Anti‐biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins

The broad‐spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio‐control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan‐rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan‐rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD‐based peptides PuroA (FPVTWRWWKWWKG‐NH₂) and Pina‐M (FSVTWRWWKWWKG‐NH₂) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG‐NH₂) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non‐pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina‐M at 2 × MIC prevented initial biomass attachment by 85–90% and inhibited >90% of 6‐h preformed biofilms of all three organisms. However Hina, with a substitution of Lys‐9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan‐rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan‐rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.     

The N-terminal 513 amino acids of the envelope glycoprotein gB of human cytomegalovirus stimulates both B- and T-cell immune responses in humans.

Host defense against human cytomegalovirus (HCMV) involves both humoral and cell-mediated immunity. In this report, human immune responses to glycoproteins encoded by the HCMV gB homolog gene have been examined by using glycoproteins purified by immunoaffinity from HCMV virions and recombinant proteins expressed by vaccinia viruses containing either the entire gB open reading frame or a C-terminal deletion mutant, gBm165, coding for the N-terminal 513 amino acids of gB. Neutralizing antibodies, helper T cells, and cytotoxic T cells reactive with epitopes on the N-terminal portion of gB were detected in some seropositive individuals, suggesting that this region of gB may be important in eliciting protective immunity during natural infection for some individuals.     

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.     

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.     

Proboscidean mitogenomics: chronology and mode of elephant evolution using mastodon as outgroup

We have sequenced the complete mitochondrial genome of the extinct American mastodon (Mammut americanum) from an Alaskan fossil that is between 50,000 and 130,000 y old, extending the age range of genomic analyses by almost a complete glacial cycle. The sequence we obtained is substantially different from previously reported partial mastodon mitochondrial DNA sequences. By comparing those partial sequences to other proboscidean sequences, we conclude that we have obtained the first sequence of mastodon DNA ever reported. Using the sequence of the mastodon, which diverged 24–28 million years ago (mya) from the Elephantidae lineage, as an outgroup, we infer that the ancestors of African elephants diverged from the lineage leading to mammoths and Asian elephants approximately 7.6 mya and that mammoths and Asian elephants diverged approximately 6.7 mya. We also conclude that the nuclear genomes of the African savannah and forest elephants diverged approximately 4.0 mya, supporting the view that these two groups represent different species. Finally, we found the mitochondrial mutation rate of proboscideans to be roughly half of the rate in primates during at least the last 24 million years.     

Semi-automatic classification of skeletal morphology in genetically altered mice using flat-panel volume computed tomography

Rapid progress in exploring the human and mouse genome has resulted in the generation of a multitude of mouse models to study gene functions in their biological context. However, effective screening methods that allow rapid noninvasive phenotyping of transgenic and knockout mice are still lacking. To identify murine models with bone alterations in vivo, we used flat-panel volume computed tomography (fpVCT) for high-resolution 3-D imaging and developed an algorithm with a computational intelligence system. First, we tested the accuracy and reliability of this approach by imaging discoidin domain receptor 2- (DDR2-) deficient mice, which display distinct skull abnormalities as shown by comparative landmark-based analysis. High-contrast fpVCT data of the skull with 200 microm isotropic resolution and 8-s scan time allowed segmentation and computation of significant shape features as well as visualization of morphological differences. The application of a trained artificial neuronal network to these datasets permitted a semi-automatic and highly accurate phenotype classification of DDR2-deficient compared to C57BL/6 wild-type mice. Even heterozygous DDR2 mice with only subtle phenotypic alterations were correctly determined by fpVCT imaging and identified as a new class. In addition, we successfully applied the algorithm to classify knockout mice lacking the DDR1 gene with no apparent skull deformities. Thus, this new method seems to be a potential tool to identify novel mouse phenotypes with skull changes from transgenic and knockout mice on the basis of random mutagenesis as well as from genetic models. However for this purpose, new neuronal networks have to be created and trained. In summary, the combination of fpVCT images with artificial neuronal networks provides a reliable, novel method for rapid, cost-effective, and noninvasive primary screening tool to detect skeletal phenotypes in mice.     

Modulation of intra-oral processing in mammals and lepidosaurs

The mammalian masticatory apparatus is distinguished from theintra-oral processing systems of other amniotes by a numberof morphological and functional features, including transversemovements of the teeth during the power stroke, precise occlusion,suspension of the teeth in the socket by a periodontal ligament,diphyodonty (reduction to two generations of teeth), a hardpalate, and the presence of a single bone (the dentary) in thelower jaw which articulates with the skull at the temporomandibularjaw joint. The evolution of these features is commonly arguedto have improved the efficiency of food processing in the oralcavity. The present aricle highlights the existence in mammalsof the fusimotor system and afferent fibers from the periodontalligament through which the CNS modulates the responses by themuscle spindles. Published data suggest that the fusimotor systemand the periodontal afferents are important components in feed-forward(or anticipatory) control of chewing behavior. We hypothesizethat this feed-forward control is used to maintain relativelyconstant cycle lengths in mammals in the face of intra-sequenceand inter-sequence variation in material properties of the food,and that this enables them to maintain a higher average chewingfrequency than that of lizards. These predictions were evaluatedusing data on mean cycle length and its variance from the literatureand from our own files. On average, mammals have less variablecycle lengths than do lizards and shorter cycle lengths thando lizards of similar size. We hypothesize that by decreasingvariance in cycle length, presumably close to the natural frequencyof their feeding systems, mammals minimize energy expenditureduring chewing, allowing them to chew for longer, thereby maintainingthe high rates of food intake required for their high metabolicrates.