Kallikrein 13 Research Articles

Novel insight into the formation and inhibition mechanism of dipeptidyl peptidase-Ⅳ inhibitory peptides from fermented mandarin fish (Chouguiyu)

Fermented foods are a potential source to produce novel dipeptidyl peptidase-IV inhibitory peptides (D4IPs). In this study, the fermented mandarin fish (Chouguiyu) was used to screen D4IPs and their formation mechanism was studied by metagenomics and peptidomics. A total of 400 D4IPs with DPP-IV inhibition structure and high hydrophobicity were identified. The correlation network map showed that Lactococcus, Bacillus, Lysobacter, Pelagivirga, Kocuria, Escherichia, Streptococcus, and Peptostreptococcus were significantly correlated with the most D4IPs. Four stable D4IPs, including KAGARALTDAETAT, GEKVDFDDIQK, VVDADEMYLKGK, and GQKDSYVGDEAQ were respectively from the precursor proteins parvalbumin, troponin, myosin, and actin, and were mainly formed by the hydrolysis of subtilisin (EC 3.4.21.62), aspartic proteinase (EC 3.4.23.1), thermolysin (EC 3.4.24.27), oligopeptidase B (EC 3.4.21.83), and proteinase P1 (EC 3.4.21.96) from Bacillus, Kocuria, Lysobacter, Lactococcus, and Peptostreptococcus. The inhibition mainly resulted from the hydrogen bond and salt bridge between D4IPs and DPP-IV enzyme. This study provides important information on the proteases and related microbial strains to directionally prepare D4IPs in Chouguiyu.

Correlation between C═O Stretching Vibrational Frequency and pKa Shift of Carboxylic Acids.

Identifying the pKa values of aspartic acid (Asp) and glutamic acid (Glu) in active sites is essential for understanding enzyme reaction mechanisms. In this study, we investigated the correlation between the C=O stretching vibrational frequency (νC=O) of protonated carboxylic acids and the pKa values using density functional theory calculations. In unsaturated carboxylic acids (e.g., benzoic acid analogues), νC=O decreases as the pKa increases (the negative correlation), whereas in saturated carboxylic acids (e.g., acetic acid analogues, Asp, and Glu), νC=O increases as the pKa increases (the positive correlation) as long as the structure of the H-bond network around the acid is identical. The negative/positive correlation between νC=O and pKa can be rationalized by the presence or absence of the C=C double bond. The pKa shift was estimated from the νC=O shift of Asp and Glu in proteins on the basis of the negative correlation derived from benzoic acids. The previous estimations should be revisited by using the positive correlation derived in this study, as demonstrated by quantum mechanical/molecular mechanical calculations of νC=O and electrostatic calculations of pKa on a key Asp85 in the proton-transfer pathway of bacteriorhodopsin.

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1.

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

Analysis of urinary kallikrein-related peptidase 13 for monitoring bladder cancer

Background Bladder cancer (BC) is one of the 10 most common types of cancer worldwide, with approximately 550,000 new cases each year. Early detection and appropriate diagnosis are important factors in successful treatment of the disease. Material and methods We used specific fluorogenic substrate for the quantitative determination of urine kallikrein 13 (KLK13) activity in healthy (n = 15) and BC (n = 54) patients. The proteolytic activity in individual urine samples was determined by fluorescence measurements. Then, immunoenzymatic analyses (ELISA, Western blot) were performed to confirm the presence of KLK13 in the tested samples. Results Urine samples from patients with G2 and G3 grade BC contained proteolytically active KLK13, as confirmed by kinetic analysis and immunochemical detection. KLK13 was not detected in the urine of patients with G1 grade BC. Discussion Previous clinical study reveals the KLK13 significance for BC prognosis as increased KLK13 expression was highlighted in bladder tumours compared to normal adjacent tissues. Our findings correlate to the report. KLK13 activity was confirmed in BC patients with G2 and G3 stage of disease development. Conclusions Using specific chromogenic/fluorogenic peptides could be useful for the non-invasive disease monitoring of BC progress.

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Kallikrein 13 serves as a priming protease during infection by the human coronavirus HKU1.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.

Site-specific rapid deamidation and isomerization in human lens αA-crystallin in vitro.

