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1.
Clin. transl. oncol. (Print) ; 24(2): 193-202, febrero 2022. ilus
Article in English | IBECS | ID: ibc-203426

ABSTRACT

During last few decades, role of microbiota and its importance in several diseases has been a hot topic for research. The microbiota is considered as an accessory organ for maintaining normal physiology of an individual. These microbiota organisms which normally colonize several epithelial surfaces are known to secrete several small molecules leading to local and systemic effects on normal biological processes. The role of microbiota is also established in carcinogenesis as per several recent findings. The effects of microbiota on cancer is not only limited to their contribution in oncogenesis, but the overall susceptibility for oncogenesis and its subsequent progression, development of coinfections, and response to anticancer therapy is also found to be affected by microbiota. The information about microbiota and subsequent contributions of microbes in anticancer response motivated researchers in development of microbes-based anticancer therapeutics. We provided current status of microbiota contribution in oncogenesis with special reference to their mechanistic implications in different aspects of oncogenesis. In addition, the mechanistic implications of bacteria in anticancer therapy are also discussed. We conclude that several mechanisms of microbiota-mediated regulation of oncogenesis is known, but approaches must be focused on understanding contribution of microbiota as a community rather than single organisms-mediated effects.


Subject(s)
Humans , Health Sciences , Carcinogenesis , Infections , Microbiota , Gastrointestinal Microbiome , Neoplasms , Cell Enlargement
2.
Clin Transl Oncol ; 24(2): 193-202, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34387847

ABSTRACT

During last few decades, role of microbiota and its importance in several diseases has been a hot topic for research. The microbiota is considered as an accessory organ for maintaining normal physiology of an individual. These microbiota organisms which normally colonize several epithelial surfaces are known to secrete several small molecules leading to local and systemic effects on normal biological processes. The role of microbiota is also established in carcinogenesis as per several recent findings. The effects of microbiota on cancer is not only limited to their contribution in oncogenesis, but the overall susceptibility for oncogenesis and its subsequent progression, development of coinfections, and response to anticancer therapy is also found to be affected by microbiota. The information about microbiota and subsequent contributions of microbes in anticancer response motivated researchers in development of microbes-based anticancer therapeutics. We provided current status of microbiota contribution in oncogenesis with special reference to their mechanistic implications in different aspects of oncogenesis. In addition, the mechanistic implications of bacteria in anticancer therapy are also discussed. We conclude that several mechanisms of microbiota-mediated regulation of oncogenesis is known, but approaches must be focused on understanding contribution of microbiota as a community rather than single organisms-mediated effects.


Subject(s)
Microbiota , Neoplasms/etiology , Humans , Neoplasms/microbiology
3.
Reproduction ; 147(5): 599-614, 2014 May.
Article in English | MEDLINE | ID: mdl-24446454

ABSTRACT

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.


Subject(s)
Endometrium/physiology , Pregnancy Proteins/physiology , Pregnancy, Animal/physiology , Proteomics , Sheep/physiology , Animals , Electrophoresis, Gel, Two-Dimensional , Embryonic Development/physiology , Estrus/physiology , Female , Mass Spectrometry , Pregnancy , Signal Transduction/physiology
4.
Oral Microbiol Immunol ; 23(1): 36-42, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18173796

ABSTRACT

INTRODUCTION: Fusobacterium nucleatum represents one of the predominant anaerobic species in the oral microbiota. Penicillin-resistant F. nucleatum have been isolated from intra- and extraoral infections. This study aimed to assess ampicillin resistance in F. nucleatum by investigating the synthesis of resistance-associated proteins. METHODS: Ampicillin-resistant and ampicillin-susceptible F. nucleatum isolates were obtained from 22 dental plaque samples. Two-dimensional gel electrophoresis and mass spectrometry were used to investigate bacterial protein synthesis. Proteins exhibiting statistically significant quantitative changes between sensitive and resistant isolates were identified using peptide mass mapping and matrix-assisted laser desorption/ionization - time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry. RESULTS: Twenty-three F. nucleatum isolates were recovered from plaque samples and their ampicillin minimum inhibitory concentrations ranged between 0.125 microg/ml and 256 microg/ml. Analysis of the bacterial cellular proteins by two-dimensional gel electrophoresis resolved 154-246 distinct protein spots (mean 212, n = 9). Between 32% and 83% of the protein spots were common for the F. nucleatum isolates. Comparisons of the protein profiles of sensitive and resistant isolates revealed the presence of a 29 kDa protein and significant increases in the synthesis of two proteins at 37 and 46 kDa in the ampicillin-resistant F. nucleatum isolates. These proteins were identified as a class D beta-lactamase, ATP-binding cassette (ABC) transporter ATP-binding protein and enolase, respectively. CONCLUSION: Synthesis of a class D beta-lactamase by ampicillin-resistant F. nucleatum isolates could complicate antimicrobial treatment because these enzymes might confer resistance to many classes of beta-lactam antibiotics. The differences observed in protein synthesis between ampicillin-resistant and ampicillin-susceptible F. nucleatum may contribute to the antibiotic resistance and virulence of these bacteria.


