Determination Of Amino Acid Sequence Research Articles

Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Fishing for elusive cCMP-degrading phosphodiesterases

Background The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMPspecific” PDE was isolated from disrupted L-1210 leukemia cells [1] and from rat liver [2,3], shows an extremely high 3’,5’-cCMP KM value of up to 9 mM and seems to be activated by iron [1]. This enzyme prefers 3’,5’-cCMP over other cyclic nucleotides. The other enzyme is called “multifunctional cCMP PDE” and was isolated from pig liver [3-5], rat liver [6,7] and human liver [8]. This enzyme shows a 3’,5’-cCMP KM value in the range of 100-300 μM and is additionally degrading 2’,3’-cCMP as well as the 2’,3’and 3’,5’-isomers of cUMP, cAMP, cGMP and cIMP. The multifunctional cCMP PDE is inhibited by inorganic phosphate and AMP. Both enzymes are calmodulin-insensitive, IBMX-resistant and show a low molecular weight of <35 kDa, which is uncommon for the “classic” phosphodiesterases. However, the exact amino acid sequence and identity of these proteins remain elusive. Our project aims at a clear and unequivocal determination of amino acid sequence and identity of these elusive cCMP-degrading enzymes by analyzing and purifying the cCMP degrading activity of organ extracts and biological fluids (e.g. serum). Moreover, we re-investigate wellknown “classic” PDEs in enzymatic assays to identify a potential 3’,5’-cCMP degrading activity.

Analysis of genetic variation of hemagglutinin and three internal genes of influenza B virus isolated in Zhejiang province from 1999 to 2012

To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus. A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed. The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates. Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.

MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8%) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74%) and with chymotrypsin from Metarhizium anisopliae (71%). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico

The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240pmol (∼0.635μg, ∼24.9ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.

Amino acid sequence determinants in self-assembly of insulin chiral amyloid superstructures: Role of C-terminus of B-chain in association of fibrils

Formation of chiral amyloid superstructures is a newly recognised phenomenon observed upon agitation-assisted fibrillation of bovine insulin. Here, by surveying several amyloidogenic precursors we examine whether formation of such entities is unique to bovine insulin. Our results indicate that only bovine, human, and porcine insulins are capable of chiral superstructural self-assembly. A tiny covalent perturbation consisting in reversal of ProB28-LysB29 residues in a human insulin analog is sufficient to prevent this process. Our study suggests that insulin’s dimer-forming interface – specifically the B-chain’s C-terminal fragment – may acquire the new role of a molecular velcro upon lateral alignment of individual fibrils into superstructures. Structured summary of protein interactionsBI and BIbind by infrared spectroscopy (View interaction)HI and HIbind by atomic force microscopy (View interaction)HEWL and HEWLbind by circular dichroism (View interaction)BI and BIbind by circular dichroism (View interaction)α-LAC and α-LACbind by circular dichroism (View interaction)BI and BIbind by atomic force microscopy (View interaction)α-LAC and α-LACbind by atomic force microscopy (View interaction)HI and HIbind by circular dichroism (View interaction)PI and PIbind by circular dichroism (View interaction)PI and PIbind by atomic force microscopy (View interaction)HEWL and HEWLbind by atomic force microscopy (View interaction)

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.

P1‐253: Research and development of anti‐Aß oligomers antibodies for the treatment of Alzheimer's disease