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens-specific αA-crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA-crystallin with Asn by using site-directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA-crystallin was subjected to enzymatic digestion followed by liquid chromatography-MS/MS to evaluate the ratio of modifications in Asn151-containing peptides. The Asp151Asn αA-crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit-subunit interactions between αA-crystallin and αB-crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA-crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L-Asp to D-Asp residues in vivo.

The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope.

The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target.IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

PF443 ADOPTIVE IMMUNOTHERAPY OF INVASIVE ASPERGILLOSIS BY T CELLS VACCINES AGAINST ASPERGILLOSIS IN MOUSE MODEL

Background:Invasive aspergillosis is a common cause of life‐threatening infection in immunocompromised patients, especially those who require aggressive immunosuppressive treatment, as Aspergillus fumigatus is the most common pathogen. Despite advances in early diagnosis and new antifungal agents, the majority of cases still associated with high morbidity and unacceptable mortality. The reconstitution of specific immunity against A. fumigatus during periods of severe immunosuppression has the potential to impact enormously on the health and well being of patients.Aims:To screened the amino acid sequences of A. fumigatus recombinant protein ASP f16 by bioinformation software and determine the potential CTL epitopes processed and presented by mature DCs to prime specific CTL cells.Methods:We confirmed the killing ability of Asp f16 peptides specific CTLs against A. fumigatus by conidiacidal and hyphae killing assays, which indicated that CTLs culture supernatant contains substances that killed fresh A. fumigatus conidia and CTLs have killing activity directly against the hyphae in vitro. In our study, we also accessed the evaluation of the protection against A. fumigatus in vivo by adoptive transfer peptides specific CTLs to an invasive aspergillosis model of HLA A0201 transgenic mouse.Results:The results demonstrated the adoptive transfer of ASP f16 peptides specific CTLs significantly altered the course of the Aspergillus infection, as indicated by statistical significantly extended survival hours after the infection and reduced fungal burden in the lung tissue, as opposed to mice not receiving T cells or receiving nonrelated peptides (NPM1) specific T cells, which all died faster with extensive fungal growth.Summary/Conclusion:Generate Asp f16 specific CTL clones in vitro and adoptive transfer to the recipients when they’re susceptive to or involved in IA, will be a promising potential option to protect the recipients against the fatal fungal infection.

PJ-1 The HIV-1 antisense protein ASP is a structural protein of the viral envelope

Introduction: The antisense strand of the HIV-1 genome encodes a 189-aa, highly hydrophobic protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP in HIV-1 infected individuals demonstrate its expression invivo. However, whether ASP plays a role in viral replication is still unknown. We studied the expression and subcellular localization of ASP in various chronically infected cell lines. In addition, we investigated the impact of ASP expression on viral fitness. Methods: For intracellular and nuclear stainings, we used BD Cytofix/Cytoperm and eBioscience FoxP3 kits. Flow cytometry data were acquired on a Millipore Guava and analyzed with FlowJo. Confocal and super-resolution microscopy images were acquired on Zeiss LSM 800 and Nikon N-STORMTi-E microscopes, and analyzed with Zen Blue and Nikon Elements Software Version 3.1. For virion-capture assays, antibodies against ASP and gp120 were immobilized on Protein G-dynabeads. For Fluorescence Correlation Spectroscopy (FCS) we used an ISS Q2 confocal microscope and the data were analyzed with ISS VistaVision. Results/Discussion: We generated an in-house monoclonal antibody 324.6 against an epitope located between 2 putative transmembrane domains in the ASP protein. Using this antibody in flow cytometry and confocal microscopy, we found thatASP is expressed in the nuclei of chronically infected myeloid (U1andOM-10.1), and lymphoid (H9-IIIB, H9-CC, H9-MN, H9-RF, ACH2 and 8E5) celllines, but not their parental uninfected lines. However, ASP was not detectable neither on the cell surface or in the cytoplasm of infected cell lines. ASP expression was higher in cells cultured at low density compared to cells cultured at high density, suggesting that the rate of cell division may influence ASP expression levels. Confocal microscopy showed a polarized nuclear distribution of ASP, preferentially in areas of the nucleus containing actively transcribed chromatin. However, after reactivation of HIV-1 expression with PMA, we found that ASP translocated to the cytoplasm and to the cell membrane of all infected cell lines. In addition, the ability to detect ASP on the cell surface without permeabilization indicates that the ASP epitope recognized by324.6 antibody is extracellular. Confocal microscopy showed that ASP and gp120 co-localize on the cell surface of productively infected cells, and we determined that the Manders overlap coefficient is 78%. Super-resolution microscopy analyses provided further evidence of ASP and gp-120 co-localization. These results suggest that ASP might be incorporated in the membrane of budding virions upon their release from infected cells. Indeed, the anti-ASP 324.6 antibody was able to capture HIV-1 particles with an efficiency similar to the anti-gp120 VRC01 antibody. Moreover, FCS showed that the binding efficiency of 324.6 antibody to single virions in solution was ∼28%. Altogether, these 2 assays demonstrate the presence of ASP on the surface of mature HIV-1 particles released from infected cells. To investigate whether ASP expression impacts viral replication, we mutated single nucleotides in the HIV-1NL4-3 genome thus introducing early stop codons in the ASP open reading frame without affecting the amino acid sequence of Env encoded on the opposite strand. We found that these ASP-deficient viruses displayed a ∼50% reduction in replication rate compared to wildtype virus. Conclusions: Our results indicate that ASP is expressed in the nuclei of non-productively infected cell lines. Upon viral reactivation, ASP translocates to the surface where it co-localizes with gp120. Moreover, ASP is present on the membrane of viral particles, indicating that ASP is a structural protein in the envelope of mature HIV-1 virions. Further, ASP expression promotes viral replication. Overall, our results suggest that ASP may represent a new target for therapeutic or preventive vaccine.

Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity.

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

Cancer-related proteins in serum are altered in workers occupationally exposed to polycyclic aromatic hydrocarbons: a cross-sectional study

Exposure to some polycyclic aromatic hydrocarbons (PAH) increases the risk of cancer and is common particularly for workers in occupations such as chimney sweeping. In exposed workers, screening of early cancer-related markers provides important information to identify individuals at risk. Here, we aimed to elucidate the associations between PAH exposure and serum levels of cancer-related proteins in 118 chimney sweeps and 126 occupationally unexposed controls, all non-smoking males from Sweden. Monoydroxylated metabolites of pyrene, phenanthrene, benzo[a]pyrene and benzo[a]anthracene were measured in urine using liquid chromatography coupled to tandem mass spectrometry and 90 cancer-related proteins were measured in serum using a proximity extension assay. Linear regression analysis adjusted for age and body mass index, and false discovery rate (FDR) identified 17 serum proteins that were differentially expressed (16 upregulated and 1 downregulated) in chimney sweeps compared with controls (FDR < 0.05). Concentrations of the peptidase kallikrein 13 (KLK13) showed significant positive associations with urinary concentrations of the PAH metabolites 3-hydroxybenzo[a]pyrene (3-OH-BaP) [B, 95% confidence interval (CI): 0.042, 0.008–0.076] and 3-hydroxybenzo[a]anthracene (3-OH-BaA) (B, 95% CI: 0.068, 0.002–0.134). Moreover, dose–response relationships were observed between KLK13 and 3-OH-BaP (trend test P = 0.027) and 3-OH-BaA (P = 0.035). Pathway and gene ontology analyses showed that cell movement, cell adhesion and cell migration were the predominant molecular functions associated with the top differentially expressed proteins. In conclusion, we found a number of putative cancer-related proteins differentially expressed in workers exposed to PAH. This warrants effective measure to reduce PAH exposure among workers as well as further investigation to confirm these findings.

Brief communication: maxillary lateral incisor morphology and uncommon trait expression: a case study from prehistoric Paa-ko, New Mexico

Prehistoric American Southwest exhibits a high frequency of dental morphological variability. This high variability may be the result of gene flow and subsequent genetic drift occurring in early periods (pre CE 900), though few studies report on dental variability in later periods. Morphological traits of the maxillary lateral incisors were analyzed from the Pueblo IV site of Paa-ko, New Mexico (CE 1300-1425) yielding high frequencies of four traits (shovel, double shovel, tuberculum dentale, interruption groove) and uncommon variants (barrel-shovel, triform, peg-shaped). Lateral incisor morphology is underrepresented in the literature but could be useful in determining population migration and affinity.

