DNA–DNA hybridization in blackflies (Diptera: Simuliidae)

1972 ◽  
Vol 50 (7) ◽  
pp. 931-940 ◽  
Author(s):  
Ikuko Teshima
Keyword(s):  
Dna Hybridization ◽  
Buoyant Density ◽  
Filter Method ◽  
Base Sequence ◽  
Thermal Transition ◽  
Dependent Manner ◽  
Maximal Yield ◽  
Close Relationship ◽  
Sequence Relationships ◽  
Homologous Reaction

This paper is a study of base sequence relationships in blackflies by DNA–DNA hybridization. The species used are: Simulium venustum, S. tuberosum, S. vittatum, Cnephia dacotensis, and Prosimulium multidentatum. The midpoints of thermal transition and the buoyant density in CsCl were determined for the DNA of each species and the percent G + C estimated. With the filter method of hybrid formation, maximal yield was obtained at 60 °C; 24 h was found to be a practical length of time for incubation. When the amount of DNA on filter was varied, the homologous reaction reached a plateau at about 50% of the input radioactive DNA; the heterologous reaction reached a plateau at a lesser value in a species-dependent manner. The results indicate a close relationship between S. venustum and S. tuberosum, a more distant one between these species and S. vittatum, and an intermediate one with C. dacotensis. Prosimulium multidentatum shared very little homology with members of the other genera. A precise quantitative evaluation of the results is difficult owing to the complexity of interacting factors. Interpretation would be aided by knowledge of genome sizes and intragenome repetition frequencies. Appropriate studies are underway.

scholarly journals Entamoeba histolytica Cysteine Proteinases Disrupt the Polymeric Structure of Colonic Mucin and Alter Its Protective Function

2003 ◽  
Vol 71 (2) ◽  
pp. 838-844 ◽  
Author(s):  
Darcy Moncada ◽  
Kathy Keller ◽  
Kris Chadee
Keyword(s):  
Entamoeba Histolytica ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Colonic Epithelium ◽  
Dependent Manner ◽  
Cysteine Proteinases ◽  
Protective Function ◽  
Mucous Layer ◽  
Colonic Mucin ◽  
Cscl Density Gradient

ABSTRACT The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [35S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time- and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.

scholarly journals Characterization ofHalorubrum sfaxensesp. nov., a New Halophilic Archaeon Isolated from the Solar Saltern of Sfax in Tunisia

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Hana Trigui ◽  
Salma Masmoudi ◽  
Céline Brochier-Armanet ◽  
Sami Maalej ◽  
Sam Dukan
Keyword(s):  
Dna Hybridization ◽  
Optimal Growth ◽  
Rrna Gene ◽  
Solar Saltern ◽  
Halophilic Archaeon ◽  
Biochemical Tests ◽  
Phenotypic Differentiation ◽  
The Family ◽  
Close Relationship ◽  
Extremely Halophilic Archaeon

An extremely halophilic archaeon, strain ETD6, was isolated from a marine solar saltern in Sfax, Tunisia. Analysis of the 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genusHalorubrumamong the familyHalobacteriaceae, with a close relationship toHrr. xinjiangense(99.77% of identity). However, value for DNA-DNA hybridization between strain ETD6 andHrr.xinjiangensewere about 24.5%. The G+C content of the genomic DNA was 65.1 mol% (T(m)). Strain ETD6 grew in 15–35% (w/v) NaCl. The temperature and pH ranges for growth were 20–55°C and 6–9, respectively. Optimal growth occurred at 25% NaCl, 37°C, and pH 7.4. The results of the DNA hybridization againstHrr. xinjiangenseand physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain ETD6 from otherHrr.species. Therefore, strain ETD6 represents a novel species of the genusHalorubrum, for which the nameHrr. sfaxensesp. nov. is proposed. The Genbank EMBL-EBI accession number is GU724599.

