Bacterial quantification in tissue homogenates from in vivo pharmacodynamic studies using growth curves

Abstract Aims Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. Methods and results NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. Conclusion Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

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Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y73-141 ◽

1973 ◽

Vol 51 (12) ◽

pp. 933-941 ◽

Cited By ~ 2

Author(s):

Njanoor Narayanan ◽

Jacob Eapen

Keyword(s):

Amino Acids ◽

Body Weight ◽

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Incorporation ◽

Contrast Administration ◽

Acid Incorporation ◽

Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

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Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4)

Journal of General Virology ◽

10.1099/jgv.0.001559 ◽

2021 ◽

Author(s):

Xiaohui Zou ◽

Yejing Rong ◽

Xiaojuan Guo ◽

Wenzhe Hou ◽

Bingyu Yan ◽

...

Keyword(s):

Reverse Genetics ◽

Growth Curves ◽

Dependent Manner ◽

Helper Plasmid ◽

Fowl Adenovirus ◽

Virus Adsorption ◽

One Step ◽

Step Growth ◽

Lmh Cells

Fibre is the viral protein that mediates the attachment and infection of adenovirus to the host cell. Fowl adenovirus 4 (FAdV-4) possesses two different fibre trimers on each penton capsomere, and roles of the separate fibres remain elusive. Here, we attempted to investigate the function of FAdV-4 fibres by using reverse genetics approaches. Adenoviral plasmids carrying fiber1 or fiber2 mutant genes were constructed and used to transfect chicken LMH cells. Fiber1-mutated recombinant virus could not be rescued. Such defective phenotype was complemented when a fiber1-bearing helper plasmid was included for co-transfection. The infection of fiber-intact FAdV-4 (FAdV4-GFP) to LMH cells could be blocked with purified fiber1 knob protein in a dose-dependent manner, while purifed fiber2 knob had no such function. On the contrary, fiber2-mutated FAdV-4, FAdV4XF2-GFP, was successfully rescued. The results of one-step growth curves showed that proliferative capacity of FAdV4XF2-GFP was 10 times lower than that of the control FAdV4-GFP. FAdV4XF2-GFP also caused fewer deaths of infected chicken embryos than FAdV4-GFP did, which resulted from poorer virus replication in vivo. These data illustrated that fiber1 mediated virus adsorption and was essential for FAdV-4, while fiber2 was dispensable although it significantly contributed to the virulence.

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Effect of bacterial endotoxemia on succinic dehydrogenase of liver and kidney of rabbit

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1961.201.1.16 ◽

1961 ◽

Vol 201 (1) ◽

pp. 16-18 ◽

Cited By ~ 2

Author(s):

J. Cascarano ◽

A. D. Rubin ◽

A. K. Neumann ◽

B. W. Zweifach

Keyword(s):

Succinic Dehydrogenase ◽

Significant Inhibition ◽

Frozen Sections ◽

Experimental Animals ◽

E Coli ◽

Fresh Frozen ◽

Tissue Homogenates ◽

Liver And Kidney ◽

Lethal Doses

The in vivo inhibition of liver and kidney succinic dehydrogenase by administration of lethal doses of bacterial endotoxin ( Escherichia coli and Salmonella typhosa) was investigated. Quantitative determinations conducted on tissue homogenates revealed significant inhibition of activity only in liver of rabbits injected with E. coli lipopolysaccharide. The histochemical distribution of succinic dehydrogenase in fresh frozen sections of kidney was the same in both control and experimental animals. However, the centrolobular areas of liver appeared considerably depressed in activity in both E. coli and S. typhosa endotoxin-treated animals. These data, along with those presented by other studies in the literature, suggest that the action of endotoxin appears to be restricted to certain cells.

