Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from Borrelia turicatae, a Causative Agent of Relapsing Fever

ABSTRACT Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with therep + and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designatedrepA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that repA (or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in bothBorrelia parkeri and B. turicatae. Transcriptional analyses demonstrate that repA is expressed, albeit at low levels, during in vitro cultivation ofB. turicatae. Transcriptional start site analysis revealed that repA is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of repAand its homologs suggest that RepA may play an important genus-wide role in the biology of the Borrelia.

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In Vitro Cultivation and Morphological Characterization of Phloemic Trypanosomatids Isolated from Coconut Trees

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.2009.00454.x ◽

2010 ◽

Vol 57 (1) ◽

pp. 87-93 ◽

Cited By ~ 1

Author(s):

DARLÃ G. KELLER ◽

FLÃVIO C. MIGUENS

Keyword(s):

Morphological Characterization ◽

In Vitro Cultivation

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Binding Site Determinants for the LysR-Type Transcriptional Regulator PcaQ in the Legume Endosymbiont Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.01456-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1237-1246 ◽

Cited By ~ 15

Author(s):

Allyson M. MacLean ◽

Michelle I. Anstey ◽

Turlough M. Finan

Keyword(s):

Binding Site ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Transcriptional Start Site ◽

Mesorhizobium Loti ◽

Equilibrium Dissociation ◽

Dyad Symmetry

ABSTRACT LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the β-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions −78 to −45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many α-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

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Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5α Protein

Journal of Virology ◽

10.1128/jvi.01559-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11669-11681 ◽

Cited By ~ 52

Author(s):

Alak Kanti Kar ◽

Felipe Diaz-Griffero ◽

Yuan Li ◽

Xing Li ◽

Joseph Sodroski

Keyword(s):

Variable Region ◽

Globular Structure ◽

Biophysical Characterization ◽

Immunodeficiency Virus ◽

Infected Insect ◽

Low Levels ◽

Trim Proteins

ABSTRACT The tripartite motif (TRIM) protein, TRIM5α, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5α promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5α protein (TRIM5Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.

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In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages

Parasitology ◽

10.1017/s0031182014001656 ◽

2014 ◽

Vol 142 (04) ◽

pp. 598-611 ◽

Cited By ~ 2

Author(s):

PETER H. GAUDET ◽

RICHARD J. CAWTHORN ◽

MELANIE A. BUOTE ◽

J. FRANK MORADO ◽

GLENDA M. WRIGHT ◽

...

Keyword(s):

Developmental Stages ◽

Partial Characterization ◽

Snow Crab ◽

In Vitro Cultivation ◽

Chionoecetes Opilio

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In vitro Cultivation and Partial Characterization of Lawsonia intracellularis from a Japanese Field Case of Porcine Proliferative Enteropathy

Journal of Veterinary Medical Science ◽

10.1292/jvms.68.609 ◽

2006 ◽

Vol 68 (6) ◽

pp. 609-613 ◽

Cited By ~ 2

Author(s):

Tomohiro KOYAMA ◽

Takuya HIRAI ◽

Shinya NAGAI

Keyword(s):

Partial Characterization ◽

In Vitro Cultivation ◽

Lawsonia Intracellularis ◽

Field Case ◽

Proliferative Enteropathy

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Biometry of fruit, germination and morphogenesis in vitro of citrus after asepsis of seeds in different concentrations of sodium hypochlorite

Scientific Electronic Archives ◽

10.36560/1332020831 ◽

2020 ◽

Vol 13 (3) ◽

pp. 14

Author(s):

D. Santos ◽

W. J. Pereira ◽

D. S. Miranda ◽

J. L. C. Souza ◽

L. A. Borges ◽

...