Amyloid beta oligomers (AbOs) are promising target for the treatment of Alzheimer's disease (AD) because they are believed as major pathogenic factor of AD. One of the hopeful therapeutic approaches is passive immunotherapy using anti- AbOs specific antibody. This approach has two merits, which are efficient therapeutic effect and decrease of side effects, because the antibody does not bind to amyloid precursor protein (APP) nor naturally existed Abeta. At AAIC2011 last year, we reported the screening results of the best anti-AbOs antibody by in vitro tests and in vivo test using AD model mouse. Here, the progress will be presented. Anti-AbOs antibodies were generated by immunization of antigens prepared by our original method to mouse. The initial screening was performed by ELISA and dotblot assay. The amino acid sequences of all positive clones were analyzed and sequence-redundant clones were eliminated. Then, antibodies that have higher selectivity to AbOs were selected by competitive ELISA. Next, antibodies that displayed non-binding property to normal human tissue array were chosen. Then, their affinities to AbOs were measured by SPR (Surface Plasmon Resonance) analysis, following that the antibodies were divided into high or low affinity groups. Next, passive immunization test using Tg2576 mouse was performed in several antibodies from each group. 184 antibodies were obtained in the results of first screening and subsequent determination of amino acid sequence. Then, 49 antibodies which have higher selectivity to AbOs were selected by the competitive ELISA. Moreover, dozens of antibodies were chosen from 49 antibodies by their non-binding feature to normal human tissue array. Next, these antibodies were divided into high and low affinity groups by SPR analysis. Then in vivo efficacy in several antibodies in each groups were examined by passive immunization test by using Tg2576 mouse. The results showed that high affinity antibodies have better efficacy of improvement of learning memory than low affinity ones, and also there were difference in clearance of Abeta in the brains among the high affinity antibodies. We have obtained the best antibody which has high specificity and affinity to AbOs and also has high efficacy in in vivo experiments.

Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.

Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides

Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration.

Synaptic Neurotransmitter-Gated Receptors

Since the discovery of the major excitatory and inhibitory neurotransmitters and their receptors in the brain, many have deliberated over their likely structures and how these may relate to function. This was initially satisfied by the determination of the first amino acid sequences of the Cys-loop receptors that recognized acetylcholine, serotonin, GABA, and glycine, followed later by similar determinations for the glutamate receptors, comprising non-NMDA and NMDA subtypes. The last decade has seen a rapid advance resulting in the first structures of Cys-loop receptors, related bacterial and molluscan homologs, and glutamate receptors, determined down to atomic resolution. This now provides a basis for determining not just the complete structures of these important receptor classes, but also for understanding how various domains and residues interact during agonist binding, receptor activation, and channel opening, including allosteric modulation. This article reviews our current understanding of these mechanisms for the Cys-loop and glutamate receptor families.

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC50 values of 7.89, 0.60 and 21.96μgml−1, respectively.

Silkworm Apolipophorin Protein Inhibits Staphylococcus aureus Virulence

Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding β-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.

Recombinant Expression of a Putative Amidase Cloned from the Genome of Listeria monocytogenes that Lyses the Bacterium and its Monolayer in Conjunction with a Protease.

Listeria monocytogenes is a Gram-positive, non-spore forming, catalase-positive rod that is a major bacterial food-borne disease agent associated with uncooked meats, including poultry, uncooked vegetables, soft cheeses, and unpasteurized milk. The bacterium may be carried by animals without signs of disease, can replicate at refrigeration temperatures, and is frequently associated with biofilms. There is a need to discover innovative pathogen intervention technologies for this bacterium. Consequently, bioinformatic analyses were used to identify genes encoding lytic protein sequences in the genomes of L. monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of a putative N-acetylmuramoyl-L-alanine amidase gene from L. monocytogenes strain 4b. The resultant amplification product was cloned into an expression vector, propagated in Escherichia coli Rosetta strains, and the recombinant protein was purified to homogeneity. Gene and protein sequencing confirmed that the predicted and chemically determined amino acid sequence of the recombinant protein designated PlyLM was a putative N-acetylmuramoyl-L-alanine amidase. The recombinant lytic protein was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot and MIC/MIB assays, but was not active against other bacteria beyond the genus. A microtiter plate assay was utilized to assay for the ability of the recombinant lysin protein to potentially aid with digestion of a L. monocytogenes biofilm. Protease or lysozyme digestion alone did not significantly reduce the L. monocytogenes biofilm. Although the recombinant protein alone reduced the biofilm by only 20%, complete digestion of the bacterial monolayer was accomplished in conjunction with a protease.

