Evaluation of a gas in vitro system for predicting methane production in vivo

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

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Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

Eukaryotic Cell ◽

10.1128/ec.00284-06 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2214-2221 ◽

Cited By ~ 53

Author(s):

Lois M. Douglas ◽

Li Li ◽

Yang Yang ◽

A. M. Dranginis

Keyword(s):

Saccharomyces Cerevisiae ◽

Biofilm Formation ◽

In Vitro System ◽

Glycosylphosphatidylinositol Anchor ◽

Fungal Adhesion ◽

Very High ◽

Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

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Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4746 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4746-4749 ◽

Cited By ~ 97

Author(s):

D I Chasman ◽

J Leatherwood ◽

M Carey ◽

M Ptashne ◽

R D Kornberg

Keyword(s):

Rna Polymerase Ii ◽

Fusion Proteins ◽

In Vitro System ◽

Transcription System ◽

Natural Mechanism ◽

Transcription In Vitro ◽

Rna Polymerase Ii Transcription ◽

Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

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Accurate transcription of a plant mitochondrial gene in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2035-2039.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2035-2039

Author(s):

P J Hanic-Joyce ◽

M W Gray

Keyword(s):

Mitochondrial Gene ◽

Plant Mitochondria ◽

In Vitro Transcription ◽

Dna Templates ◽

In Vitro System ◽

Transcription System ◽

Translation Initiation Codon ◽

Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

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Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽

10.1017/s0043174500055417 ◽

1980 ◽

Vol 28 (3) ◽

pp. 334-340 ◽

Cited By ~ 26

Author(s):

Luanne M. Deal ◽

J. T. Reeves ◽

B. A. Larkins ◽

F. D. Hess

Keyword(s):

Protein Synthesis ◽

Sand Culture ◽

Leucine Incorporation ◽

Insoluble Protein ◽

In Vitro System ◽

A Cell ◽

Protein Incorporation ◽

Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

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Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6726-6734.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6726-6734 ◽

Cited By ~ 71

Author(s):

Tetsuya Miyamoto ◽

Junichi Obokata ◽

Masahiro Sugiura

Keyword(s):

Rna Editing ◽

Editing Site ◽

Mrna Editing ◽

Higher Plant ◽

In Vitro System ◽

Cis Acting ◽

Biochemical Identification ◽

Editing Activity

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

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Modelling the impact of the macroalgae Asparagopsis taxiformis on rumen microbial fermentation

10.1101/2020.11.09.374330 ◽

2020 ◽

Author(s):

Rafael Muñoz-Tamayo ◽

Juana C. Chagas ◽

Mohammad Ramin ◽

Sophie J. Krizsan

Keyword(s):

Methane Production ◽

Volatile Fatty Acids ◽

Mean Squared Error ◽

Microbial Fermentation ◽

Model Structure ◽

Red Macroalgae ◽

Asparagopsis Taxiformis ◽

The Impact

AbstractBackgroundThe red macroalgae Asparagopsis taxiformis is a potent natural supplement for reducing methane production from cattle. A. taxiformis contains several anti-methanogenic compounds including bromoform that inhibits directly methanogenesis. The positive and adverse effects of A. taxiformis on the rumen microbiota are dose-dependent and operate in a dynamic fashion. It is therefore key to characterize the dynamic response of the rumen microbial fermentation for identifying optimal conditions on the use of A. taxiformis as a dietary supplement for methane mitigation. Accordingly, the objective of this work was to model the effect of A. taxiformis supplementation on the rumen microbial fermentation under in vitro conditions. We adapted a published mathematical model of rumen microbial fermentation to account for A. taxiformis supplementation. We modelled the impact of A. taxiformis on the fermentation and methane production by two mechanisms, namely (i) direct inhibition of the growth rate of methanogenesis by bromoform and (ii) hydrogen control on sugars utilization and on the flux distribution towards volatile fatty acids production. We calibrated our model using a multi-experiment estimation approach that integrated experimental data with six macroalgae supplementation levels from a published in vitro study assessing the dose-response impact of A. taxiformis on rumen fermentation.Resultsour model captured satisfactorily the effect of A. taxiformis on the dynamic profile of rumen microbial fermentation for the six supplementation levels of A. taxiformis with an average determination coefficient of 0.88 and an average coefficient of variation of the root mean squared error of 15.2% for acetate, butyrate, propionate, ammonia and methane.Conclusionsour results indicated the potential of our model as prediction tool for assessing the impact of additives such as seaweeds on the rumen microbial fermentation and methane production in vitro. Additional dynamic data on hydrogen and bromoform are required to validate our model structure and look for model structure improvements. We are working on model extensions to account for in vivo conditions. We expect this model development can be useful to help the design of sustainable nutritional strategies promoting healthy rumen function and low environmental footprint.

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A new in vitro system for studying secondary palate development

Development ◽

10.1242/dev.34.2.485 ◽

1975 ◽

Vol 34 (2) ◽

pp. 485-495

Author(s):

L. Brinkley ◽

G. Basehoar ◽

A. Branch ◽

J. Avery

Keyword(s):

Gas Permeation ◽

Present System ◽

Embryonic Mouse ◽

Fluid Medium ◽

In Vitro System ◽

Palate Development ◽

Fixed Orientation ◽

The Brain

An in vitro system was devised which supports palate development in partially dissected embryonic mouse heads. The heads were suspended in the culture chamber so that they were not held in a fixed orientation and were constantly surrounded with a fluid medium. Under these circumstances the developing palate must effect closure without the aid of gravitational forces. The culture medium was constantly circulated, gassed with 95% O2, 5% CO2 using hollow fiber gas permeation devices, and kept at 34°C. Swiss-Webster mouse embryos of 12 days 12–18 h (ca. 48 h prior to expected in vivo closure) or 13 days 8–14 h (ca. 24 h prior to closure) were used to test the ability of the system to support palatal development. Embryonic heads were dissected in one of two ways before culture: brain and tongue removed, or brain, tongue and mandible removed. After 24 h in culture, preparations of either age with only the brain and tongue removed had made substantially greater progress than their counterparts with the brain, tongue and mandible removed. With only the brain and tongue removed, the palatal shelves were contacting, adhered or fused in 67 % of the older embryos, whereas most of the embryos of the same age cultured with the brain, tongue and mandible removed had shelves that were not fully elevated and still separated by a moderate gap. Thus for maximal progress in the present system, the oral cavity must be intact except for the tongue.

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