Subject(s)
Ampicillin Resistance , Bacterial Proteins/analysis , Fusobacterium nucleatum/metabolism , Proteome/analysis , ATP-Binding Cassette Transporters/analysis , Ampicillin/pharmacology , Carrier Proteins/analysis , Dental Plaque/microbiology , Electrophoresis, Gel, Two-Dimensional , Fusobacterium nucleatum/classification , Fusobacterium nucleatum/drug effects , Humans , Microbial Sensitivity Tests , Molecular Weight , Peptide Mapping , Phosphopyruvate Hydratase/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , beta-Lactamases/analysis
5.
J Food Prot ; 70(6): 1475-82, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17612079

ABSTRACT

A colorimetric method, reverse transcriptase PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was evaluated for ease of use, reliability, and sensitivity when detecting known human pathogenic virus present in shellfish, using a traditional polyethylene precipitation or immunocapture virus concentration method. The newly developed ELISA method could successfully detect enteroviruses and noroviruses in artificially and naturally contaminated shellfish. Overall, ELISA was shown to be a robust and sensitive method, which had a detection limit of 10 to 100 50% tissue culture infective dose enterovirus per gram of Crassostrea gigas (Pacific oyster) digestive gland and whole Mytilus edulis (common blue mussel). The technique was easily established in a new laboratory and required no specialized equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Mollusca/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Shellfish/virology , Animals , Consumer Product Safety , Enterovirus/isolation & purification , Humans , Norovirus/isolation & purification , Sensitivity and Specificity
6.
J Virol Methods ; 132(1-2): 92-6, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16221496

ABSTRACT

A new method, termed RT-PCR-ELISA, was evaluated for ease of use, reliability and sensitivity when detecting infectious pancreatic necrosis virus (IPNV) present in trout kidney tissue. The method had comparable sensitivity to existing PCR assays and could successfully detect 1.5 x 10(4) pfu IPNV in artificially contaminated trout kidney samples. The technique was easily established in a new laboratory and required no specialised equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. The RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.


Subject(s)
Birnaviridae Infections/veterinary , Enzyme-Linked Immunosorbent Assay/methods , Fish Diseases/diagnosis , Infectious pancreatic necrosis virus/isolation & purification , Oncorhynchus mykiss/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Birnaviridae Infections/diagnosis , Birnaviridae Infections/virology , Fish Diseases/virology , Kidney/virology , Reproducibility of Results , Sensitivity and Specificity
7.
Antonie Van Leeuwenhoek ; 81(1-4): 33-42, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12448703

ABSTRACT

Bacteria and archaea occupy a considerable diversity of niches that vary with respect to the physical conditions. Survival and colonisation requires the capacity to sense, and adapt to, environmental change. In this short review we consider the issues of adaptation to acidic conditions, in particular the mechanisms that might be employed by different bacteria to respond to the specific challenges of their niche. We lay particular emphasis on the protection of the cytoplasm during alterations of the cytoplasmic pH and, in the Gram negative bacteria, on recent work that suggests that protection of the periplasm is critical for survival of exposure to extreme acid. Finally, we discuss potential mechanisms by which pH might be sensed and consider the insights gained from proteins that sense and respond specifically to changes in pH.


Subject(s)
Adaptation, Physiological , Bacteria/growth & development , Gene Expression Regulation, Bacterial , Signal Transduction , Bacteria/metabolism , Bacterial Physiological Phenomena , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hydrogen-Ion Concentration
8.
9.
Pharm Dev Technol ; 6(2): 255-61, 2001.
Article in English | MEDLINE | ID: mdl-11417000

ABSTRACT

PURPOSE: BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. METHODS: SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. RESULTS: Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. CONCLUSION: Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.


