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2.
J Cardiothorac Vasc Anesth ; 34(12): 3398-3408, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31982292

RESUMO

Experimental and clinical heart transplantation programs in the USSR, Russia, and post-Soviet states are described, including information about early experimental studies conducted by Russian and Soviet scientists in the early to mid-20th century. The novel research of V.P. Demikhov, a Soviet transplantologist famous for performing the first experimental heart transplantations and coronary artery bypass surgeries in the world is highlighted. In addition, the preparation and implementation of the USSR's first clinical heart transplantations during the pre-cyclosporine era (1960-1970s) are described, and the features of anesthesia, cardiopulmonary bypass, and graft protection, as well as causes of heart failure are analyzed. Furthermore, information about the first successful heart transplantation performed by V.I. Shumakov (1987), the first successful heart transplantation programs in the USSR and Russia during the cyclosporine era (after 1980), and anesthesiologic developments for heart transplantation are presented. The current status, modern research, and prospects of heart transplantation in Russia and the implementation of heart transplantation programs in Lithuania, Ukraine, Latvia, Republic of Belarus, and Kazakhstan also are described.

3.
J Cardiothorac Vasc Anesth ; 33(5): 1407-1419, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30228053

RESUMO

Natriuretic peptides, predominantly B-type, are widely used in cardiology as prognostic and diagnostic biomarkers or, much less often, as a substantive treatment tool. They are hormones that are produced mainly in the myocardium in response to overload and ischemia, and their level quite accurately reflects the degree of myocardial dysfunction. Although their use in cardiac anesthesia and intensive care setting seems to be very beneficial for assessing the risk of acute disturbance of myocardial function or its laboratory monitoring, the actual significance of natriuretic peptides in this area is not yet recognized. This is due to the lack of clear diagnostic and prognostic values for these biomarkers supported by high-quality researches. On the basis of the available data, main advantages, existing difficulties, and most effective ways of using natriuretic peptides for determining the risk of heart surgery and assessing the severity of sepsis, pneumonia, and other critical conditions have been discussed in this review. In addition, the expediency of using natriuretic peptides as target parameters for goal-oriented therapy and as a substantive tool for treatment is considered.


Assuntos
Anestesia em Procedimentos Cardíacos/métodos , Procedimentos Cirúrgicos Cardíacos/métodos , Cuidados Críticos/métodos , Peptídeos Natriuréticos/sangue , Complicações Pós-Operatórias/sangue , Anestesia em Procedimentos Cardíacos/efeitos adversos , Biomarcadores/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Humanos , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Prognóstico
4.
J Cardiothorac Vasc Anesth ; 33(12): 3358-3365, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30072269

RESUMO

Anesthesiology, the branch of medicine concerning anesthesia and management of the vital functions of patients undergoing surgery, has played an important role in the development of cardiac surgery. In the middle of the last century, medical professionals had little experience in the treatment of congenital and acquired heart diseases. Progress of cardiac anesthesiology in Russia, as well as in countries across the globe, was due to requests to increase the safety of surgical procedures and to improve survival rates for the increasing number of patients with complex heart diseases. The development of cardiac surgery and anesthesiology in Russia evolved in 2 directions simultaneously in the mid-1950s. Some surgeons widely accepted the use of perfusionless hypothermia (hypothermia caused by surface cooling without perfusion); others were in favor of cardiopulmonary bypass technology. This review focuses on major historic milestones of cardiac anesthesiology in Russia, including its current status and the major problems it faces today.


Assuntos
Anestesia/história , Anestesiologia/história , Procedimentos Cirúrgicos Cardíacos/história , Cardiologia/história , História do Século XX , História do Século XXI , Humanos , Federação Russa
5.
Chem Biol ; 22(8): 1122-33, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26256476

RESUMO

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Metaloproteases/química , Metaloproteases/metabolismo , Sequência de Aminoácidos , Catálise , Humanos , Hidrólise , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Peptídeos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
6.
FEBS J ; 281(11): 2487-502, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24698179

RESUMO

Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases.


Assuntos
Bacteroides fragilis/enzimologia , Ilhas Genômicas/genética , Metaloproteases/genética , Bacteroides fragilis/genética , Metaloproteases/química
7.
J Biol Chem ; 288(48): 34956-67, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24145028

RESUMO

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.


Assuntos
Bacteroides fragilis/genética , Inflamação/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Bacteroides fragilis/patogenicidade , Ilhas Genômicas/genética , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloendopeptidases/genética , Microbiota , Neoplasias/genética , Neoplasias/patologia , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Proteólise , Especificidade por Substrato
8.
PLoS One ; 8(1): e54290, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23335997

RESUMO

BACKGROUND: There is a growing appreciation of the role of proteolytic processes in human health and disease, but tools for analysis of such processes on a proteome-wide scale are limited. Furin is a ubiquitous proprotein convertase that cleaves after basic residues and transforms secretory proproteins into biologically active proteins. Despite this important role, many furin substrates remain unknown in the human proteome. METHODOLOGY/PRINCIPAL FINDINGS: We devised an approach for proteinase target identification that combines an in silico discovery pipeline with highly multiplexed proteinase activity assays. We performed in silico analysis of the human proteome and identified over 1,050 secretory proteins as potential furin substrates. We then used a multiplexed protease assay to validate these tentative targets. The assay was carried out on over 3,260 overlapping peptides designed to represent P7-P1' and P4-P4' positions of furin cleavage sites in the candidate proteins. The obtained results greatly increased our knowledge of the unique cleavage preferences of furin, revealed the importance of both short-range (P4-P1) and long-range (P7-P6) interactions in defining furin cleavage specificity, demonstrated that the R-X-R/K/X-R ↓ motif alone is insufficient for predicting furin proteolysis of the substrate, and identified ≈ 490 potential protein substrates of furin in the human proteome. CONCLUSIONS/SIGNIFICANCE: The assignment of these substrates to cellular pathways suggests an important role of furin in development, including axonal guidance, cardiogenesis, and maintenance of stem cell pluripotency. The novel approach proposed in this study can be readily applied to other proteinases.