Cover Picture: Proteomics – Clinical Applications 7–8/11

AbstractA line in the sand: Late summer starting point for workflow designSecurity is handled by the rising tide. Remily‐Wood et al. developed a procedure for high‐throughput quantitative analysis for cancer research and clinical studies. The process begins with some bench and library work to define the target to be quantitated by determining amino acid sequences, isoforms, post‐translational modifications, and Western blots. Data go into QuAD, a JavaServer supplying other resources (Swiss‐Prot, etc.) and summary statistics of the target proteins. Target proteins and pathways must have ≥3 distinctive peptides, quantitative assays and have cultured cell line equivalents. Heavy isotope‐labeled synthetic peptides and cultured cell sequences are used to follow the LC‐Multiple Reaction Monitoring. The molecular weight equivalent SDS‐PAGE region is excised for screening peptides after in‐gel digestion. The screening conditions use minimal amounts of labeled peptides.Remily‐Wood, E. R. et al., Proteomics Clin. Appl. 2011, 5, 383–396.Getting your ducks in a row: A tough herding jobFrench medical practices require testing all newborns for sickle cell disease. There is a standard method for the test – isoelectric focusing (IEF) followed by HPLC on a cation exchange column. The HPLC ducks can be readily automated and labor costs minimized, but not so for IEF. It is tedious, uses expensive materials and is generally disliked in the lab, especially when many samples need to be run. Hachani et al. report on the development of a MALDI‐MS method that deals with most of problems. Previous research work had been incomplete for a variety of reasons. The new method used the same sample cards, a robotic commercial sample prep method, and automated analysis of samples using a MALDI‐TOF/TOF instrument. Failures of the new method against IEF were informative – most were premature or heterozygous neonatal infants. Conclusion: automated herding of 1000 samples/day looks feasible.Hachani, J. et al., Proteomics Clin. Appl. 2011, 5, 405–414.Double or nothing for the kidney?It is a sucker's bet on some streets in New York and serious business for patients waiting for a kidney transplant. Chronic allograft nephropathy (CAN) is the leading cause of kidney graft failure in the first year after transplant, being responsible for a 50–80% loss rate. The ability to reduce the failure rate by early recognition and treatment of CAN would markedly increase the number of kidneys available. Johnston et al. report here an improved urine protein marker pattern that can distinguish normal from CAN urine protein patterns. The principal element of the pattern (SELDI‐TOF‐MS hardware) was the amount of β2 microglobulin (11.7 kDa). A single β2 microglobulin point is insufficiently discriminating so further work will have to be done in a more complex setting.Johnston, O. et al., Proteomics Clin. Appl. 2011, 5, 422–431.

In this issue

AbstractA line in the sand: Late summer starting point for workflow designpp. 383–396Security is handled by the rising tide. Remily‐Wood et al. developed a procedure for high‐throughput quantitative analysis for cancer research and clinical studies. The process begins with some bench and library work to define the target to be quantitated by determining amino acid sequences, isoforms, post‐translational modifications, and Western blots. Data go into QuAD, a JavaServer supplying other resources (Swiss‐Prot, etc.) and summary statistics of the target proteins. Target proteins and pathways must have ≥3 distinctive peptides, quantitative assays and have cultured cell line equivalents. Heavy isotope‐labeled synthetic peptides and cultured cell sequences are used to follow the LC‐Multiple Reaction Monitoring. The molecular weight equivalent SDS‐PAGE region is excised for screening peptides after in‐gel digestion. The screening conditions use minimal amounts of labeled peptides. magnified imageGetting your ducks in a row: A tough herding jobpp. 405–414French medical practices require testing all newborns for sickle cell disease. There is a standard method for the test – isoelectric focusing (IEF) followed by HPLC on a cation exchange column. The HPLC ducks can be readily automated and labor costs minimized, but not so for IEF. It is tedious, uses expensive materials and is generally disliked in the lab, especially when many samples need to be run. Hachani et al. report on the development of a MALDI‐MS method that deals with most of problems. Previous research work had been incomplete for a variety of reasons. The new method used the same sample cards, a robotic commercial sample prep method, and automated analysis of samples using a MALDI‐TOF/TOF instrument. Failures of the new method against IEF were informative – most were premature or heterozygous neonatal infants. Conclusion: automated herding of 1000 samples/day looks feasible. magnified imageDouble or nothing for the kidney?pp. 422–431It is a sucker's bet on some streets in New York and serious business for patients waiting for a kidney transplant. Chronic allograft nephropathy (CAN) is the leading cause of kidney graft failure in the first year after transplant, being responsible for a 50–80% loss rate. The ability to reduce the failure rate by early recognition and treatment of CAN would markedly increase the number of kidneys available. Johnston et al. report here an improved urine protein marker pattern that can distinguish normal from CAN urine protein patterns. The principal element of the pattern (SELDI‐TOF‐MS hardware) was the amount of β2 microglobulin (11.7 kDa). A single β2 microglobulin point is insufficiently discriminating so further work will have to be done in a more complex setting. magnified image