Subject(s)
Anti-Inflammatory Agents/chemistry , Peptides/chemistry , Drug Stability , Hydrogen-Ion Concentration , Oncostatin M , Trypsin Inhibitors/pharmacology
10.
Aust J Rural Health ; 9(6): 280-5, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11998262

ABSTRACT

This paper reports on a descriptive study into family violence in rural Victoria. Focus groups were held in a number of areas across rural Victoria with a total of 24 community nurse participants. The focus groups were audio-taped and the tapes transcribed to enable the clustering of themes. The dominant themes were: picking up cues, helping and helplessness, holding secrets and quiet resistance. Underpinning all these themes however, was the notion of 'risky business'. All nurses in the study gave examples of situations that they encountered; their ways of helping; of working around a system that is unhelpful; and the ways in which their work while skilled, thoughtful and wise, is also costly in terms of the emotional wounds they carry. Rural nurses work with considerable risk and courage as they engage in the care and support of women experiencing family violence.


Subject(s)
Community Health Nursing/methods , Domestic Violence/prevention & control , Rural Health Services , Anecdotes as Topic , Confidentiality , Continuity of Patient Care , Female , Focus Groups , Humans , Male , Nonverbal Communication , Nurse's Role , Nurse-Patient Relations , Victoria
11.
J Pathol ; 192(3): 280-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11054709

ABSTRACT

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins.


Subject(s)
Cardiovascular Diseases/diagnosis , Neoplasms/diagnosis , Nervous System Diseases/diagnosis , Proteome , Biomarkers , Biomarkers, Tumor , Computational Biology , Electrophoresis, Gel, Two-Dimensional , Humans , Image Processing, Computer-Assisted , Mass Spectrometry , Sequence Analysis, Protein
12.
Electrophoresis ; 21(6): 1187-201, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10786891

ABSTRACT

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.


Subject(s)
Bacterial Proteins/analysis , Proteome/analysis , Drug Resistance, Microbial , Genetic Variation , Genome, Bacterial , Humans
13.
Electrophoresis ; 20(11): 2259-68, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10493130

ABSTRACT

Streptococcus pneumoniae is a significant human pathogen which is an important cause of pneumonia and bacteraemia. Over the past few years the incidence of antibiotic resistance among clinical isolates of S. pneumoniae has increased. Penicillin resistance is now widespread and the frequency of isolates that are resistant to erythromycin has risen. Erythromycin resistance in S. pneumoniae follows two basic patterns. The MLS erythromycin-resistant phenotype is due to the enzymatic methylation of ribosomal RNA that blocks erythromycin binding to the ribosome. Alternatively, in isolates of the M phenotype, a more recently documented mechanism, resistance is associated with an active efflux process that reduces intracellular levels of erythromycin. We used two-dimensional electrophoresis to examine the proteins synthesised by erythromycin-susceptible and -resistant S. pneumoniae. Erythromycin-resistant S. pneumoniae with the M phenotype showed a significantly increased synthesis of a 38,500 Dalton (pI 6.27) protein compared to susceptible isolates. Peptide mass mapping was used to identify the 38,500 Dalton protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was demonstrated that S. pneumoniae synthesised at least three forms of GAPDH that differed in their isoelectric points. The form of GAPDH possessing the most basic pI showed the increased synthesis in the erythromycin-resistant S. pneumoniae isolates. Alterations in the synthesis of GAPDH were only found for those erythromycin-resistant isolates possessing the M phenotype. S. pneumoniae isolates with the MLS phenotype were indistinguishable from the susceptible strains using the analytical conditions employed for the current study. The possible role of GAPDH in erythromycin resistance of S. pneumoniae is considered.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Erythromycin/pharmacology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/metabolism , Drug Resistance, Microbial , Electrophoresis, Gel, Two-Dimensional/methods , Glyceraldehyde-3-Phosphate Dehydrogenases/analysis , Humans , Peptide Mapping/methods , Streptococcus pneumoniae/isolation & purification
14.
Electrophoresis ; 18(8): 1472-82, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9298661

ABSTRACT

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.


