Control of chondroitin sulphate biosynthesis. β-d-Xylopyranosides as substrates for UDP-galactose: d-xylose transferase from embryonic-chicken cartilage

Embryonic-chicken epiphyseal cartilage was incubated in vitro with a variety of beta-xylosides and the amount of [3H]acetate incorporation into chondroitin sulphate was determined under conditions when normal protein core production was inhibited by cycloheximide. The ability of the different beta-xylosides to relieve thea cycloheximide-mediated inhibition of chondroitin sulphate synthesis was influenced by the nature of the aglycan group of te xyloside. beta-Xylosides with apolar and uncharged aglycan groups were most effective and produced a severalfold stimulation of chondroitin sulphate biosynthesis. beta-Xylosides with charged aglycan groups were less effective initiators of chondroitin sulphate synthesis. The rate of galactose transfer from UDP-galactose to each of the beta-xylosides, catalysed by a cell-free microsomal preparation from embryonic cartilage, was measured. This study showed that the nature of the aglycan group of the beta-xyloside was a factor determining the capacity of the xyloside to act as an acceptor for galactosyltransferase I, the enzyme that catalyses the first galactose transfer reaction of chondroitin sulphate synthesis. The aglycan group of the xyloside also appeared to influence other steps leading to chondroitin sulphate chain initiation in vitro.

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Initiation of chondroitin sulphate synthesis by β-d-galactosides. Substrates for galactosyltransferase II

Biochemical Journal ◽

10.1042/bj2270805 ◽

1985 ◽

Vol 227 (3) ◽

pp. 805-814 ◽

Cited By ~ 6

Author(s):

J A Robinson ◽

H C Robinson

Keyword(s):

Chick Embryo ◽

Glucuronic Acid ◽

Chondroitin Sulphate ◽

Acetate Incorporation ◽

Glycosidic Linkage ◽

Microsomal Preparation ◽

Galactose Residue ◽

Chain Synthesis ◽

Chondroitin Sulphate Chain

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.

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The sulphation of chondroitin sulphate in embryonic chicken cartilage

Biochemical Journal ◽

10.1042/bj1130543 ◽

1969 ◽

Vol 113 (3) ◽

pp. 543-549 ◽

Cited By ~ 19

Author(s):

H. C. Robinson

Keyword(s):

Ionic Strength ◽

Incubation Time ◽

Chondroitin Sulphate ◽

Soluble Enzyme ◽

Enzyme Preparations ◽

Embryonic Chicken ◽

Inorganic Sulphate ◽

Effects Of Ph ◽

Embryonic Cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [35S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1·5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3′-phosphate 5′-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.

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The effect of β-d-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors

Biochemical Journal ◽

10.1042/bj1620217 ◽

1977 ◽

Vol 162 (2) ◽

pp. 217-233 ◽

Cited By ~ 34

Author(s):

K D Gibson ◽

B J Segen ◽

T K Audhya

Keyword(s):

Protein Synthesis ◽

Incubation Medium ◽

Chondroitin Sulphate ◽

Gel Filtration ◽

Protein Synthesis Inhibitors ◽

Serum Factors ◽

Embryonic Chicken ◽

Low Concentrations ◽

Normal Human

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3′,5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

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The effect of D-xylose, β-D-xylosides and β-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage

Biochemical Journal ◽

10.1042/bj1480025 ◽

1975 ◽

Vol 148 (1) ◽

pp. 25-34 ◽

Cited By ~ 114

Author(s):

H C Robinson ◽

M J Brett ◽

P J Tralaggan ◽

D A Lowther ◽

M Okayama

Keyword(s):

Incubation Medium ◽

Chondroitin Sulphate ◽

Polysaccharide Chain ◽

Embryonic Chicken ◽

Chain Synthesis

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, xβ-D-xylosides and β-D-galactosides to the incubation medium. β-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, β-D-xylosides and β-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

10.26434/chemrxiv.10299164.v1 ◽

2019 ◽

Author(s):

Candace E. Benjamin ◽

Zhuo Chen ◽

Olivia Brohlin ◽

Hamilton Lee ◽

Stefanie Boyd ◽

...