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The effects of a dietary excess of leucine on the synthesis of nicotinamide nucleotides in the rat

British Journal Of Nutrition ◽

10.1079/bjn19830041 ◽

1983 ◽

Vol 49 (3) ◽

pp. 321-329 ◽

Cited By ~ 18

Author(s):

Bahieldin I. Magboul ◽

David A. Bender

Keyword(s):

Significant Inhibition ◽

Nucleotide Synthesis ◽

Tryptophan Oxygenase ◽

Rate Limiting Step ◽

Minimum Requirements ◽

Tissue Homogenates ◽

Rate Limiting ◽

Competitive Nature ◽

Precipitating Factor

1. In order to test the suggestion that a dietary excess of leucine may be a precipitating factor in pellagra, rats were fed on diets that provided 15 g leucine/kg in excess of requirements for 7 weeks from weaning. This led to a significant reduction in the concentrations of nicotinamide nucleotides in liver and blood. The effect was only apparent when the diets provided less than a minimally adequate amount of nicotinamide, so that the animals were dependent on the synthesis of nicotinamide nucleotides from tryptophan to meet all or part of their requirements.2. Urinary excretion of N1-methyl nicotinamide was not a useful indicator of tissue concentrations of nicotinamide nucleotides, and seemed not to be adequately sensitive to differentiate between minimal adequacy and marginal deficiency, as demonstrated by changes in concentrations of nicotinamide nucleotides in liver and blood.3. The addition of leucine to incubation media for the measurement of enzyme activity in tissue homogenates at concentrations within the physiological range, led to a significant activation of tryptophan oxygenase (L-tryptophan: oxygen oxidoreductase (decyclizing), EC 1.13.11.11) and significant inhibition of kynureninase (L-kynurenine hydrolase, EC 3.7.1.3). The effect on tryptophan oxygenase may not be physiologically significant, in view of the considerable range of activity of this enzyme under normal conditions. However, the inhibition of kynureninase, which was primarily competitive with respect to the substrate, probably is physiologically significant, and was enough for this enzyme to become a probable rate-limiting step in tryptophan metabolism and nicotinamide nucleotide synthesis. Other enzymes of the tryptophan – nicotinamide nucleotide pathway were not affected by the addition of leucine to the incubation medium.4. Feeding 15 g leucine/kg diet in excess of minimum requirements had no effect on the activities of tryptophan oxygenase or kynureninase in liver homogenates. This may reflect the reversible competitive nature of the inhibition of kynureninase by leucine, and hence be an artefact of the incubation procedure. Rats fed on the high-leucine diets excreted significantly more kynurenine than did control animals, which is evidence of inhibition of kynureninase in vivo.5. It appears that a dietary excess of leucine, of the order of 15 g/kg above requirements, may be a precipitating factor in pellagra when there is reliance on the synthesis of nicotinamide nucleotides from tryptophan to meet a part or all of the requirements, but not when minimally adequate niacin is available from the diet.

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Autographa californica Nucleopolyhedrovirus orf69 Encodes an RNA Cap (Nucleoside-2′-O)-Methyltransferase

Journal of Virology ◽

10.1128/jvi.77.6.3430-3440.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3430-3440 ◽

Cited By ~ 19

Author(s):

Xiaofeng Wu ◽

Linda A. Guarino

Keyword(s):

Late Phase ◽

Growth Curves ◽

Immunoblot Analysis ◽

Single Step ◽

Coding Region ◽

Methyltransferase Activity ◽

Autographa Californica Nucleopolyhedrovirus ◽

High Conservation ◽

Step Growth

ABSTRACT The AcNPV orf69 gene encodes a protein that contains an S-adenosylmethionine (AdoMet)-dependent methyltransferase signature motif. More significantly, ORF69 shows high conservation at residues diagnostic for (nucleoside 2′-O)-methyltransferase activity. To analyze the function of this protein, which was renamed MTase1, it was overexpressed in Escherichia coli and purified to homogeneity. Photo cross-linking experiments showed that MTase1 bound AdoMet, and functional assays demonstrated cap 0-dependent methyltransferase activity. In vivo expression assays in insect cells showed that MTase1 was synthesized during the late phase of infection and that its expression was dependent on viral DNA replication. Primer extension analysis identified a late promoter motif, ATAAG, at the transcription start site. A mutant virus was constructed by inserting the lacZ gene into the coding region of mtase1. Immunoblot analysis confirmed that MTase1 was not synthesized in these cells, and single-step growth curves revealed that the rate of virus replication in tissue culture was not affected by the absence of MTase1.