Subject(s)
Bacterial Proteins/isolation & purification , Databases, Factual , Electrophoresis, Gel, Two-Dimensional/methods , Haemophilus influenzae/chemistry , Bacterial Proteins/genetics , Electrophoresis, Gel, Two-Dimensional/statistics & numerical data , Gene Expression , Genome, Bacterial , Haemophilus/chemistry , Haemophilus/genetics , Haemophilus influenzae/genetics , Humans , Image Processing, Computer-Assisted , Mutation , Peptide Mapping/methods , Peptide Mapping/statistics & numerical data , Species Specificity , Superoxide Dismutase/genetics , Superoxide Dismutase/isolation & purification
15.
J Neurovirol ; 3(1): 76-82, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9147825

ABSTRACT

Enteroviruses have been implicated in persistent infections of the nervous system and in certain paralytic motor neuron syndromes. Enteroviral persistence may depend on defective transcription, resulting in the abnormal production of equal amounts of genomic and template RNA strands rather than the normal ratio of 60-100:1. An in vitro model of a persistent coxsackie virus in human skeletal muscle cells was investigated using in situ hybridisation and a semiquantitative parallel, complementary, reverse transcriptase polymerase chain reaction. The ratio of genomic to template RNA was found to be approximately 60:1. We conclude that enteroviral persistence in this in vitro model is not dependent on altered transcription. In vivo, other viral and host factors should be considered.


Subject(s)
Enterovirus B, Human/genetics , Gene Expression Regulation, Viral , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , Transcription, Genetic , Enterovirus B, Human/physiology , Genome, Viral , Humans , In Situ Hybridization , Muscle, Skeletal/virology , Polymerase Chain Reaction , Rhabdomyosarcoma/pathology , Templates, Genetic , Tumor Cells, Cultured , Virus Latency , Virus Replication
16.
Nurs Inq ; 4(4): 246-56, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9437961

ABSTRACT

Nurses live and work in complex practice worlds; worlds of shrinking resources and expanding needs. Reflection through journaling offers unique opportunities to gain insight into practice. What might we learn from one's journal? A reflective journal can be a source of interplay between the self as written and the self as other. Likewise, the journal may act to situate ourselves in practice, while at the same time enabling us to illuminate how and in what ways our understandings have become distorted. The extent to which one's journal is educative depends upon the manner in which one chooses to use it as a transformative tool, a tool that might well be described as a process of healing and enlightenment. In order to illustrate the reflexive nature of journaling, this paper is presented as a play reading, where a conversation about practice stories between the different aspects of the nurse's self is depicted. In adopting a play reading, an alternative pedagogical tool is used to illustrate different methodologies exemplifying the emergence of how and in what ways we develop and reconstruct our understanding in nursing.


Subject(s)
Nurses/psychology , Nursing Process , Self Concept , Thinking , Writing , Humans , Nurse-Patient Relations , Nursing Methodology Research , Nursing Theory
17.
Pept Res ; 9(6): 327-31, 1996.
Article in English | MEDLINE | ID: mdl-9048428

ABSTRACT

An 18-residue-long fragment of vasoactive intestinal polypeptide [VIP(11-28)-NH2] that is known to be analgesic was synthesized by solid-phase t-Boc methodology on a 4-methylbenzhydrylamine resin. Circular dichroism spectroscopy gave evidence that the peptid acquires about 60% helical structure in 50/50 methanol/phosphate buffer, pH 6.0, and 65% (+/-5%) helicity in 80/20 methanol/phosphate buffer pH 7.0, A 2.0 mM solution of VIP (11-28) NH2 in 80% methanol, 20% phosphate buffer pH 7.0 was subjected to 2-dimensional nuclear magnetic resonance (NMR) studies The NMR results suggested formation of an extended helical structure extending from residue 11 to 27 essentially the same region found to be helical in a VIP(1-28)-NH2 and log. This finding suggests that the sequence required for analgesia assumes a helical structure at the receptor.


Subject(s)
Analgesics/chemistry , Peptide Fragments/chemistry , Vasoactive Intestinal Peptide/chemistry , Amino Acid Sequence , Circular Dichroism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Protein Conformation , Protein Structure, Secondary , Vasoactive Intestinal Peptide/pharmacology
18.
Clin Diagn Lab Immunol ; 3(3): 280-6, 1996 May.
Article in English | MEDLINE | ID: mdl-8705669