Apparent α-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay

1992 ◽  
Vol 134 (3) ◽  
pp. 341-352 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies ◽  
G. Baxter ◽  
R. Webb ◽  
A. S. McNeilly
Keyword(s):  
Buoyant Density ◽  
Subcellular Fractionation ◽  
Subcellular Fractions ◽  
Corpora Lutea ◽  
Dependent Manner ◽  
Tissue Extracts ◽  
Variable Activity ◽  
Luteal Tissue ◽  
Porcine Granulosa Cell ◽  
Dose Dependent

ABSTRACT Immunoreactive α-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1–26 amino acid sequence of the N-terminus of the α chain of porcine inhibin (p1–26 α-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1–26 α-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1·15–1·21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (Mr 45 000) which immunoblotted against p1–26 α-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1–26 α-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate α-inhibin material, and immunocytochemical localization studies of α-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of α-inhibin (Mr similar to that of α-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for α-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay. Journal of Endocrinology (1992) 134, 341–352

Relations Among Ringtail Possums (Marsupialia, Pseudocheiridae) Based on Dna-Dna Hybridization

1992 ◽  
Vol 40 (4) ◽  
pp. 423 ◽  
Author(s):  
MS Springer ◽  
GM Mckay ◽  
KP Aplin ◽  
JAW Kirsch
Keyword(s):  
Dna Hybridization ◽  
Phylogenetic Analyses ◽  
Single Copy ◽  
Chromatography Method ◽  
Dna Evolution ◽  
Dna Hybridisation ◽  
Close Relationship ◽  
Copy Dna ◽  
The Stability ◽  
Single Copy Dna

Comparison among eight pseudocheirid species and two outgroup petaurids were made by means of the hydroxyapatite chromatography method of DNA hybridisation. Matrices of DELTAT(m) and DELTAT(m)H-C values were analysed with the FITCH algorithm in Felsenstein's PHYLIP (Version 3.3). Jackknifing and bootstrapping were applied to determine the stability of resulting topologies. All the phylogenetic analyses produced trees that support (1) the monophyly of the Pseudocheirus herbertensis complex, (2) the monophyly of Pseudocheirus, (3) a close relationship between Hemibelideus and Petauroides, and (4) a close relationship between Pseudochirops archeri and Pseudochirops cupreus. Rates of single-copy DNA evolution are slightly faster in Pseudocheirus, Hemibelideus, and Petauroides than in Pseudochirops. Hybridisation evidence also provides a framework for understanding the timing of the pseudocheirid radiation and suggests that the divergence between extant genera dates back to about 36 million years ago.

Proteomic analysis of exosomes reveals an association between cell invasiveness and exosomal bioactivity on endothelial and mesenchymal cell migration in vitro

2018 ◽  
Vol 132 (18) ◽  
pp. 2029-2044 ◽  
Author(s):  
Shayna Sharma ◽  
Mona Alharbi ◽  
Miharu Kobayashi ◽  
Andrew Lai ◽  
Dominic Guanzon ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Buoyant Density ◽  
Tumour Microenvironment ◽  
Ovarian Cancer Cells ◽  
Dependent Manner ◽  
Cell Of Origin ◽  
Invasive Capacity

Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity.

scholarly journals Proposal of Viridibacillus gen. nov. and reclassification of Bacillus arvi, Bacillus arenosi and Bacillus neidei as Viridibacillus arvi gen. nov., comb. nov., Viridibacillus arenosi comb. nov. and Viridibacillus neidei comb. nov.

2007 ◽  
Vol 57 (12) ◽  
pp. 2729-2737 ◽  
Author(s):  
Richard A. Albert ◽  
Julieta Archambault ◽  
Melissa Lempa ◽  
Beth Hurst ◽  
Christine Richardson ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Sequence Similarity ◽  
Single Species ◽  
Rrna Gene ◽  
New Taxon ◽  
Clear Cut ◽  
Close Relationship ◽  
Chemotaxonomic Markers ◽  
Sequence Similarities