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A Novel In Vivo Animal Model for Human Multiple Myeloma Based on Bioluminescence Imaging of Tumor Cell Growth.

Blood ◽

10.1182/blood.v106.11.3452.3452 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3452-3452

Author(s):

Anton C. Martens ◽

Henk Rozemuller ◽

Ellen van der Spek ◽

Lijnie Bogers-Boer ◽

Niels van de Donk ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Growth ◽

Quantitative Evaluation ◽

Bioluminescence Imaging ◽

Growth Curves ◽

Population Doubling ◽

Tumor Mass ◽

In Vivo Animal Model

Abstract Preclinical testing of new therapeutical strategies for the treatment of multiple myeloma (MM) requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. Models that meet both requirements have thus far not been described. Here we present a novel in vivo MM model by engraftment of MM U266 or RPMI-8226/S cells, both of human origin, into RAG2γc double knock-out mice. These mice are totally immune deficient because they lack T-, B and NK cells and the mice easily accept human cells (van Rijn et al., Blood 2003, Rozemuller et al., 2004). After intravenous injection of 2x106 MM cells engraftment and outgrowth occurred in all mice but was limited to the bone marrow compartment only. Flow cytometry (FCM) confirmed the presence of human CD45/38/138 positive MM cells in femur, spine, tibia and sternum bone specimens. Infiltration into other organs was not observed. In a next step MM cells were stably transduced using a retroviral vector encoding both the Green Fluorescent Protein (GFP) and firefly Luciferase (fLuc) marker genes. Technical advances in recent years in optical imaging by Bioluminescence Imaging (BLI) techniques allow visualization and quantification of bioluminescent light by detecting photons that are transmitted through mammalian tissue. When luciferase converts the substrate luciferin, photons are emitted that can be registered by using sensitive CCCD cameras. The absolute number of photons that are produced correlates, in our application, with local tumor mass. Mice were injected i.v. with 2x106 GFP-fLuc transduced MM cells (U266 or RPMI8226/S) and imaged weekly using BLI. Within 2 weeks after injection significant BLI signals were detectable. Per mouse 5-10 foci showed luciferase activity, predominantly in the pelvic region, skull, limbs, sternum, ribs and the spine. This low frequency of engraftment is in line with earlier reports on RPMI8226/S (Mitsiades et al., Cancer Res 2003). At 9 weeks the first mice developed hind leg paralysis which could be attributed to tumor associated spinal lesions. After 12 weeks the last mouse was sacrificed. BLI revealed that the intensity of light production at the various sites of tumor growth within individual mice as well as between mice showed a similar increase. This reflects an increase in tumor mass. Quantitative analysis of subsequent BLI images allowed construction of tumor growth curves of the total tumor mass per mouse as well as for the individual foci of MM growth in individual mice. We typically observed exponential growth, with growth curves running parallel with an average population doubling time of approximately 4–5 days. The range in which tumor growth could be monitored (and as a consequence also the response to treatment) spans 3–4 decades. In contrast with previously reported murine models for human MM where -next to bone marrow homing- also extra-skeletal tumors were observed our model almost exclusively shows homing of MM cells to the BM and is therefore more consistent with the clinical manifestation in myeloma patients. The major advantage of the model is the option for quantitative evaluation of the effect of a given treatment on the tumorload. Currently we are studying the efficacy of newly developed geranyl-geranyl-transferase inhibitors (GGTI). In conclusion, we have developed a novel in vivo model to study the characteristics of homing and outgrowth of MM and for quantitative evaluation of the efficacy of the therapeutic intervention applied.