Keyword(s):

Sodium Hypochlorite ◽

In Vitro Cultivation ◽

Sodium Hypochlorite Solution ◽

Citrus Aurantium ◽

Federal Institute ◽

Growth Room ◽

Morphogenesis In Vitro ◽

Morphological Processes

In tissue culture it is necessary to use seeds of good quality and free of pathogens in order to avoid damages in the work to be carried out in the laboratory. Therefore, strict cleaning of the seeds intended for in vitro cultivation is essential. Thus, the objective of this study was to evaluate fruit biometry, germination and morphogenesis of 'Cravo' (Citrus limonia Osbeck) and Persia Lima (Citrus aurantium) seeds in vitro submitted to different concentrations of NaClO for asepsis. Fruits of 'Cravo' lemon and Lima of Persia. Subsequently, they were transported to the Biotechnology Laboratory of the Goiano Federal Institute Campus Urutaí-GO, where they performed the biometric characterization of the fruits. The seeds of these fruits were submitted to treatment with sodium hypochlorite solution in different concentrations: 0,0; 1.0; 1.5; 2.0 and 2.5% for 10 minutes, constituting then five treatments for each variety and placed in an acclimatized growth room to analyze the results. The variables evaluated were: biometry of fruits and seedlings; oxidation indexes; germination; contamination; and types of contamination. NaClO doses were compared from 95% confidence intervals. The analyzes were performed using the glm () function of software R version 3.4.1. It was verified that the aseptic treatment with NaClO for lime and lemon was promising for the morphological processes of germination and development of seedlings, as well as for the control of contaminating agents in vitro.

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In Vitro Cultivation and Electron Microscopy Characterization of Trachipleistophora anthropophthera Isolated From the Cornea of an AIDS Patient

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.2005.00024.x ◽

2005 ◽

Vol 52 (3) ◽

pp. 179-190 ◽

Cited By ~ 10

Author(s):

SANDRA I. JUAREZ ◽

CHATURONG PUTAPORNTIP ◽

SOMCHAI JONGWUTIWES ◽

AKITOYO ICHINOSE ◽

TETSUO YANAGI ◽

...

Keyword(s):

Electron Microscopy ◽

In Vitro Cultivation ◽

Microscopy Characterization

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Production and characterization of antisera to synthetic thyrotropin-releasing hormone (TRH).

Journal of Histochemistry & Cytochemistry ◽

10.1177/30.9.6813375 ◽

1982 ◽

Vol 30 (9) ◽

pp. 926-931 ◽

Cited By ~ 12

Author(s):

Y F Chen ◽

V D Ramirez

Keyword(s):

Ethylenediaminetetraacetic Acid ◽

Double Diffusion ◽

Pituitary Hormones ◽

Thyrotropin Releasing Hormone ◽

Releasing Hormone ◽

Undiluted Serum ◽

Hypothalamic Tissue ◽

Low Levels

To generate anti-thyrotropin-releasing hormone (TRH) antibodies TRH was rendered antigenic presumably by reaction of its histidine residue with bis-diazotized benzidine (BDB) coupled to bovine serum albumin (BSA). Six California white rabbits were each injected with 330 micrograms protein/ml of emulsified immunogen by the multisite intradermal immunization technique. Seven repeated injections were given at 30-day intervals using half of the original quantity of antigen. Antibodies binding 125I-TRH appeared in the serum of four of the six rabbits three months after the first injection. Five months later the sera of two rabbits bound 50% of the labeled TRH at 1:6000 final dilution. Using this antiserum a radioimmunoassay for TRH was developed in which as little as 10 pg/300 microliter unlabeled TRH can be detected. The linear range of detectable TRH was 10 to 10000 pg. No cross-reaction with various hypothalamic and pituitary hormones, neurotransmitters, neuropeptides, and BSA were detected in this immunoassay. Extracts from rat and frog hypothalami produced 125I-TRH-binding inhibition curves parallel to synthetic TRH. Samples from elutes of rat medio-basal hypothalami superfused in vitro were examined by using this antiserum. Serial dilution of superfusate showed a similar inhibitions curve. Stimulatory effect of K+ depolarization on TRH release from superfused hypothalami was inhibited in Ca2+-free ethylenediaminetetraacetic acid medium. Ouchterlony double diffusion test of the TRH antisera revealed that low levels of antibodies against albumin but not immunoglobulin G or ovalbumin were also produced. However, the immunoprecipitates could only be detected with undiluted serum. In conclusion, this antiserum generated one of the most sensitive TRH radioimmunoassay currently available in the literature. The anti-TRH serum produced by this method can be used to examine both content of TRH from several tissues as well as release from hypothalamic tissue in vitro and might be useful to trace brain TRH pathways by immunocytochemistry.

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