The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly

Various epigenetic marks at the HIV-1 5’LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3’LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5’LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5’LTR and proviral latency through the PRC2 pathway.

Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes.

The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD, and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces, resulting in the capacity to accurately segregate chromosomes.

Bovine Meat Proteins as Potential Precursors of Biologically Active Peptides - a Computational Study based on the BIOPEP Database

The aim of the present study was to perform an in silico evaluation of bovine meat proteins as potential precursors of biologically active peptides, as well as to determine whether such peptides can be released by selected proteolytic enzymes. The sequences of 19 bovine meat proteins were processed using the BIOPEP database and program. The profiles of potential biological activity of protein fragments were determined and the following parameters were calculated: the frequency of occurrence of fragments with given activity (A), the frequency of release of fragments with given activity by selected enzymes (A(E)), and the relative frequency of release of fragments with given activity by selected enzymes (W). Among the examined proteins, collagen and elastin appear to be the richest potential source of bioactive peptides, in particular of angiotensin-converting enzyme inhibitors, antithrombotic fragments, inhibitors of dipeptidyl peptidase IV and peptides regulating gastric mucosal activity. The high number of bioactive fragments in collagen and elastin is associated with a high content of glycine and proline, amino acids that are most abundant in biologically active fragments. Of the two investigated proteolytic enzymes, Proteinase K - an enzyme with broad specificity (e.g., against peptide bonds formed by the carboxyl groups of proline) can release considerably more biologically active fragments than Proteinase P1 - an enzyme with narrow specificity, not including proline residues.

Unusual fragmentation of β-linked peptides by ExD tandem mass spectrometry.

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) represent a powerful tool for biological analysis. ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β(3)-type amino acid that has an additional methylene group in the backbone, forming a C(α)-C(β) bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a C(α)-C(β) bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N-C(β) and C(α)-C(β) bond cleavages were rare, providing few c and z• type fragments, which was attributed to the instability of the C(β) radical. Instead, the electron capture resulted primarily in the formation of a• and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the β amino acid residues.

Investigating protein features contribute to salt stability of halolysin proteins

We used various screening techniques, clustering, decision tree and generalized rule induction (association) (GRI) models and molecular phylogenic relationship to search for patterns of halophi-licy and to find features contribute to halolysin salt stability. We found Met was the sole N-terminal amino acid in halolysin proteins, whereas other amino acids found at this position of oth-er proteases and termitase. Eighty-three protein features were shown to be important in feature selection modeling, and just one peer group with an anomaly index of 2.42 declined to 1.87 after being run using only important selected features. The depth of the trees generated by various de-cision tree models varied from 1 to 5 branches. The number of peer groups in clustering models was reduced significantly (p<0.05) compared to datasets without feature selection. In most deci-sion tree models, the frequency of Gly - Gly was the most important feature for decision tree rule sets; and this feature was used in antecedent to support the rules in most GRI association rules. Significant differences (p < 0.001) found in charged amino acids between halolysin and other pro-teins with more Asp and Glu in halolysin proteins, while more hydrophobic residues and aliphatic amino acids found in other proteases.

Changes in chemical composition in male turkeys during growth

In growing animals, requirements for many nutrients (and energy) are determined by the retention of these nutrients. During growth, this retention changes in an absolute way and also between nutrients and energy, resulting in changing nutrient requirements. The objective of this study was to describe the changes in chemical composition in male growing turkeys. The serial slaughter technique was used to determine the composition of amino acids, lipid, ash, and water in feather-free body (FFB) and feathers in male turkeys offered feed ad libitum from 1 to 15 wk of age. Allometric relations were used to describe changes in body composition. The feather content in the body decreased from 6% at 1 wk of age to less than 3% at 15 wk of age. The water and protein content in FFB decreased with increasing FFB mass, with allometric scalars (b) of, respectively, 0.967 and 0.970, whereas the lipid content increased with increasing FFB mass (b = 1.388). The water, protein, and ash content in fat-free FFB was constant and represented, respectively, 71.6, 24.2, and 4.2% of the fat-free FFB mass. The amino acid content of FFB protein was relatively constant and only the Cys content decreased between 1 and 15 wk of age, whereas the Ile content increased. Feathers were mostly composed of protein, and the protein content did not change during growth. During growth, the Lys, Met, Trp, His, Tyr, Asp, and Glu contents in feather protein decreased, whereas the Cys, Val, and Ser contents increased. The contribution of feathers to whole-body amino acid retention ranged from 5% for His to 33% for Cys. On average, the weight gain of FFB contained 21.3% protein and 12.7% lipid, corresponding to an energy content of 10.1 kJ/g. The weight gain of feathers contained 87.4% protein, corresponding to an energy content of 20.4 kJ/g. The results of the present study can be used in a factorial approach to determine nutrient requirements in growing turkeys.