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Combined structural, biochemical and cellular evidence demonstrates that both FGDF motifs in alphavirus nsP3 are required for efficient replication

Open Biology ◽

10.1098/rsob.160078 ◽

2016 ◽

Vol 6 (7) ◽

pp. 160078 ◽

Cited By ~ 20

Author(s):

Tim Schulte ◽

Lifeng Liu ◽

Marc D. Panas ◽

Bastian Thaa ◽

Nicole Dickson ◽

...

Keyword(s):

Viral Replication ◽

Semliki Forest Virus ◽

Protein Structures ◽

Growth Curves ◽

Structural Protein ◽

Binding Studies ◽

Efficient Replication ◽

Hierarchical Interaction

Recent findings have highlighted the role of the Old World alphavirus non-structural protein 3 (nsP3) as a host defence modulator that functions by disrupting stress granules, subcellular phase-dense RNA/protein structures formed upon environmental stress. This disruption mechanism was largely explained through nsP3-mediated recruitment of the host G3BP protein via two tandem FGDF motifs. Here, we present the 1.9 Å resolution crystal structure of the NTF2-like domain of G3BP-1 in complex with a 25-residue peptide derived from Semliki Forest virus nsP3 (nsP3-25). The structure reveals a poly-complex of G3BP-1 dimers interconnected through the FGDF motifs in nsP3-25. Although in vitro and in vivo binding studies revealed a hierarchical interaction of the two FGDF motifs with G3BP-1, viral growth curves clearly demonstrated that two intact FGDF motifs are required for efficient viral replication. Chikungunya virus nsP3 also binds G3BP dimers via a hierarchical interaction, which was found to be critical for viral replication. These results highlight a conserved molecular mechanism in host cell modulation.

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Supplementation of Perkinsus marinus Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.421-431.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 421-431 ◽

Cited By ~ 23

Author(s):

Christopher G. Earnhart ◽

Mary Ann Vogelbein ◽

Gwynne D. Brown ◽

Kimberly S. Reece ◽

Stephen L. Kaattari

Keyword(s):

Cell Size ◽

Expression Profiles ◽

Tissue Homogenate ◽

Perkinsus Marinus ◽

Oyster Species ◽

Tissue Homogenates ◽

Protease Secretion ◽

Protease Expression

ABSTRACT The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.

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Quantification of the Relationship between Bacterial Kinetics and Host Response for Monkeys Exposed to AerosolizedFrancisella tularensis

Applied and Environmental Microbiology ◽

10.1128/aem.01190-10 ◽

2010 ◽

Vol 77 (2) ◽

pp. 485-490 ◽

Cited By ~ 11

Author(s):

Yin Huang ◽

Charles N. Haas

Keyword(s):

Survival Data ◽

Count Data ◽

Host Response ◽

Growth Curves ◽

Bacterial Count ◽

Extensive Literature ◽

Schu S4 ◽

The Relationship ◽

Dose Dependent

ABSTRACTFrancisella tularensiscan be disseminated via aerosols, and once inhaled, only a few microorganisms may result in tularemia pneumonia. Effective responses to this threat depend on a thorough understanding of the disease development and pathogenesis. In this study, a class of time-dose-response models was expanded to describe quantitatively the relationship between the temporal probability distribution of the host response and thein vivobacterial kinetics. An extensive literature search was conducted to locate both the dose-dependent survival data and thein vivobacterial count data of monkeys exposed to aerosolizedF. tularensis. One study reporting responses of monkeys to four different sizes of aerosol particles (2.1, 7.5, 12.5, and 24.0 μm) of the SCHU S4 strain and three studies involving fivein vivogrowth curves of various strains (SCHU S4, 425, and live vaccine strains) initially delivered to hosts in aerosol form (1 to 5 μm) were found. The candidate models exhibited statistically acceptable fits to the time- and dose-dependent host response and provided estimates for the bacterial growth distribution. The variation pattern of such estimates with aerosol size was found to be consistent with the reported pathophysiological and clinical observations. The predicted growth curve for 2.1-μm aerosolized bacteria was highly consistent with the available bacterial count data. This is the first instance in which the relationship between thein vivogrowth ofF.tularensisand the host response can be quantified by mechanistic mathematical models.

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