ABSTRACT

The neutralizing and enhancing activities of respiratory syncytial virus (RSV)-specific antibodies were examined. These two biological activities were measured for a panel of six monoclonal antibodies (MAbs) specific to the RSV surface F and G glycoproteins. Four MAbs specific for the F protein possessed both neutralizing and enhancing activities. One MAb (11-2-D2), specific to the G protein, enhanced RSV infection of U937 cells, a human macrophage cell line, but did not neutralize virus infectivity. One MAb (11-3-A3), specific to the F protein, efficiently neutralized virus infectivity but did not enhance RSV infection of U937 cells. MAb 11-3-A3 neutralized representative strains of the two antigenic subtypes of RSV. Assays performed with mixtures of MAbs showed that high concentrations of MAb 11-3-A3 masked the enhancing activity of MAb 11-2-D2. The assay of mixtures of two MAbs possessing only enhancing activities demonstrated that this response was synergistic. The role of neutralizing and enhancing antibodies in determining the outcome of RSV infection was examined for infants from whom cord blood serum samples were collected at birth. There was no significant difference in the magnitude of the serum-enhancing activities between infants who were hospitalized with RSV infections and a group of age- and sex-matched control infants with no reported respiratory illness requiring hospitalization. However, the results indicated a possible correlation between RSV infection of the infants and the occurrence of in vitro antibody-dependent enhancement of the cord blood sera at a serum dilution of 10(-2). A significant inverse correlation was found between the plaque-neutralizing and enhancing activities of the cord blood sera from infants, irrespective of subsequent RSV infection. These data are discussed in relation to the possible contribution of antibody-dependent enhancement to the normal course of RSV pathology in vivo.


Subject(s)
Antibodies, Viral/immunology , Respiratory Syncytial Virus, Human/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Viral/analysis , Antibody Specificity , Antibody-Dependent Enhancement , Fetal Blood/immunology , Humans , Infant, Newborn , Neutralization Tests
19.
Antimicrob Agents Chemother ; 40(3): 646-51, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8851587

ABSTRACT

Rifampin-resistant (Rifr) Neisseria meningitidis strains are known to have single point mutations in the central conserved regions of the rpoB gene. We have demonstrated two distinct resistance phenotypes in strains with identical mutations in this region, an intermediate level of resistance in Rifr clinical isolates and a high level of resistance in mutants selected in vitro. The possible role of membrane permeability in the latter was investigated by measuring MICs in the presence of Tween 80; values for high-level-resistance mutants were reduced to intermediate levels, whereas those for intermediate-level-resistance strains were unaffected. The highly resistant mutants were also found to have increased resistance to Triton X-100 and gentian violet. Sequencing of the meningococcal mtrR gene and its promoter region (which determine resistance to hydrophobic agents in Neisseria gonorrhoeae) from susceptible or intermediate strains and highly resistant mutants generated from them showed no mutation within this region. Two-dimensional gel electrophoresis of two parent and Rif mutant strains showed identical shifts in the pI of one protein, indicating that differences between the parent and the highly Rifr mutant are not confined to the rpoB gene. These results indicate that both permeability and rpoB mutations play a role in determining the resistance of N. meningitidis to rifampin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cell Membrane Permeability/drug effects , Neisseria meningitidis/drug effects , Rifampin/pharmacology , Base Sequence , DNA, Bacterial/analysis , Drug Resistance, Microbial , Electrophoresis, Polyacrylamide Gel , Genes, Bacterial , Microbial Sensitivity Tests , Molecular Sequence Data , Neisseria meningitidis/genetics , Neisseria meningitidis/ultrastructure , Phenotype , Polymerase Chain Reaction
20.
J Chromatogr A ; 698(1-2): 203-24, 1995 Apr 28.
Article in English | MEDLINE | ID: mdl-7773364

ABSTRACT

High-resolution two-dimensional electrophoresis (2DE) can resolve many hundreds of proteins present in complex mixtures depending on the method of detection. These proteins can be characterised qualitatively, with respect to their electrophoretic mobilities (i.e. charge and apparent molecular mass) and quantitatively, using densitometry, to determine their amounts. There has been a widespread application of 2DE in the analysis and characterisation of protein mutations for a range of organisms. This review presents examples of the use of 2DE to study naturally occurring protein mutations and polymorphisms as well as the characterisation of induced protein mutations in prokaryotes and eukaryotes. Examples are presented to illustrate the use of 2DE to detect mutations affecting the electrophoretic mobility and biosynthesis of individual proteins as well as mutations leading to global alterations in cellular protein synthesis. The advantages and disadvantages of 2DE in the detection of protein mutations are discussed.


Subject(s)
Electrophoresis, Gel, Two-Dimensional , Mutation , Proteins/chemistry , Proteins/genetics , Animals , Humans
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