Assuntos
Furina/química , Furina/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína , Proteólise , Reprodutibilidade dos Testes , Especificidade por Substrato
9.
PLoS One ; 7(6): e37441, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22701568

RESUMO

We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.


Assuntos
DNA Complementar/genética , Hepacivirus/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Fosfotransferases/metabolismo , Proteômica/métodos , DNA Complementar/metabolismo , Análise em Microsséries/métodos , Peptídeos/metabolismo , Fosforilação , Especificidade por Substrato , Proteínas não Estruturais Virais/metabolismo
10.
PLoS One ; 7(4): e35759, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558217

RESUMO

BACKGROUND: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle. CONCLUSIONS/SIGNIFICANCE: Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.


Assuntos
Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/análise , Peptídeos/síntese química , Poliproteínas/química , Processamento de Proteína Pós-Traducional , Proteólise , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
11.
Comb Chem High Throughput Screen ; 11(1): 24-35, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18220541

RESUMO

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.


Assuntos
Bioensaio/métodos , Endopeptidases/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sequência de Aminoácidos , Sequência de Bases , Biotina/química , Biotina/metabolismo , DNA/química , DNA/metabolismo , Endopeptidases/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Histidina/química , Histidina/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soluções/química , Especificidade por Substrato
12.
Nucleosides Nucleotides Nucleic Acids ; 26(10-12): 1353-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18066782

RESUMO

We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.


Assuntos
DNA/química , Oligonucleotídeos/química , Peptídeo Hidrolases/análise , Peptídeos/química , Fosfotransferases/análise , Peptídeo Hidrolases/química , Fosfotransferases/química , Especificidade por Substrato
13.
Chembiochem ; 8(8): 933-42, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17471478

RESUMO

We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.


Assuntos
Técnicas de Sonda Molecular , Proteínas Quinases/química , Extratos Celulares , DNA/química , Ativação Enzimática , Células HeLa , Humanos , Marcação por Isótopo , Oligonucleotídeos/química , Peptídeos/química , Fosforilação , Fosfosserina/química , Fosfotreonina/química , Fosfotirosina/química , Proteínas Quinases/análise , Sensibilidade e Especificidade , Soluções/química , Especificidade por Substrato
14.
J Chromatogr Sci ; 45(4): 207-11, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17504569

RESUMO

The retention of histidine-containing peptides in immobilized metal-affinity chromatography is studied using several hundred modeled peptides. Retention is driven primarily by the number of histidine residues; however, the amino acid composition in the immediate vicinity plays a significant role. Specifically, the arginine and tryptophan content has to be taken into consideration. During the course of this study, an alternative tag that can be used similarly to a polyhistidine tag is discovered.


Assuntos
Cromatografia de Afinidade/instrumentação , Histidina/química , Peptídeos/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Níquel
15.
J Virol ; 81(9): 4501-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17301157

RESUMO

The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1'-P2' positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1' or P2'. Structure-based modeling suggests that Arg(76) and Pro(131)-Thr(132) limit the P1'-P2' subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu(76) and Lys(131)-Pro(132) widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3'-P4' positions were fixed and the P1' and P2' positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.


Assuntos
Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese , RNA Helicases/genética , RNA Helicases/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato/genética , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/genética
16.
Biochem J ; 401(3): 743-52, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17067286

RESUMO

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.


Assuntos
Vírus da Dengue/enzimologia , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sítios de Ligação , Mutação , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
17.
Comb Chem High Throughput Screen ; 9(6): 481-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16842230

RESUMO

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.


Assuntos
Bioensaio/métodos , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Caspases/química , Caspases/genética , Estrutura Molecular , Peptídeos/química , Soluções/química , Especificidade por Substrato , Fatores de Tempo
18.
Nucleosides Nucleotides Nucleic Acids ; 24(5-7): 1037-41, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16248087

RESUMO

A key factor influencing the quality of long oligonucleotides is the choice of controlled pore glass (CPG) which is used as a solid support during oligonucleotide synthesis. We studied the influence of CPG pore size on the quality of 75-mer oligonucleotides. Using electrophoresis and HPLC, we demonstrated failure modes that can occur at certain oligo lengths with 1000A pore size, and compared yield and purity of 75-mer oligos using 1000A and larger pore size CPG. We showed that oligonucleotides with much better quality are obtained using CPG with pore sizes of 1400A and larger. We also identified the key characteristics for CPG selection that lead to the best CPG performance.


Assuntos
Biologia Molecular/instrumentação , Nucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/síntese química , Cromatografia Líquida de Alta Pressão , DNA/química , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Vidro , Biologia Molecular/métodos , Nucleosídeos/química , Nucleotídeos/química
19.
Biopolymers ; 73(5): 621-30, 2004 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-15048786

RESUMO

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.


Assuntos
Imunoconjugados/química , Oligonucleotídeos/síntese química , Proteínas/análise , Anticorpos/química , Imunoensaio/métodos , Oligonucleotídeos/química
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