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC50; <10μg/mL). Various ACEI peptides derived from β-casein [(f(193–205), f(193–207), and f(193–209)] and αS1-casein [f(1–15), f(1–22), f(14–23), and f(24–34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.

Research and development of anti-Abeta oligomers antibodies for the treatment of Alzheimer's disease

Amyloid beta oligomers (AbOs) are a promising target for treatment of Alzheimer's disease (AD) because they are considered as major pathogenic factor of AD. One of the hopeful therapeutic approaches targeted to AbOs is passive immunotherapy using anti-AbOs specific antibody. This approach has two merits, which are efficient therapeutic effect and decrease of side effects, because the antibody does not bind to amyloid precursor protein (APP) nor naturally existed Abeta. We generated a number of anti-AbOs antibodies and performed screening to select the best one by in vitro tests and in vivo test using AD model mouse. These results will be presented. Anti-AbOs antibodies were generated by immunization of antigens prepared by our original method to mouse. The initial screening was performed by ELISA and dotblot assay. The amino acid sequences of all positive clones were analyzed and sequence-redundant clones were eliminated. Then, antibodies that have higher selectivity to AbOs were selected by competitive ELISA using Abeta monomer or AbOs as competitors. Next, antibodies that displayed non-binding property to normal human tissue array were chosen. Then, their affinities to AbOs were measured by SPR (Surface Plasmon Resonance) analysis, following that the antibodies were divided into high or low affinity groups. Finally, passive immunization test using Tg2576 mouse was performed in several antibodies from each group. Behavioral tests and biochemical analysis were used for the evaluation. 184 antibodies were obtained in the results of first screening and subsequent determination of amino acid sequence. Then, 49 antibodies which have higher selectivity to AbOs were selected by the competitive ELISA. Moreover, dozens of antibodies were chosen from 49 antibodies by their non-binding feature to normal human tissue array. Next, these antibodies were divided into high and low affinity groups by SPR analysis. Finally, in vivo efficacy in several antibodies in each groups were examined by passive immunization test by using Tg2576 mouse. The results showed that high affinity antibodies have better efficacy of improvement of learning memory than low affinity ones. And also there were difference in clearance of Abeta in the brains among the high affinity antibodies. The screening to select the best antibody, which has high specificity and affinity to AbOs and also has in vivo efficacy, was finished.

Identification of an antifungal peptide from Trapa natans fruits with inhibitory effects on Candida tropicalis biofilm formation

Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studies have been focused on screening of plant peptides to find novel compounds having antifungal activities. Here, a novel antifungal plant peptide, with molecular mass of 1230Da was purified from fruits of Trapa natans by reverse phase high performance liquid chromatography using 300SB-C18 column and named as Tn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed the inhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentration dependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCR analysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by a unique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) of this peptide for the development of more effective antimycotic compounds.