Keyword(s):

Cellular Uptake ◽

Rhodamine B ◽

Virus Like Particle ◽

A Cell ◽

Viral Nanoparticles ◽

The Stability ◽

Open Question ◽

Viral Surface ◽

Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

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Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

Nuklearmedizin ◽

10.3413/nukmed-0450-11-12 ◽

2012 ◽

Vol 51 (05) ◽

pp. 179-185 ◽

Cited By ~ 3

Author(s):

M. Wendisch ◽

D. Aurich ◽

R. Runge ◽

R. Freudenberg ◽

J. Kotzerke ◽

...

Keyword(s):

Cell Model ◽

Low Energy ◽

Thyroid Cells ◽

Low Energy Electrons ◽

A Cell ◽

Vitro Model ◽

Uptake Studies ◽

Radiobiological Effects ◽

Radioactivity Retention

SummaryTechnetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, 99mTc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much 99mTc can be present in exposed cells and which radiobiological effects could be estimated in 99mTc-overloaded cells. Methods: Sodium iodine symporter (NIS)- positive thyroid cells were used. 99mTc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyro - phosphate kit solution or as a standard 99mTc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. Results: Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of 99mTc and is followed by further pertechnetate influx to an unexpectedly high 99mTc level. The uptake of 99mTc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3–5% to >80%. The maximum possible cellular uptake of 99mTc was 90 Bq/cell. Compared with nearly pure extracellular irradiation from routine 99mTc complexes, cell survival was reduced by 3–4 orders of magnitude after preincubation with stannous pyrophosphate. Conclusions: Intra cellular 99mTc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.

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Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-87-90 ◽

2019 ◽

Vol 35 (6) ◽

pp. 87-90

Author(s):

S.V. Nikulin ◽

V.A. Petrov ◽

D.A. Sakharov

Keyword(s):

Impedance Spectroscopy ◽

Cell Monolayer ◽

Microfluidic Chips ◽

Russian Science ◽

Electric Capacitance ◽

A Cell ◽

Static Conditions ◽

Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

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Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells

BMJ Open Respiratory Research ◽

10.1136/bmjresp-2020-000830 ◽

2021 ◽

Vol 8 (1) ◽

pp. e000830

Author(s):

Souichi Yamada ◽

Shuetsu Fukushi ◽

Hitomi Kinoshita ◽

Makoto Ohnishi ◽

Tadaki Suzuki ◽

...

Keyword(s):

Virus Isolation ◽

Laboratory Diagnostics ◽

Viral Isolation ◽

Viral Loads ◽

A Cell ◽

Blind Passage ◽

Upper Respiratory ◽

Novel Coronavirus ◽

Respiratory Specimens

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

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Identifying volatile in vitro biomarkers for oral bacteria with proton-transfer-reaction mass spectrometry and gas chromatography–mass spectrometry

Scientific Reports ◽

10.1038/s41598-021-96287-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kajsa Roslund ◽

Markku Lehto ◽

Pirkko Pussinen ◽

Kari Hartonen ◽

Per-Henrik Groop ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Proton Transfer ◽

Transfer Reaction ◽

Proton Transfer Reaction ◽

Oral Bacteria ◽

Gas Chromatography Mass Spectrometry ◽

Volatile Analysis ◽

Bacterial Volatiles

AbstractWe have measured the volatile fingerprints of four pathogenic oral bacteria connected to periodontal disease and dental abscess: Porphyromonas gingivalis (three separate strains), Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia. Volatile fingerprints were measured in vitro from the headspace gas of the bacteria cultured on agar. Concrete identification of new and previously reported bacterial volatiles were performed by a combination of solid phase microextraction (SPME) and offline gas chromatography–mass spectrometry (GC–MS). We also studied the effect of the reduced electric field strength (E/N) on the fragmentation patterns of bacterial volatiles in online proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS). We aimed to discover possible new biomarkers for the studied oral bacteria, as well as to validate the combination of GC–MS and PTR-MS for volatile analysis. Some of the most promising compounds produced include: 1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), indole, and a cascade of sulphur compounds, such as methanethiol, dimethyl disulphide (DMDS) and dimethyl trisulphide (DMTS). We also found that several compounds, especially alcohols, aldehydes and esters, fragment significantly with the PTR-MS method, when high E/N values are used. We conclude that the studied oral bacteria can be separated by their volatile fingerprints in vitro, which could have importance in clinical and laboratory environments. In addition, using softer ionization conditions can improve the performance of the PTR-MS method in the volatile analysis of certain compounds.

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