterization of protein digests, particularly for the detection of small structural differences between homologous proteins. The classical thin-layer strategy, which exploits differences in charge and hydrophobicity, has been realized as a method based on reversed-phase high-performance liquid chromatography. An initial fractionation at pH 7.2 with 100 mM potassium phosphate, followed by chromatography with 0.1% trifluoroacetic acid, has been applied to chymotryptic digests of cytochromes c. The use of UV-transparent and (in the final stage) volatile solvents allows detection and rapid recovery of nanomole amounts of peptides suitable for sequence determination. As an example of the application of this method we report the comparison of two variants of cytochrome c from the butterfly, Pieris brassicae, one being the wild type and the other a spontaneous mutant isolated from a laboratory colony. The single residue difference was easily detected and identified.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25128/1/0000561.pd

Escherichia coli O157 Exposure in Wyoming and Seattle: Serologic Evidence of Rural Risk

We tested the hypothesis that rural populations have increased exposure to Escherichia coli O157:H7. We measured circulating antibodies against the O157 lipopolysaccharide in rural Wyoming residents and in blood donors from Casper, Wyoming, and Seattle, Washington, by enzyme immunoassay (EIA). EIA readings were compared by analysis of variance and the least squares difference multiple comparison procedure. Rural Wyoming residents had higher antibody levels to O157 LPS than did Casper donors, who, in turn, had higher levels than did Seattle donors (respective least squares means: 0.356, 0.328, and 0.310; p<0.05, Seattle vs. Casper, p<0.001, rural Wyoming vs. either city). Lower age was significantly correlated with EIA scores; gender; and, in rural Wyoming, history of bloody diarrhea, town, duration of residence, and use of nontreated water at home were not significantly correlated. These data suggest that rural populations are more exposed to E. coli O157:H7 than urban populations

Cloning and Sequence Analysis of the Structural Gene for the bc 1 - Type Rieske Iron-Sulfur Protein from Thermus thermophilus HB8

The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc 1 or b 6 f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus . Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al ., 1996 Structure 4 , 567–579).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44801/1/10863_2004_Article_409077.pd

Amino acid sequence of the active site of human serum cholinesterase from usual, atypical, and atypical-silent genotypes

Active-site tryptic peptides were isolated from three genetic types of human serum cholinesterase. The active-site peptide was identified by labeling the active-site serine with [ 3 H] diisopropylfluorophosphate. Peptides were purified by high-performance liquid chromatography. Amino acid composition and sequence analysis showed that the peptide from the usual genotype contained 29 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu-Leu-Ser-Pro-Gly-Ser-His-Ser-Leu-Phe-Thr-Arg. The active-site serine was the eighth residue from the N- terminal. The peptide containing the active-site serine from the atypical genotype contained 22 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu-Leu-Ser-Pro-Gly. The peptide from the atypical-silent genotype contained eight residues with the sequence Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser. Thus, the sequences of the atypical and atypical-silent active-site peptides were identical to the corresponding portions of the usual peptide.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44153/1/10528_2004_Article_BF00499101.pd

Priming of plane-rotated objects depends on attention and view familiarity

Three experiments investigated the role of attention in visual priming across rotations in the picture plane. Experiment 1 showed that naming latencies increased with the degree of misorientation for objects commonly seen in an upright view (base objects) but not for objects seen familiarly from many views (no-base objects). In Experiment 2, no-base objects revealed a priming pattern identical to that observed previously for left-right reflections (Stankiewicz, Hummel, & Cooper, 1998): Attended objects primed themselves in the same and rotated views, whereas ignored images primed themselves only in the same view, with additive effects of attention and orientation. In Experiment 3 ignored base objects only primed themselves in a familiar (upright) view, indicating that priming only obtains when that image makes contact with object memory. These data challenge theories of object recognition that rely on any single representation of shape and contribute to evidence suggesting holistic (view-like) representations for ignored and analytic (view-insensitive) representations for attended objects

Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis

Background Necrotizing enterocolitis (NEC) is a catastrophic disease of preterm infants, and microbial dysbiosis has been implicated in its pathogenesis. Studies evaluating the microbiome in NEC and preterm infants lack power and have reported inconsistent results. Methods and results Our objectives were to perform a systematic review and meta-analyses of stool microbiome profiles in preterm infants to discern and describe microbial dysbiosis prior to the onset of NEC and to explore heterogeneity among studies. We searched MEDLINE, PubMed, CINAHL, and conference abstracts from the proceedings of Pediatric Academic Societies and reference lists of relevant identified articles in April 2016. Studies comparing the intestinal microbiome in preterm infants who developed NEC to those of controls, using culture-independent molecular techniques and reported α and β-diversity metrics, and microbial profiles were included. In addition, 16S ribosomal ribonucleic acid (rRNA) sequence data with clinical meta-data were requested from the authors of included studies or searched in public data repositories. We reprocessed the 16S rRNA sequence data through a uniform analysis pipeline, which were then synthesized by meta-analysis. We included 14 studies in this review, and data from eight studies were available for quantitative synthesis (106 NEC cases, 278 controls, 2944 samples). The age of NEC onset was at a mean ± SD of 30.1 ± 2.4 weeks post-conception (n = 61). Fecal microbiome from preterm infants with NEC had increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes prior to NEC onset. Alpha- or beta-diversity indices in preterm infants with NEC were not consistently different from controls, but we found differences in taxonomic profiles related to antibiotic exposure, formula feeding, and mode of delivery. Exploring heterogeneity revealed differences in microbial profiles by study and the target region of the 16S rRNA gene (V1-V3 or V3-V5). Conclusions Microbial dysbiosis preceding NEC in preterm infants is characterized by increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes. Microbiome optimization may provide a novel strategy for preventing NEC

Sensory, microbiological and chemical changes in vacuum-packaged Blue Spotted Emperor (Lethrinus sp), Saddletail Snapper (Lutjanus malabaricus), Crimson Snapper (Lutjanus erythropterus), Barramundi (Lates calcarifer) and Atlantic Salmon (Salmo salar) fillets stored at 4°C

Quality assessment of finfish fillets during storage is important to be able to predict the shelf life of the fresh product during distribution. Microbial, chemical (pH, TMA, and TVB‐N), and sensory (Quality index assessment QIA, Torry scheme) changes in vacuum‐packaged blue‐spotted emperor (Lethrinus sp), saddletail (Lutjanus malabaricus), crimson snapper (Lutjanus erythropterus), barramundi (Lates calcarifer), and Atlantic salmon (Salmo salar) fillets stored at 4°C were evaluated for 5 days. Microbiological study included evaluation of TVC (total viable counts), total psychrotrophic organisms, and H2S‐producing bacteria. Numbers increased during storage time and reached an average of 8.5, 8.5, and 9.2 log10 cfu/g, respectively, for the five different fish species. These levels were above accepted microbiological limits for fish fillets. Although the sensory analyses showed a decrease in quality, none of the finfish fillets were considered unacceptable at the end of the storage trial. Chemically, there was a slight pH increase, but trimethylamine (TMA) levels remained low. However, total volatile basic nitrogen (TVB‐N) levels increased over time, reaching levels above 35 mg/100 g for blue spotted emperor, saddletail snapper, and crimson snapper by the end of the storage period. Results show that the deterioration of finfish fillet quality is a complex event of biochemical, sensory, and microbial factors, and multiple analyses may be required to define acceptability