A polyphasic study was undertaken to clarify the taxonomic position of endospore-forming strains 433-D9, 433-E17 and 121-X1. BOX-PCR-generated fingerprints indicated that they may be members of a single species. 16S rRNA gene sequence similarity demonstrated that a representative of this group, 433-D9, is affiliated closely with Bacillus arvi DSM 16317T (100 %), Bacillus arenosi DSM 16319T (99.8 %) and Bacillus neidei NRRL BD-87T (97.1 %). Sequence similarities revealed Bacillus pycnus NRRL NRS-1691T and several Kurthia species as the next nearest relatives. DNA–DNA hybridization results showed that strain 433-D9 is a member of B. arvi. Detection of l-Lys–d-Asp-based peptidoglycan in strain 433-D9, B. arvi DSM 16317T and B. arenosi DSM 16319T was in agreement with their close relationship, but differentiated these strains from B. neidei NRRL BD-87T and B. pycnus NRRL NRS-1691T, for which l-Lys–d-Glu was reported. A similar quinone system was detected in strains 433-D9, 433-E17, 121-X1, B. arvi DSM 16317T, B. arenosi DSM 16319T and B. neidei NRRL BD-87T. This system, unusual for bacilli, consisted of the major compound menaquinone MK-8 (69–80 %) and moderate amounts of MK-7 (19–30 %). This observation was in contrast to the predominance of MK-7 of the closest relative B. pycnus NRRL NRS-1691T, as also reported for representatives of the closely related non-endospore-forming genus Kurthia. Strains 433-D9, B. arvi DSM 16317T and B. arenosi DSM 16319T exhibited homogeneous and discriminative polar lipid profiles and fatty acid profiles consisting of major acids i-C15 : 0 and ai-C15 : 0 and moderate amounts of i-C17 : 1 ω10c and i-C17 : 1 I/ai-C17 : 1 B that discriminated them from closely related strains such as B. neidei NRRL BD-87T. On the basis of clear-cut discriminative chemotaxonomic markers, we propose strains 433-D9, 433-E17 and 121-X1, B. arvi DSM 16317T, B. arenosi DSM 16319T and B. neidei NRRL BD-87T to be reclassified within a separate genus. For this new taxon, we propose the name Viridibacillus gen. nov., and we propose the reclassification of Bacillus arvi, Bacillus arenosi and Bacillus neidei as Viridibacillus arvi gen. nov., comb. nov. (the type species of Viridibacillus, with the type strain DSM 16317T =LMG 22165T), Viridibacillus arenosi comb. nov. (type strain DSM 16319T =LMG 22166T) and Viridibacillus neidei comb. nov. (type strain NRRL BD-87T =DSM 15031T =JCM 11077T).

scholarly journals Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis

Gut ◽  
2020 ◽  
pp. gutjnl-2020-320777
Author(s):  
Yunben Yang ◽  
Lili Li ◽  
Chunjing Xu ◽  
Yunke Wang ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Cell Activation ◽  
Tumour Microenvironment ◽  
Interleukin 17 ◽  
Dependent Manner ◽  
Microbial Composition ◽  
Transcriptional Signature ◽  
Close Relationship ◽  
Enhanced Secretion ◽  
Underlying Mechanisms

ObjectiveMacrophages are among the most abundant cells in the colon tumour microenvironment, and there is a close relationship among monocytes, macrophages and the gut microbiota. Alterations in the gut microbiota are involved in tumour development, but the underlying mechanisms remain unclear. We aim to elucidate the temporal changes in macrophage subsets and functions, and how these dynamics are regulated by microbial cues in the initiation of colitis-associated cancer.DesignA mouse model of colitis-associated tumourigenesis was established to determine macrophage dynamics. The role of monocyte-like macrophage (MLM) was confirmed by targeting its chemotaxis. The effects of the gut microbiota were assessed by antibiotic treatment and faecal microbiota transplantation.ResultsA selective increase in MLMs was observed in the initial stages of colitis-associated cancer, with an enhanced secretion of inflammatory cytokines. MLM accumulation was regulated by CCL2 expression of colonic epithelial cells, which was influenced by bacteria-derived lipopolysaccharide (LPS). LPS further stimulated interleukin 1β production from MLMs, inducing interleukin-17-producing T-helper cell activation to promote inflammation. These observations were also supported by altered microbial composition associated with human colitis and colorectal cancer, evolving transcriptional signature and immune response during human colitis-associated tumourigenesis.ConclusionsThe gut microbiota uses LPS as a trigger to regulate MLM accumulation in a chemokine-dependent manner and generate a precancerous inflammatory milieu to facilitate tumourigenesis.

scholarly journals Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor

1994 ◽  
Vol 14 (11) ◽  
pp. 7245-7255
Author(s):  
K A Schandel ◽  
D D Jenness
Keyword(s):  
Cell Surface ◽  
Direct Evidence ◽  
Buoyant Density ◽  
Structural Features ◽  
Dependent Manner ◽  
Extracellular Proteases ◽  
Surface Receptors ◽  
Alpha Factor ◽  
Carboxy Terminal ◽  
Vacuolar Membranes