Hors d'oeuvre: Science, the Short Film, and &lt;i&gt;The Perception of Life&lt;/i&gt;

I no longer know if I'm looking with my naked eye at a starry sky or at a drop of water in a microscope. Blaise Cendrars, Profound Today (1917) Hors d'oeuvre, French for of the work, commonly brings to mind small dishes that come prior to an entree or, more formally, between the soup and entree. An hors d'oeuvre performs a modest act of seduction, whetting the appetite, delicately stoking desire, and making overtures or ouvertures (openings) for what is to come. The culinary defi nition is but one of a number of meanings of the term, and is actually a more recent usage. In its most literal sense, hors d'oeuvre refers to supplemental elements of an architectural structure, an artwork, or a piece of literature- an ornamental or decorative fl ourish or an appendix that is supposed to be inessential to a work's integrity, lying outside of what is proper to it, and vestigial to the vitality of its corpus.1 The hors d'oeuvre is often playful (I write this hors d'oeuvre to divert you) but it can also signify extraordinary objects or events that are [the] customary place or time or of the ordinary course.2 The hors d'oeuvre announces, prefaces, and can even initiate or spur the oeuvre. It comes before. It starts a specifi ed course (the main course) without necessarily belonging to its sequence, succession, or telos. It stands outside of or exterior to the work (even when physically within it), and from this vantage can introduce eccentric and heterogeneous possibilities into the work or draw them out of it. Typically small (or at least relatively smaller) in scale, the hors d'oeuvre is incidental but not inconsequential. It presents a beginning, an outside, a diversion, and an uncommon, even slightly untimely event or phenomenon. The science fi lm has historically played the role of hors d'oeuvre, in its multiple senses, to narrative, documentary, and experimental cinemas. As a broad descriptor, the science fi lm can refer to the highly specialized use of the cinematograph as a visual prosthesis and recording instrument for research within a laboratory context, to educational fi lms used to demonstrate and teach scientifi c techniques and materials, and to pop u lar or documentary fi lms with scientifi c subjects intended for general audiences.3 In most accounts of the history of the cinematographic apparatus, the chronophotographic and cinematic motion studies produced in the physiological, zoological, and psychiatric laboratories by Etienne- Jules Marey, Georges Demeny, and Albert Londe, to name but three researchers, produced a set of experiments that inaugurated the principle technologies and techniques for fi lm- providing the cinema with noble ancestors- while largely remaining circumscribed from its commercial development. In their polemic defi nitions of documentary fi lm written in the early 1930s, John Grierson and Paul Rotha echo this perspective, amplifying it by explicitly excluding scientifi c, wildlife, and avant- garde fi lms from the purview and vocation of documentary due to a perceived lack of narrative and rhetorical structure as well as for their unruly visual excesses and indulgences.4 Such fi lms were fi ne pre ce dents, according to Grierson and Rotha (in a rare agreement), but they could not be given admission into the proper corpus of the documentary. In the early years of cinema, science fi lms were frequently exhibited both as a fairground attraction and in the beginning portions of cinema programs, serving as a visually titillating amuse- bouche-or amuse- oeil-prior to the main course of the feature fi lm's feast for the eyes. Since such fi lms were projected prior to the feature, however, they often suffered from their status as an expendable part of the program. The critic Emile Vuillermoz, writing in 1917, lamented the fact that the documentaries and science fi lms at the beginning of programs were, for the most part, neglected and even disdained. …