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.

scholarly journals Identification, Structural Characterization and Gene Expression Analysis of Members of the Nuclear Factor-Y Family in Chickpea (Cicer arietinum L.) under Dehydration and Abscisic Acid Treatments

2018 ◽  
Vol 19 (11) ◽  
pp. 3290 ◽  
Author(s):  
Ha Chu ◽  
Kien Nguyen ◽  
Yasuko Watanabe ◽  
Dung Le ◽  
Thu Pham ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
In Silico ◽  
Major Gene ◽  
Expression Patterns ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Nuclear Factor Y ◽  
Fundamental Information ◽  
Close Relationship ◽  
In Silico Analyses

In plants, the Nuclear Factor-Y (NF-Y) transcription factors (TFs), which include three distinct types of NF-YA, NF-YB, and NF-YC TFs, have been identified to play key roles in the regulation of various plant growth and developmental processes under both normal and environmental stress conditions. In this work, a total of 40 CaNF-Y-encoding genes, including eight CaNF-YAs, 21 CaNF-YBs, and 11 CaNF-YCs, were identified in chickpea, and their major gene and protein characteristics were subsequently obtained using various web-based tools. Of our interest, a phylogenetically-based analysis predicted 18 CaNF-Ys (eight CaNF-YAs, seven CaNF-YBs, and three CaNF-YCs) that potentially play roles in chickpea responses to dehydration according to their close relationship with the well-characterized GmNF-Ys in soybean. These results were in good agreement with the enrichment of drought-responsive cis-regulatory motifs and expression patterns obtained from in silico analyses using publically available transcriptome data. Most of the phylogenetically predicted drought-responsive CaNF-Y genes (15 of 18) were quantitatively validated to significantly respond to dehydration treatment in leaves and/or roots, further supporting the results of in silico analyses. Among these CaNF-Y genes, the transcript levels of CaNF-YA01 and CaNF-YC10 were the most highly accumulated in leaves (by approximately eight-fold) and roots (by approximately 18-fold), respectively, by dehydration. Furthermore, 12 of the 18 CaNF-Y genes were found to be responsive to the most well-known stress hormone, namely abscisic acid (ABA), in leaves and/or roots, suggesting that these genes may act in chickpea response to dehydration in ABA-dependent manner. Taken together, our study has provided a comprehensive and fundamental information for further functional analyses of selected CaNF-Y candidate genes, ultimately leading to the improvement of chickpea growth under water-limited conditions.

scholarly journals Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood

2006 ◽  
Vol 52 (6) ◽  
pp. 1054-1061 ◽  
Author(s):  
Isabelle Giuliani ◽  
François Rieunier ◽  
Catherine Larue ◽  
Jean-François Delagneau ◽  
Claude Granier ◽  
...  
Keyword(s):  
Heart Failure ◽  
New York ◽  
Natriuretic Peptide ◽  
Heart Association ◽  
Cross Reactivity ◽  
Accurate Estimation ◽  
Dependent Manner ◽  
Close Relationship ◽  
New York Heart Association ◽  
Set Up

Abstract Background: B-Type natriuretic peptide (BNP1–32) as well as the N-terminal fragment of the prohormone containing residues 1–76 (NT-proBNP1–76), both cleavage products of the precursor proBNP1–108, are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to develop an immunoassay that specifically measures solely proBNP1–108 in plasma. Methods: After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP1–108, an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP1–108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP1–76 or synthetic BNP1–32. By combining mAb Hinge76 with a polyclonal antibody directed against BNP1–32, we were able to set up a proBNP1–108-specific sandwich immunoassay able to confirm the presence of proBNP1–108 in blood samples. Results: From a cohort of 50 healthy persons and 170 patients with congestive heart failure (CHF), our assay was able to differentiate healthy individuals from CHF patients (P <0.005). Interestingly, plasma proBNP1–108 concentrations were correlated with New York Heart Association classification. Moreover, a close relationship between proBNP1–108 and BNP1–32 concentrations may exist, as a good correlation (r2 = 0.89) was obtained when their respective concentrations were compared. Conclusion: mAb Hinge76 is the first proBNP1–108-specific mAb produced that allows accurate estimation of proBNP1–108 concentrations in plasma.

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